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WO2021047775A1 - Use of inhibitors of tgfb/activinb signaling pathway for the treatment of patients suffering from medulloblastoma group 3 - Google Patents

Use of inhibitors of tgfb/activinb signaling pathway for the treatment of patients suffering from medulloblastoma group 3 Download PDF

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Publication number
WO2021047775A1
WO2021047775A1 PCT/EP2019/074309 EP2019074309W WO2021047775A1 WO 2021047775 A1 WO2021047775 A1 WO 2021047775A1 EP 2019074309 W EP2019074309 W EP 2019074309W WO 2021047775 A1 WO2021047775 A1 WO 2021047775A1
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Prior art keywords
group
activinb
pathway
tgfp
expression
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PCT/EP2019/074309
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French (fr)
Inventor
Celio POUPONNOT
Magalie LARCHER
Morgane MORABITO
Alain EYCHENE
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique (Cnrs)
Institut Curie
Université Paris-Saclay
Universite De Paris
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Priority to PCT/EP2019/074309 priority Critical patent/WO2021047775A1/en
Publication of WO2021047775A1 publication Critical patent/WO2021047775A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is in the field of medicine, in particular oncology.
  • Medulloblastoma a cerebellar tumor
  • MB Medulloblastoma
  • Current therapy associates surgery, chemotherapy, and radiotherapy.
  • This aggressive regimen allowed an increase in the overall survival rate up to 70-80% but induces dramatic long-term side effects (Martin et al, 2014).
  • the overall survival rate of high-risk patients is far below (Holgado et al, 2017; Wang et al, 2018). It is therefore crucial to identify new treatments that decrease side effects and improve efficacy.
  • Genomic and transcriptomic approaches allowed the stratification of MB patients into 4 different molecular groups: WNT (Wingless), SHH (Sonic Hedgehog), Group 3, and Group 4 (Northcott et al, 2012a; Taylor et al, 2012). These groups display differences in terms of cell of origin, transcriptional, epigenetic, and mutational signatures. They also differ in their clinical characteristics such as histology, overall survival rate, and presence of metastases. Recently, intragroup heterogeneity has been further uncovered, allowing their division into subtypes with some specific clinical parameters as well as genomic alterations (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017).
  • the WNT group represents 10% of all MBs and is driven by constitutive activation of the WNT/p-catenin pathway with patients showing the best prognosis.
  • the SHH group accounts for 20-25% of MB and is characterized by mutations involving different mediators of the SHH pathway. It is considered of intermediate prognosis.
  • recent sub-classifications identified SHH subtypes with poorer outcomes (Cavalli et al, 2017a; Schwalbe et al, 2017).
  • Group 3 and Group 4 are far less characterized due to their genetic and clinical heterogeneity.
  • Group 3 represents 20-25% of MB patients and is associated with bad prognosis. This group is highly metastatic and characterized by MYC overexpression, which can be explained in 15-20% of cases by its amplification. However, MYC overexpression is not sufficient to induce Group 3 MB and requires additional cooperating oncogenic events (Kawauchi et al, 2012; Pei et al, 2012). Some of them have been identified, such as GFI1 and GFI1B that are highly expressed in a subset of Group 3 through enhancer hijacking (Northcott et al, 2014). These transcription factors have been demonstrated to drive Group 3 MB tumorigenesis in animal models when associated with MYC overexpression (Northcott et al, 2014).
  • Group 3 is characterized by the expression of a photoreceptor program defined by genes whose expression is highly restricted to the retina (Kool et al, 2008; Cho et al, 2011). We recently uncovered that this program defines a subtype within Group 3 tumors, which exhibits a functional dependency to this ectopic program through its two main drivers, the retina-specific transcription factors NRL and CRX (Garancher et al, 2018). Thus, Group 3 can be subdivided into 2-3 different subtypes according to the different studies (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017).
  • Cavalli et al (2017a) have identified 3 subtypes, one is composed of tumors with high MYC expression including those with amplification of this gene, named G3y. This subtype has the worse prognosis.
  • the second subtype, t3b is overrepresented by tumors with GFI1 alterations, and the last one G3a, by tumors expressing photoreceptor genes in which few amplifications of mediators of the TGFp/Activin pathway can be found (Cavalli et al, 2017a). Since Group 3 displays the worse prognosis, targeted therapies are actively searched.
  • OTX2 has been demonstrated to be a target gene of this signaling pathway (Jia et al, 2009), it has been proposed WO 2021/047775 PCT/EP2019/074309 that OTX2 amplifications could represent a mechanism by which the pathway is also deregulated downstream (Northcott et al, 2012b).
  • the putative significance of this signaling pathway in Group 3 was reinforced by two subsequent studies, one involving sequencing in a large cohort of MB (Northcott et al, 2017) and the other showing that several components of this signaling pathway could also be deregulated at their expression level, through Group 3- specific enhancers (Lin et al, 2016). Although these studies might indicate a potential deregulation of the Smad2/3 signaling pathway, this could account for only a modest proportion of Group 3 tumors.
  • the TGFP superfamily is a large family of cytokines divided into two distinct groups of ligands: the TGFPs/Activins and the BMPs.
  • TGFp/Activin ligands signal through Smad2/3. These ligands bring together two types of serine/threonine kinase receptors, the type I and the type II, which are specific for a set of ligands.
  • the TGFPs (TGFpi, TGFP2, and TGFP3) signal through the TGFBR1 type I and TGFBR2 type II receptors.
  • Activin encoded by 4 different genes, INHBA, INHBB, INHBC, and INHBE, can activate different couples of receptors including the ACVR2A and ACVR2B type II and ACVR1A (ALK4) and ACVR1C (ALK7) type I receptors.
  • INHA encoding inhibin-a, is an inhibitor of the Activin ligands.
  • Activin and TGFP ligands lead to the phosphorylation and activation of the same intracellular mediators, Smad2 and Smad3, which then associate with the co-Smad, Smad4.
  • the hetero-complex translocates to the nucleus, where it activates the transcription of target genes with the help of DNA binding partners (Levy & Hill, 2006; Ross & Hill, 2008).
  • TGFp/ Activin signaling displays pleiotropic functions depending on the cellular and environmental context. Its implication in cancer has been well documented, mainly through TGFP ligands, although BMPs and Activins ligands can be also involved (Seoane & Gomis, 2017).
  • the role of the TGFP signaling pathway in cancer is complex, acting either as a tumor suppressor pathway in some instances or as a tumor promoter in others (Massague, 2008; Seoane & Gomis, 2017). Its oncogenic role is mainly associated with an autocrine (or paracrine) stimulation, due to the strong expression of TGFP ligands.
  • TGFP pathway has been shown to promote cell proliferation in specific context such as in Glioblastoma (Bruna et al, 2007) and cancer stem cell maintenance (Penuelas et al, 2009; Anido et al, 2010; Lonardo et al, 2011).
  • Studies on the role of Activin ligands in cancer are much more scarce (Wakefield & Hill, 2013).
  • Smad2/3 By activating the same mediators Smad2/3, a parallel can be drawn between TGFP and Activin.
  • Activins act both as tumor suppressors and tumor promoters (Chen et al, 2002; WO 2021/047775 PCT/EP2019/074309
  • Their pro- tumorigenic role has been validated in animal models in which deletion of the activin inhibitor, INHA, led to gonadal tumors in mice as well as cachexia-like syndrome (Matzuk et al, 1994; Vassalli et al, 1994).
  • ActivinB has also been shown to play a role in cancer stem cell maintenance (Lonardo et al, 2011) and in cell dedifferentiation in an insulinoma mouse model and deletion of INHBB encoding ActivinB increases survival (Ripoche et al, 2015).
  • the present invention relates to use of inhibitors of the TGFp/ActivinB signaling pathway for the treatment of patients suffering from medulloblastoma group 3.
  • Medulloblastoma is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFp/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. The inventors showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation.
  • the first object of the present invention relates to a method of treating a patient suffering from medulloblastoma group 3 comprising administering to the patient a therapeutically effective amount of an inhibitor of the TGFp/ActivinB signaling pathway.
  • medulloblastoma has its general meaning in the art and refers to a fast-growing, aggressive, high-grade brain tumor. Regardless of the subtype, medulloblastoma always occurs in the cerebellum of the brain, and more specifically, within the posterior fossa of the cerebellum. The term is also known as cerebellar primitive neuroectodermal tumor (PNET). Symptoms of medulloblastoma include, but are not limited to, behavioral changes, changes in appetite, and symptoms of increased pressure on the brain (e.g., headache, nausea, vomiting, and drowsiness, as well as problems with coordination (e.g. clumsiness, problems with handwriting, and visual problems)).
  • PNET cerebellar primitive neuroectodermal tumor
  • Unusual eye movements may also occur. If the cancer has spread to the spinal cord, symptoms may include back pain, trouble walking, and/or problems controlling bladder and bowel functions.
  • Genomic and transcriptomic approaches allowed the stratification of MB patients into 4 different molecular groups: WNT (Wingless), SHH (Sonic Hedgehog), Group 3, and Group 4 (Northcott et al, 2012a; Taylor et al, 2012).
  • the inhibitors of the TGFp/ActivinB signaling pathway are particularly suitable for the treatment of patients belonging to group 3, or event patients belonging to the subtype Group 3a according to the classification of Cavalli et al (2017a).
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • induction regimen or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high WO 2021/047775 PCT/EP2019/074309 level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • the phrase "maintenance regimen” or “maintenance period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • continuous therapy e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.
  • intermittent therapy e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the TGFp/ActivinB signaling pathway is well known and is part of the common general knowledge.
  • Activin ligands encoded by 4 different genes, INHBA, INHBB, INHBC, and INHBE can activate different couples of receptors including the ACVR2A and ACVR2B type II and ACVR1A (ALK4) and ACVR1C (ALK7) type I receptors.
  • Activin ligands lead to the phosphorylation and activation of the intracellular mediators, Smad2 and Smad3, which then associate with the co-Smad, Smad4.
  • the hetero-complex translocates to the nucleus, where it activates the transcription of target genes with the help of DNA binding partners (Levy & Hill, 2006; Ross & Hill, 2008).
  • the term “inhibitor of the TGFp/ActivinB signaling pathway” refers to a compound that partially or fully blocks, inhibits, or neutralizes the TGFp/ActivinB signaling pathway.
  • the inhibitor can be a molecule of any type that interferes with the TGFp/ActivinB signaling pathway in a cell, for example, either by decreasing transcription or translation of neuropilin-encoding nucleic acid, or by inhibiting or blocking the activity or expression of any molecule involved in said pathway, such Activin B, ACVR2A and ACVR2B type II receptors as well as ACVR1 A (ALK4) and ACVR1C (ALK7) type I receptors but also smad2 and smad3 or even PMEPA1.
  • inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, aptamers, antibodies, small molecules, and peptides.
  • inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, aptamers, antibodies, small molecules, and peptides.
  • Inhibitor of the TGFp/ActivinB signaling pathway are well known in the art are typically described in: de Gramont A, Faivre S, Raymond E. Novel TGF-b inhibitors ready for prime time in onco-immunology. Oncoimmunology. 2016 Dec 7;6(l):el257453. doi: 10.1080/2162402X.2016.1257453. eCollection 2017. Review. PubMed PMID: 28197376; PubMed Central PMCID: PMC5283641.
  • inhibitors include but are not limited to LY-580276, LY-364947, LY- 2109761, LY-2157299, LY-573636, SB- 505124, SB-431542, SB-525234, SD-208, SD-093, Ki-26894, NPC-30345, SX-007, IN-1130, EW-7203, EW-7195, EW-7197 and GW6604.
  • inhibitor of the present invention may be selected from the group consisting of:
  • TGF-p RI 616458 TGF-b RI Kinase Inhibitor VII - CAS666729-57-3, l-(2-((6,7-
  • TGF-b RI 616463 TGF-b RI Kinase Inhibitor IX, 4-((4-((2,6- Dimethylpyridin-3-
  • Galunisertib 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazol-3-
  • R 268712 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-lH- pyrazol-4-yl]phenyl]-lH- pyrazole- 1 -ethanol
  • ITD 1 4-[l,r-Biphenyl]-4-yl-l,4,5,6,7,8-hexahydro-2,7,7- trimethyl-5-oxo-3- quinolinecarboxylic acid ethyl ester
  • the inhibitor of the present invention is Galunisertib as described above. In some embodiments, the inhibitor of the present invention is an antibody having specificity for Activin B.
  • antibody is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • VH and VL regions can be further subdivided into regions of hypervariability, WO 2021/047775 PCT/EP2019/074309 termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the antibodies of the invention may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen -binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
  • SMIPs small modular immunopharmaceuticals
  • the inhibitor of the TGFp/ActivinB signaling pathway is an inhibitor of expression, in particular an inhibitor that will reduce or inhibit the expression of WO 2021/047775 PCT/EP2019/074309
  • an “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the target, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the target can be synthesized, e.g., by conventional phosphodiester techniques.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs siRNAs
  • siRNAs can also function as inhibitors of expression for use in the present invention.
  • the targeted gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference or RNAi
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing the target.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • the endonuclease is CRISPR-cas.
  • the endonuclease is CRISPR-cas9, which is from Streptococcus pyogenes.
  • the CRISPR/Cas9 system has been WO 2021/047775 PCT/EP2019/074309 described in US 8697359 B1 and US 2014/0068797.
  • the endonuclease is CRISPR-Cpfl, which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • the inhibitors of the TGFp/ActivinB signaling pathway are suitable for the treatment of patients characterized by high level of Smad2 phosphorylation, high expression level of INHBB (i.e. inhibin subunit beta B, Gene ID: 3625), and high expression level PMEPA1 (i.e. prostate transmembrane protein, androgen induced 1 gene, Gene ID: 56937).
  • INHBB i.e. inhibin subunit beta B, Gene ID: 3625
  • PMEPA1 i.e. prostate transmembrane protein, androgen induced 1 gene, Gene ID: 56937.
  • the levels of said markers may be measured according to any routine techniques and typically are determined as described in the EXAMPLE.
  • the method of the present invention thus comprises the step of determining the level of Smad2 phosphorylation, the level of INHBB expression and the level PMEPA1 expression in a tumor sample obtained from the patient.
  • each determined level is compared to a predetermined reference value wherein when the three determined levels are higher than their respective predetermined reference value, the patient is thus eligible to a treatment with the inhibitor of the TGFp/ActivinB signaling pathway.
  • the predetermined reference values are determined by any routine method. For example, retrospective measurement of the level in properly banked historical patient samples may be used in establishing the predetermined reference value.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in WO 2021/047775 PCT/EP2019/074309 the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • a therapeutically effective amount of a therapeutic compound may decrease tumour size, or otherwise ameliorate symptoms in a subject.
  • An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the inhibitor of the present invention is administered to the subject in the form of a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • the inhibitor of the present invention is administered to the patient in combination with chemotherapy.
  • chemotherapy has its general meaning in the art and refers to the treatment that consists in administering to the patient a chemotherapeutic agent.
  • Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; du
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, WO 2021/047775 PCT/EP2019/074309 caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolin
  • the inhibitor of the present invention is administered to the patient in combination with radiotherapy.
  • radiation therapies include, but are not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron WO 2021/047775 PCT/EP2019/074309 therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like.
  • external beam radiotherapy such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron WO 2021/047775 PCT/EP2019/
  • Radiotherapy is used for the treatment of diseases of oncological nature with irradiation corresponding to ionizing radiation.
  • Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow.
  • Radiotherapy may be used to treat localized solid tumors cancers of the skin, tongue, larynx, brain, breast, lung or uterine cervix.
  • One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body.
  • radiotherapy may be proton radiotherapy or proton minibeam radiation therapy.
  • Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun 1;104(2):266-271.
  • Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.
  • FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015 ; 31 : 121-123. DOI: 10.1051/medsci/20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016/j.ijrobp.2018.06.403. Epub 2018 Jul 11). WO 2021/047775 PCT/EP2019/074309
  • the inhibitor of the present invention is administered to the patient during or after surgery.
  • a further object of the present invention relates to a method for determining whether a patient suffering from a medulloblastoma will achieve a response with an inhibitor of the TGFp/ActivinB signaling pathway comprising determining the expression level of PMEPA1, wherein said expression level correlates with the response of the patient to the treatment.
  • the method comprises the steps of i) determining the expression level of PMEPA1, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will achieve a response when the level determined at step i) is higher than the predetermined reference value.
  • the method is thus particularly suitable for discriminating responder from non responder.
  • responder in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or improved.
  • the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after the therapy.
  • a non-responder or refractory patient includes patients for whom the cancer does not show reduction or improvement after the therapy.
  • the term “non responder” also includes patients having a stabilized cancer.
  • the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set.
  • the standard may be the profile of a patient who is known to be a responder or non responder or alternatively may be a numerical value.
  • Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media.
  • the methods further comprises the steps consisting of determining the level of p-Smad2 and the expression level of INHBB, comparing said levels with their corresponding predetermined reference value, and concluding that the patient will achieve a response when the determined level are higher than their predetermined reference value.
  • FIGURES are a diagrammatic representation of FIGURES.
  • TGFp/ActivinB signaling promotes cell proliferation in Group 3 MB cell lines
  • A-H 1603MED (A-D) or D283 (E-H) cells were treated with DMSO (vehicle), with LY364947, or with SB431542.
  • a and E Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2, and b-actin upon inhibition of TGFp/Activin signaling using LY364947 and SB431542 inhibitors for 24 h. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to b-actin.
  • B and F Growth curve experiments showing cell proliferation upon TGtRb/Aohn ⁇ h signaling inhibition.
  • C and G Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labeling at 48 h upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S, and G2/M phases).
  • D and H Percentage of apoptotic cells measured by FACS analysis of cleaved caspase-3 48 h after TGtRb/Aohn ⁇ h signaling inhibition. The P-values were determined by unpaired t-test and two-way ANOVA for (B and F). *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. Bars represent the mean ⁇ SD. Number of replicates is n > 3.
  • the rectangle represents Galunisertib treatment duration, while the dotted lines represent the 3 Cisplatin administrations.
  • Normalized primary medulloblastoma gene expression data (763 samples) and samples affiliation published in Cavalli et al (2017a) were used to generate scatter plots and gene expression boxplots per subgroup and subtype for the genes of interest.
  • Normalized primary medulloblastoma protein levels data 45 samples
  • Wilcoxon rank-sum tests were performed between subgroups and subtypes. Spearman rank correlation coefficients were computed between the INHBB gene expression values and all other genes for Group 3 samples. The gene pairs were ranked according to the Spearman correlation values.
  • HD-MB03 (named HDMB03) obtained from Dr. Milde (Milde et al, 2012), D458MED (named D458) obtained from Dr. Bigner (He et al, 1991), UW228 (Keles et al, 1995), ONS-76, and DAOY MB cell lines (ATCC) were cultured as described in Garancher et al (2016). 1603MED obtained from Dr.
  • Raso (Raso et al, 2008) and D283MED (ATCC) (named D283) cell lines were maintained in DMEM condition supplemented with 12% fetal bovine serum (GIBCO), 50 units/ml penicillin and streptomycin (Invitrogen) and 0.1 mM non-essential amino WO 2021/047775 PCT/EP2019/074309 acids and sodium pyruvate. 1603MED cell lines were also supplemented with 2 mM L- glutamine. All cells were cultured at 37°C in a humidified atmosphere containing 5% C02. LY363947 and SB431532 resuspended in DMSO (selleckchem) were used at a final concentration of 5 mM for 24 h.
  • Stimulations with TGFB1 and ActivinB were performed for 1 or 24 h at 10 ng/ml.
  • Inhibitions with a recombinant blocking antibody against ActivinB (R&D systems) or recombinant follistatin (R&D systems) were performed for 24 h at 5 and 0.2 pg/ml, respectively.
  • Receiving cells were plated at 1.5 x 10 5 cells/well in 6-wells plates. 1603MED and HDMB03 conditioned media were obtained by 18 h of incubation at 1 x 10 6 cells/ml. Non-conditioned media was obtained in the same conditions in absence of cells. Media were collected, filtered, and incubated with PBS as control or blocking antibody against ActivinB (5 pg/ml) for 2 h at 4°C with rotation. Cells were treated with 1 ml of media for 1 h, and cell extracts were collected for WB analysis.
  • Transfection assays were performed in either 96- or 6-well plates. siRNA transfection was performed according to the manufacturer's instructions (Dharmacon). DharmaFECT 3 transfection reagent was used at 0.15 and 4 m1/100 m ⁇ of transfection medium for D283 and 1603MED cell lines, respectively. D283 cells were plated at 5 x 10 5 cells/ml and siRNA were used at a final concentration of 25 nM. 1603MED cells were plated at a concentration of 1 x 10 6 cells/ml with 10 mM final of siRNA. Transfection assay efficiency was assessed using siGlo (D001630-01-05).
  • siRNA smartpool CTRL D-001810-00-1005
  • smartpool GNHBB L- 011702-00-0010
  • smartpool PMEPA1 L-010501-00-0020
  • ON -TARGETplus individual siRNA PMEPA1#1 L-010501-05
  • PMEPA1#2 L-010501-08
  • 1603MED and D283 cell lines were plated at 8 x 10 5 and 2.5 x 10 5 cell/ml, respectively.
  • Apoptosis was assessed at day 2 using cleaved caspase-3 staining with Apoptosis Kit, APC (BD Bioscience).
  • Cell cycle was analyzed at day 2 using APC BrdU flow Kit (BD Bioscience).
  • Experiments were performed using FACS Kanto (BD Bio science) and analyzed with FlowJo software (Tree Star).
  • PDXs were obtained, maintained, dissociated, and cultured as described in Garancher et al (2016).
  • PDX3, PDX4, and PDX7 correspond to ICN-MB-PDX-3, ICN-MB-PDX-4, and ICN-MB-PDX-7, respectively. All in vitro treatments were performed as described for cell lines.
  • mice were obtained from Janvier Laboratory. Experiments were performed on 7-8 weeks old female mice after 1 week of acclimation in animal facility of Curie Institute. Mice were housed under a controlled temperature and 12 h/12 h light-dark cycle with access to food and water ad libitum in conventional animal facility. For the animal welfare, mice are maintained in social groups with enrichment. Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved WO 2021/047775 PCT/EP2019/074309 by the ethics committee of the Institut Curie CEEA-IC #118 (Authorization 02383.02 given by National Authority) in compliance with the international guidelines.
  • mice Nude female mice (Janvier labs) were orthotopically grafted directly in the cerebellum at 7 weeks with 3 x 105 cells/5 m ⁇ of ICN-MB-PDX-4 cells as described in Garancher et al (2016). After 3 days, mice were administrated 300 m ⁇ of LY2157299 (Galunisertib, AbMole Bioscience) orally at a dose of 75 mg/kg in 12% DMSO, 30% PEG, and water. Mice were treated 7 days a week twice a day until day 30. Mice were injected with Cisplatin (Sigma) in saline solution at a dose of 2 mg/kg intra-peritoneally at days 4, 8, and 12 post-grafting. Mice were euthanized when scientific and clinical end points were reached and brains were collected and fixed.
  • Cisplatin Cisplatin
  • mice per group received ice-cold PBS and 4% formaldehyde/PBS via intracardiac perfusions. Brains were collected and fixed overnight in 4% formaldehyde/PBS at 4°C. IHC was performed on 12-pm-thick sections with the following primary antibodies: anti-PhosphoSmad2 (CST, CS138D4, 1/300), Ki67 (CST, CS9161, 1/500), and cleaved caspase-3 (eBioscience, #14-5698-82, 1/500). Image acquisitions were performed on a Zeiss microscope. Tumor size and IHC staining were assessed using ImageJ software.
  • TGFp/ActivinB signaling pathway is active in Group 3 MB
  • Non-Group 3 cell lines showed an increase in P- Smad2 signals in response to TGFP stimulation, while no modulation was observed upon Activin stimulation (data not shown).
  • Group 3 MB cell lines showed the complete opposite profile: P-Smad2 signal was increased upon Activin stimulation, while it remained unchanged upon TGFP stimulation (data not shown).
  • 1603MED displayed a very high basal level of P-Smad2 which is constitutive. The reason for which G3 cell lines respond to Activin but not to TGFP is currently unknown.
  • PMEPA1 is implicated in ActivinB promotion of cell growth
  • PMEPA1 which scored as the top gene, is a well-established Smad2/3 target gene in different cell types including P19 cells stimulated by Activin (Coda et al, 2017). Accordingly, we found that PMEPA1 expression level was enriched in Group 3 MB (data not shown) and correlated with INHBB expression in MB (data not shown). This correlation is highest in G3 as compared to the other groups (data not shown). Accordingly, we observed a good correspondence between P-Smad2 overall level and PMEPA1 protein expression in patient samples by Western blot analysis (data not shown). We next tested whether PMEPA1 is also a target of the Smad2 signaling in MB by modulating pathway activation (data not shown).
  • MYC and OTX2 are key players in Group 3 MB and are also known as Smad2/3 target genes in other cell types (Jia et al, 2009; Brown et al, 2011; Coda et al, 2017). Therefore, we investigated whether their expression could be modulated by this pathway in Group 3 MB cell lines.
  • Galunisertib a pharmacological inhibitor currently in clinical trial for Glioblastoma, Cisplatin as described in Niklison-Chirou et al (2017), or a combination.
  • Galunisertib is described as a TGFP type I inhibitor but, since TGFP and Activin type I receptors are very similar, it also inhibits very efficiently ActivinB- induced Smad2 activation (data not shown).
  • Galunisertib recapitulated the main in vitro data obtained with LY364947 and SB431542 (data not shown).
  • Galunisertib -treated mice survived longer as compared to controls (Fig 2A), demonstrating the benefit of such treatment in tumors displaying high level of activation of the pathway. Accordingly, Galunisertib -treated mice displayed smaller tumors with less P-Smad2 (Fig 2B).
  • NRL and CRX control photoreceptor genes expression and define a subset of Group 3 tumors We recently reported that NRL and CRX control photoreceptor genes expression and define a subset of Group 3 tumors (Garancher et al, 2018). We found that alike INHBB, NRL is highly expressed in the G3a subtype (data not shown). This identifies Group 3a as the subtype that expresses high level of INHBB and high photoreceptor genes.
  • Group 3 is the most aggressive MB group with patients showing the poorest prognosis.
  • Several genomic alterations have been identified, including those targeting the TGFp/Activin pathway at very low frequency. Indeed, SCNA analyses have identified uncommon gains and/or amplifications of genes encoding receptors of the TGFp/Activin pathway. Activation of the cognate Smad2/3 pathway in Group 3 tumors has never been investigated, neither its potential biological consequences nor its potential therapeutic targeting. Using patient samples, PDXs, and cell lines, we showed that, beside these infrequent genomic alterations, the WO 2021/047775 PCT/EP2019/074309
  • TGFp/Activin pathway is also activated in a specific subtype of Group 3, through an autocrine mechanism involving ActivinB. This pathway is involved in MB growth and represents an interesting therapeutic target.
  • TGFP pathway activation involves autocrine loops, due to the high expression of genes encoding the different TGFP ligands (Rodon et al, 2014).
  • TGFPs and Activins activate the same Smad pathway (Smad2/3)
  • TGFPs autocrine mechanisms have been much more frequently described to be implicated in cancer progression than Activins (Chen et al, 2002; Wakefield & Hill, 2013), highlighting a singularity of Group 3 MBs. Since Activin is involved in developmental processes (Wu & Hill, 2009), its implication in Group 3 MB instead of TGFP may relate to the pediatric nature of these tumors or to their cell of origin. In support of the latter and according to brain atlas data, INHBB displays a very cell-specific and dynamic profile during cerebellar development.
  • ActivinB induces PMEPA1 expression and promotes cell cycle progression
  • the TGFp/ Activin pathway is highly pleiotropic and sometimes displays antagonistic functions during carcinogenic processes. For example, it can promote either cell cycle arrest or proliferation, depending on the context. This opposite role has been well illustrated in Glioblastoma in which the epigenetic status of the cells, in particular its DNA methylation profile, is responsible for this duality (Bruna et al, 2007). In agreement with this pro-mitogenic activity, we found that pathway inhibition decreased cell proliferation in Group 3 MB, while ActivinB stimulation increased it by consistently promoting cell cycle progression.
  • MYC and OTX2 two genes known to promote cell proliferation in Group 3 MB, are target genes of the Smad2/3 pathway in other contexts (Jia et al, 2009; Brown et al, 2011; Coda et al, 2017). In general, this signaling pathway reduces MYC expression (Warner et al, 1999; Seoane et al, 2001), although it can be induced in human embryonic stem cells (Brown et al, 2011).
  • OTX2 Since OTX2 has been demonstrated to be a major Smad2/3 target gene in the nervous system (Jia et al, 2009), it has been proposed to be a Smad2/3 inducible gene in Group 3 MB (Ferrucci et al, 2018) and considered as part of this signaling pathway in MB (Northcott et al, 2012b). We did not detect any consistent changes in MYC and OTX2 expression upon modulation of the Activin pathway, suggesting that this signaling pathway does not regulate these two genes in Group 3 tumors and promotes tumor growth through other mechanisms.
  • PMEPA1 whose expression is induced by TGFP or Activin signaling in many different contexts (Coda et al, 2017), is also an Activin-regulated gene in Group 3 MB. Indeed, inhibition or activation of the Activin signaling pathway modulated PMEPA1 expression accordingly. This regulation is likely to be relevant in patients since INHBB and PMEPA1 expression is correlated in human MB samples. PMEPA1 is the top correlated gene with INHBB within Group 3 MB, showing that their expression is strongly linked in this group. In all MB samples, the correlation is lower than within Group 3 samples. Indeed, PMEPA1 expression is higher in WO 2021/047775 PCT/EP2019/074309
  • This subtype is characterized by the lack of MYC amplification and, as shown in this study, an overall moderate to low MYC expression level.
  • This subtype displays high photoreceptor gene expression (Cavalli et al, 2017a), including those of the two master regulators of this program, NRF and CRX. Accordingly, we recently showed that their expression defines a specific subtype within Group 3 (Garancher et al, 2018). Our data may suggest that the expression of INHBB could lead to Smad2/3 activation in this subtype. Indeed, we found that PMEPA1, whose expression can be considered as a readout of Smad2/3 activation (see above), is significantly higher in Group 3a subtype as compared to other Group 3 subtypes.
  • the TGFp/ Activin signaling pathway is activated through an ActivinB autocrine mechanism in a subset of Group 3 MB subtype. Not only this pathway is activated, but it also plays a growth -promoting role and constitutes an important driver of therapeutic interest in these tumors.
  • high levels of INHBB, PMEPA1 expression, and Smad2 phosphorylation might constitute biomarkers for potential Group 3 patients to be eligible to treatment with inhibitors of the TGFp/Activin signaling pathway, in particularly Galunisertib.
  • TGF-b receptor inhibitors target the CD44(high)/Idl(high) glioma-initiating cell population in human glioblastoma. Cancer Cell 18: 655-668.
  • HD-MB03 is a novel Group 3 medulloblastoma model demonstrating sensitivity to histone deacetylase inhibitor treatment. J Neurooncol 110: 335-348.
  • TAp73 is a marker of glutamine addiction in medulloblastoma. Genes Dev 31: 1738-1753.
  • TGF-beta increases glioma-initiating cell self -renewal through the induction of LIF in human glioblastoma. Cancer Cell 15: 315-327.
  • TGF-b determines the pro -migratory potential of bFGF signaling in medulloblastoma. Cell Rep 23: 3798-3812. e8.
  • TGF-beta growth factor-beta
  • TMEPAI Transforming growth factor-beta (TGF-beta) -inducible gene TMEPAI converts TGF-beta from a tumor suppressor to a tumor promoter in breast cancer. Cancer Res 70: 6377-6383.

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Abstract

Medulloblastoma (MB) is a pediatric tumor of the cerebellum arising at a median age of 7 years. MB is a heterogeneous disease classified in four groups, with the poorly characterized Group 3 showing the worst prognosis. The inventors now show that a subset of Group 3 MBs displays activation of the TGFp/Activin pathway. In contrast to carcinomas where TGFPs are the main driver of activation of this pathway, the data established that this activation is mainly due to an autocrine stimulation involving ActivinB. These tumors express high levels of INHBB (encoding ActivinB) and display high expression of PMEPA1, a well-known target gene of this signaling pathway. Functionally, the pathway sustains cell proliferation by inducing the expression of PMEPA1. Importantly, treatment with Galunisertib, an inhibitor of this pathway currently tested in clinical trials for Glioblastoma patients, increases the survival of mice orthotopically grafted with Group 3 MB-PDX. Thus, the present invention relates to use of inhibitors of the TGFp/ ActivinB signaling pathway for the treatment of patients suffering from medulloblastoma group 3.

Description

WO 2021/047775 PCT/EP2019/074309
USE OF INHIBITORS OF TGFB/ACTIVINB SIGNALING PATHWAY FOR THE TREATMENT OF PATIENTS SUFFERING FROM MEDULLOBLASTOMA GROUP
3
FIELD OF THE INVENTION:
The present invention is in the field of medicine, in particular oncology.
BACKGROUND OF THE INVENTION:
Medulloblastoma (MB), a cerebellar tumor, is one of the most common malignant brain tumors in children (Holgado et al, 2017; Wang et al, 2018). Current therapy associates surgery, chemotherapy, and radiotherapy. This aggressive regimen allowed an increase in the overall survival rate up to 70-80% but induces dramatic long-term side effects (Martin et al, 2014). In addition, the overall survival rate of high-risk patients is far below (Holgado et al, 2017; Wang et al, 2018). It is therefore crucial to identify new treatments that decrease side effects and improve efficacy.
Genomic and transcriptomic approaches allowed the stratification of MB patients into 4 different molecular groups: WNT (Wingless), SHH (Sonic Hedgehog), Group 3, and Group 4 (Northcott et al, 2012a; Taylor et al, 2012). These groups display differences in terms of cell of origin, transcriptional, epigenetic, and mutational signatures. They also differ in their clinical characteristics such as histology, overall survival rate, and presence of metastases. Recently, intragroup heterogeneity has been further uncovered, allowing their division into subtypes with some specific clinical parameters as well as genomic alterations (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017). Although the existence of subdivisions within the different groups is clear, the outlines of the different subtypes have not completely reached a consensus so far. The WNT group represents 10% of all MBs and is driven by constitutive activation of the WNT/p-catenin pathway with patients showing the best prognosis. The SHH group accounts for 20-25% of MB and is characterized by mutations involving different mediators of the SHH pathway. It is considered of intermediate prognosis. However, recent sub-classifications identified SHH subtypes with poorer outcomes (Cavalli et al, 2017a; Schwalbe et al, 2017). On the other side, Group 3 and Group 4 are far less characterized due to their genetic and clinical heterogeneity. They display some degrees of overlap with a few samples (-10%) being difficult to specifically assign to either Group. They share some clinical characteristics, such as a high WO 2021/047775 PCT/EP2019/074309 propensity to metastasis and genetic alterations such as OTX2 amplifications or KBTBD4 mutations (Northcott et al, 2017). In contrast to SHH and WNT groups, no deregulation of a given signaling pathway has been yet reported. Group 4 represents 35-40% of all MB patients and shows, in few cases, MYCN and CDK6 amplifications and KDM6A mutations. Recently, it has been shown that genomic alterations involving enhancer hijacking induce PRDM6 overexpression in 15-20% of Group 4 (Northcott et al, 2017). Group 3 represents 20-25% of MB patients and is associated with bad prognosis. This group is highly metastatic and characterized by MYC overexpression, which can be explained in 15-20% of cases by its amplification. However, MYC overexpression is not sufficient to induce Group 3 MB and requires additional cooperating oncogenic events (Kawauchi et al, 2012; Pei et al, 2012). Some of them have been identified, such as GFI1 and GFI1B that are highly expressed in a subset of Group 3 through enhancer hijacking (Northcott et al, 2014). These transcription factors have been demonstrated to drive Group 3 MB tumorigenesis in animal models when associated with MYC overexpression (Northcott et al, 2014). At the transcriptomic level, Group 3 is characterized by the expression of a photoreceptor program defined by genes whose expression is highly restricted to the retina (Kool et al, 2008; Cho et al, 2011). We recently uncovered that this program defines a subtype within Group 3 tumors, which exhibits a functional dependency to this ectopic program through its two main drivers, the retina-specific transcription factors NRL and CRX (Garancher et al, 2018). Thus, Group 3 can be subdivided into 2-3 different subtypes according to the different studies (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017). Cavalli et al (2017a) have identified 3 subtypes, one is composed of tumors with high MYC expression including those with amplification of this gene, named G3y. This subtype has the worse prognosis. The second subtype, t3b, is overrepresented by tumors with GFI1 alterations, and the last one G3a, by tumors expressing photoreceptor genes in which few amplifications of mediators of the TGFp/Activin pathway can be found (Cavalli et al, 2017a). Since Group 3 displays the worse prognosis, targeted therapies are actively searched. Different actionable targets have been proposed mainly based on genomic data, including the TGFP signaling, which has been suggested to be deregulated in few Group 3 MB, although no functional data have been reported so far. A study on structural genomic variations across over 1,000 MB has first described few amplifications of different mediators of the TGFp/Activin pathway in Group 3 MB (Northcott et al, 2012b). They include ACVR2A and ACVR2B, two type II receptors for Activin, as well as TGFBR1, a type I receptor for TGFp, highlighting a potential deregulation of Smad2/3 signaling (see below). Additionally, since OTX2 has been demonstrated to be a target gene of this signaling pathway (Jia et al, 2009), it has been proposed WO 2021/047775 PCT/EP2019/074309 that OTX2 amplifications could represent a mechanism by which the pathway is also deregulated downstream (Northcott et al, 2012b). The putative significance of this signaling pathway in Group 3 was reinforced by two subsequent studies, one involving sequencing in a large cohort of MB (Northcott et al, 2017) and the other showing that several components of this signaling pathway could also be deregulated at their expression level, through Group 3- specific enhancers (Lin et al, 2016). Although these studies might indicate a potential deregulation of the Smad2/3 signaling pathway, this could account for only a modest proportion of Group 3 tumors.
The TGFP superfamily is a large family of cytokines divided into two distinct groups of ligands: the TGFPs/Activins and the BMPs. TGFp/Activin ligands signal through Smad2/3. These ligands bring together two types of serine/threonine kinase receptors, the type I and the type II, which are specific for a set of ligands. The TGFPs (TGFpi, TGFP2, and TGFP3) signal through the TGFBR1 type I and TGFBR2 type II receptors. Activin, encoded by 4 different genes, INHBA, INHBB, INHBC, and INHBE, can activate different couples of receptors including the ACVR2A and ACVR2B type II and ACVR1A (ALK4) and ACVR1C (ALK7) type I receptors. INHA, encoding inhibin-a, is an inhibitor of the Activin ligands. Activin and TGFP ligands lead to the phosphorylation and activation of the same intracellular mediators, Smad2 and Smad3, which then associate with the co-Smad, Smad4. The hetero-complex translocates to the nucleus, where it activates the transcription of target genes with the help of DNA binding partners (Levy & Hill, 2006; Ross & Hill, 2008).
TGFp/ Activin signaling displays pleiotropic functions depending on the cellular and environmental context. Its implication in cancer has been well documented, mainly through TGFP ligands, although BMPs and Activins ligands can be also involved (Seoane & Gomis, 2017). The role of the TGFP signaling pathway in cancer is complex, acting either as a tumor suppressor pathway in some instances or as a tumor promoter in others (Massague, 2008; Seoane & Gomis, 2017). Its oncogenic role is mainly associated with an autocrine (or paracrine) stimulation, due to the strong expression of TGFP ligands. The TGFP pathway has been shown to promote cell proliferation in specific context such as in Glioblastoma (Bruna et al, 2007) and cancer stem cell maintenance (Penuelas et al, 2009; Anido et al, 2010; Lonardo et al, 2011). Studies on the role of Activin ligands in cancer are much more scarce (Wakefield & Hill, 2013). By activating the same mediators Smad2/3, a parallel can be drawn between TGFP and Activin. Indeed, Activins act both as tumor suppressors and tumor promoters (Chen et al, 2002; WO 2021/047775 PCT/EP2019/074309
Antsiferova & Wemer, 2012; Marino et al, 2013; Wakefield & Hill, 2013). Their pro- tumorigenic role has been validated in animal models in which deletion of the activin inhibitor, INHA, led to gonadal tumors in mice as well as cachexia-like syndrome (Matzuk et al, 1994; Vassalli et al, 1994). ActivinB has also been shown to play a role in cancer stem cell maintenance (Lonardo et al, 2011) and in cell dedifferentiation in an insulinoma mouse model and deletion of INHBB encoding ActivinB increases survival (Ripoche et al, 2015).
Several observations pinpoint to a potential role of the Smad2/3 signaling pathway in Group 3 MB but no published data have confirmed the deregulation of this signaling pathway, nor its functional involvement in Group 3 biology.
SUMMARY OF THE INVENTION:
As defined by the claims, the present invention relates to use of inhibitors of the TGFp/ActivinB signaling pathway for the treatment of patients suffering from medulloblastoma group 3.
DETAILED DESCRIPTION OF THE INVENTION:
Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFp/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. The inventors showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, the inventors demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB-PDX. The data demonstrate that the TGFp/ Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted. WO 2021/047775 PCT/EP2019/074309
Accordingly, the first object of the present invention relates to a method of treating a patient suffering from medulloblastoma group 3 comprising administering to the patient a therapeutically effective amount of an inhibitor of the TGFp/ActivinB signaling pathway.
As used herein, the term “medulloblastoma” has its general meaning in the art and refers to a fast-growing, aggressive, high-grade brain tumor. Regardless of the subtype, medulloblastoma always occurs in the cerebellum of the brain, and more specifically, within the posterior fossa of the cerebellum. The term is also known as cerebellar primitive neuroectodermal tumor (PNET). Symptoms of medulloblastoma include, but are not limited to, behavioral changes, changes in appetite, and symptoms of increased pressure on the brain (e.g., headache, nausea, vomiting, and drowsiness, as well as problems with coordination (e.g. clumsiness, problems with handwriting, and visual problems)). Unusual eye movements may also occur. If the cancer has spread to the spinal cord, symptoms may include back pain, trouble walking, and/or problems controlling bladder and bowel functions. Genomic and transcriptomic approaches allowed the stratification of MB patients into 4 different molecular groups: WNT (Wingless), SHH (Sonic Hedgehog), Group 3, and Group 4 (Northcott et al, 2012a; Taylor et al, 2012). According to the present invention, the inhibitors of the TGFp/ActivinB signaling pathway are particularly suitable for the treatment of patients belonging to group 3, or event patients belonging to the subtype Group 3a according to the classification of Cavalli et al (2017a).
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high WO 2021/047775 PCT/EP2019/074309 level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
The TGFp/ActivinB signaling pathway is well known and is part of the common general knowledge. Activin ligands encoded by 4 different genes, INHBA, INHBB, INHBC, and INHBE, can activate different couples of receptors including the ACVR2A and ACVR2B type II and ACVR1A (ALK4) and ACVR1C (ALK7) type I receptors. Activin ligands lead to the phosphorylation and activation of the intracellular mediators, Smad2 and Smad3, which then associate with the co-Smad, Smad4. The hetero-complex translocates to the nucleus, where it activates the transcription of target genes with the help of DNA binding partners (Levy & Hill, 2006; Ross & Hill, 2008).
Accordingly, the term “inhibitor of the TGFp/ActivinB signaling pathway” refers to a compound that partially or fully blocks, inhibits, or neutralizes the TGFp/ActivinB signaling pathway. The inhibitor can be a molecule of any type that interferes with the TGFp/ActivinB signaling pathway in a cell, for example, either by decreasing transcription or translation of neuropilin-encoding nucleic acid, or by inhibiting or blocking the activity or expression of any molecule involved in said pathway, such Activin B, ACVR2A and ACVR2B type II receptors as well as ACVR1 A (ALK4) and ACVR1C (ALK7) type I receptors but also smad2 and smad3 or even PMEPA1. Examples of inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, aptamers, antibodies, small molecules, and peptides. WO 2021/047775 PCT/EP2019/074309
Inhibitor of the TGFp/ActivinB signaling pathway are well known in the art are typically described in: de Gramont A, Faivre S, Raymond E. Novel TGF-b inhibitors ready for prime time in onco-immunology. Oncoimmunology. 2016 Dec 7;6(l):el257453. doi: 10.1080/2162402X.2016.1257453. eCollection 2017. Review. PubMed PMID: 28197376; PubMed Central PMCID: PMC5283641.
Isaka Y. Targeting TGF-b Signaling in Kidney Fibrosis bit J Mol Sci. 2018 Aug 27; 19(9). pii: E2532. doi: 10.3390/ijms 19092532. Review. PubMed PMID: 30150520; PubMed Central PMCID: PMC6165001.
Zhang S, Sun WY, Wu JJ, Wei W. TGF-b signaling pathway as a pharmacological target in liver diseases. Pharmacol Res. 2014 Jul;85: 15-22. doi:
10.1016/j.phrs.2014.05.005. Epub 2014 May 17. Review. PubMed PMID: 24844437.
Fields SZ, Parshad S, Anne M, Raftopoulos H, Alexander MJ, Sherman ML, Laadem A, Sung V, Terpos E. Activin receptor antagonists for cancer-related anemia and bone disease. Expert Opin Investig Drugs. 2013 Jan;22(l):87-101. doi: 10.1517/13543784.2013.738666. Epub 2012 Nov 6. Review. PubMed PMID: 23127248.
Jindal A, Thadi A, Shailubhai K. Hepatocellular Carcinoma: Etiology and Current and Future Drugs. J Clin Exp Hepatol. 2019 Mar-Apr;9(2):221-232. doi: 10.1016/j.jceh.2019.01.004. Epub 2019 Jan 25. Review. PubMed PMID: 31024205; PubMed Central PMCID: PMC6477125.
Examples of inhibitors include but are not limited to LY-580276, LY-364947, LY- 2109761, LY-2157299, LY-573636, SB- 505124, SB-431542, SB-525234, SD-208, SD-093, Ki-26894, NPC-30345, SX-007, IN-1130, EW-7203, EW-7195, EW-7197 and GW6604.
More specifically, the inhibitor of the present invention may be selected from the group consisting of:
Agent Alternative Name
LY-364947 616451, TGF-b RI Kinase Inhibitor I, CAS 396129-53-6, [3-(Pyridin-2- yl)-4-(4-quinonyl)]-lH-pyrazole, ALK5 Inhibitor I, LY-364947, HTS- 466284 WO 2021/047775 PCT/EP2019/074309
Repsox 616452, TGF-b RI Kinase Inhibitor II, CAS 446859-33-2, 2-(3-(6-
Methylpyridin-2-yl)- lH-pyrazol-4-yl)- 1 ,5-naphthyridine
SB-505124 616453, TGF-b RI Kinase Inhibitor III, CAS 356559- 13-22-(5-
Benzo[l,3]dioxol-4-yl-2-tert-butyl-lH- imidazol-4-yl)-6- methylpyridine, HC1, ALK5 Inhibitor III, SB-505124, HC1
A-83-01 616454, TGF-b RI Kinase Inhibitor IV - CAS 909910-43-6, 3-(6-
Methylpyridin-2-yl)-4-(4-quinolyl)- 1 - phenylthiocarbamoyl- 1H- pyrazole, A-83-01, ALK5 Inhibitor IV
SD-208 616456, TGF-b RI Kinase Inhibitor V, CAS 627536-09-8, 2-(5-Chloro-
2-fluorophenyl)pteridin-4- yl)pyridin-4-yl amine, SD-208, ALK5 Inhibitor V
SB-431542 616461, TGF-b RI Kinase Inhibitor VI, SB431542 - CAS 301836-41-9,
4-[4-(3,4- Methylenedioxyphenyl)-5-(2-pyridyl)- lH-imidazol-2- yl]benzamide, Dihydrate, 4-[4-(l,3-Benzodioxol-5-yl)-5-(2-pyridyl)- lH-imidazol-2-yl]benzamide, dihydrate
TGF-p RI 616458, TGF-b RI Kinase Inhibitor VII - CAS666729-57-3, l-(2-((6,7-
Kinase Inhibitor Dimethoxy-4- quinolyl)oxy)-(4,5-dimethylphenyl)-l-ethanone, ALK5
VII Inhibitor VII
SB-525334 616459, TGF-b RI Kinase Inhibitor VIII - CAS356559-20-1, SB-
525334, 6-(2-tert-Butyl-5-(6- methyl -p yridin-2-yl)- lH-imidazol-4-yl)- quinoxaline, ALK5 Inhibitor VIII
TGF-b RI 616463, TGF-b RI Kinase Inhibitor IX, 4-((4-((2,6- Dimethylpyridin-3-
Kinase Inhibitor yl)oxy)pyridin-2- yl)amino)benzenesulfonamide, ALK5 Inhibitor IX
IX
GW788388 4-(4-(3-(pyridin-2-yl)-lH-pyrazol-4-yl)pyridin-2-yl)- N-(tetrahydro-2H- pyran-4-yl)benzamide
LY2109761 7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6- dihydro-4H- pyrrolo[ 1 ,2-b]pyrazol-3-yl)quinoline
Galunisertib 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazol-3-
(LY2157299) yl)quinoline-6- carboxamide
EW-7197 N-(2-fhiorophenyl)-5-(6-methyl-2-pyridinyl)-4-[l,2,4]triazolo[l,5- a]pyridin-6-yl- 1 H-imidazole-2- methanamine
Pirfenidone 5-methyl- 1 -phenyl- 1 ,2-dihydropyridin-2-one WO 2021/047775 PCT/EP2019/074309
K02288 3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3- pyridinyl]phenol
D 4476 4-[4-(2, 3-Dihydro- l,4-benzodioxin-6-yl)-5-(2- pyridinyl)-lH-imidazol-
2-yl]benzamide
R 268712 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-lH- pyrazol-4-yl]phenyl]-lH- pyrazole- 1 -ethanol
ITD 1 4-[l,r-Biphenyl]-4-yl-l,4,5,6,7,8-hexahydro-2,7,7- trimethyl-5-oxo-3- quinolinecarboxylic acid ethyl ester
SIS3 l,2,3,4-Tetrahydro-6,7-dimethoxy-2-[(2E)-3-(l- phenyl-lH- pyrrolo[2,3-b]pyridin-3-yl)-l-oxo-2- propenyl] -isoquinoline hydrochloride
A77-01 4-[5-(6-methylpyridin-2-yl)-lH-pyrazol-4- yl]quinoline
SM16 4-(5-(benzo[d][l,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-lH-imidazol-
2-yl)bicyclo[2.2.2]octane-l- carboxamide Trx-xFoxHIb Smad-interacting peptide aptamers
Trx-Lefl Distertide (pI44)
PI
LSKL
In some embodiments, the inhibitor of the present invention is Galunisertib as described above. In some embodiments, the inhibitor of the present invention is an antibody having specificity for Activin B.
The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, WO 2021/047775 PCT/EP2019/074309 termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antibodies of the invention (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “anti gen -binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain- specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
In some embodiments, the inhibitor of the TGFp/ActivinB signaling pathway is an inhibitor of expression, in particular an inhibitor that will reduce or inhibit the expression of WO 2021/047775 PCT/EP2019/074309
Activin B, Activin type I receptor, Smad2, Smad3 or PMEPA1. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the target, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the target can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. The targeted gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing the target. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor of expression is an endonuclease. In a particular embodiment, the endonuclease is CRISPR-cas. In some embodiment, the endonuclease is CRISPR-cas9, which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been WO 2021/047775 PCT/EP2019/074309 described in US 8697359 B1 and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl, which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
More particularly, the inhibitors of the TGFp/ActivinB signaling pathway are suitable for the treatment of patients characterized by high level of Smad2 phosphorylation, high expression level of INHBB (i.e. inhibin subunit beta B, Gene ID: 3625), and high expression level PMEPA1 (i.e. prostate transmembrane protein, androgen induced 1 gene, Gene ID: 56937). Typically, the levels of said markers may be measured according to any routine techniques and typically are determined as described in the EXAMPLE.
In some embodiments, the method of the present invention thus comprises the step of determining the level of Smad2 phosphorylation, the level of INHBB expression and the level PMEPA1 expression in a tumor sample obtained from the patient. In some embodiments, each determined level is compared to a predetermined reference value wherein when the three determined levels are higher than their respective predetermined reference value, the patient is thus eligible to a treatment with the inhibitor of the TGFp/ActivinB signaling pathway. Typically, the predetermined reference values are determined by any routine method. For example, retrospective measurement of the level in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in WO 2021/047775 PCT/EP2019/074309 the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of drug employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. A therapeutically effective amount of a therapeutic compound may decrease tumour size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. WO 2021/047775 PCT/EP2019/074309
Typically, the inhibitor of the present invention is administered to the subject in the form of a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
In some embodiments, the inhibitor of the present invention is administered to the patient in combination with chemotherapy. As used herein, the term “chemotherapy” has its general meaning in the art and refers to the treatment that consists in administering to the patient a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, WO 2021/047775 PCT/EP2019/074309 caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In some embodiments, the inhibitor of the present invention is administered to the patient in combination with radiotherapy. Suitable examples of radiation therapies include, but are not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron WO 2021/047775 PCT/EP2019/074309 therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like. As used herein, the term "radiotherapy" is used for the treatment of diseases of oncological nature with irradiation corresponding to ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. Radiotherapy may be used to treat localized solid tumors cancers of the skin, tongue, larynx, brain, breast, lung or uterine cervix. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiotherapy. Gamma rays are another form of photons used in radiotherapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, radiotherapy may be proton radiotherapy or proton minibeam radiation therapy. Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun 1;104(2):266-271. doi: 10.1016/j.ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1): 16479. doi: 10.1038/s41598-018-34796-8). Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015 ; 31 : 121-123. DOI: 10.1051/medsci/20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016/j.ijrobp.2018.06.403. Epub 2018 Jul 11). WO 2021/047775 PCT/EP2019/074309
In some embodiments, the inhibitor of the present invention is administered to the patient during or after surgery.
A further object of the present invention relates to a method for determining whether a patient suffering from a medulloblastoma will achieve a response with an inhibitor of the TGFp/ActivinB signaling pathway comprising determining the expression level of PMEPA1, wherein said expression level correlates with the response of the patient to the treatment.
In some embodiments, the method comprises the steps of i) determining the expression level of PMEPA1, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will achieve a response when the level determined at step i) is higher than the predetermined reference value.
The method is thus particularly suitable for discriminating responder from non responder. As used herein the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or improved. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after the therapy. A non-responder or refractory patient includes patients for whom the cancer does not show reduction or improvement after the therapy. According to the invention the term “non responder” also includes patients having a stabilized cancer. Typically, the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a patient who is known to be a responder or non responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media. When it is concluded that the patient is a non responder, the physician could take the decision to stop the therapy to avoid any further adverse sides effects.
In some embodiments, the methods further comprises the steps consisting of determining the level of p-Smad2 and the expression level of INHBB, comparing said levels with their corresponding predetermined reference value, and concluding that the patient will achieve a response when the determined level are higher than their predetermined reference value. WO 2021/047775 PCT/EP2019/074309
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. TGFp/ActivinB signaling promotes cell proliferation in Group 3 MB cell lines
A-H 1603MED (A-D) or D283 (E-H) cells were treated with DMSO (vehicle), with LY364947, or with SB431542. (A and E) Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2, and b-actin upon inhibition of TGFp/Activin signaling using LY364947 and SB431542 inhibitors for 24 h. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to b-actin. (B and F). Growth curve experiments showing cell proliferation upon TGtRb/Aohnίh signaling inhibition. (C and G) Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labeling at 48 h upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S, and G2/M phases). (D and H) Percentage of apoptotic cells measured by FACS analysis of cleaved caspase-3 48 h after TGtRb/Aohnίh signaling inhibition. The P-values were determined by unpaired t-test and two-way ANOVA for (B and F). *P < 0.05, **P < 0.01, ***P < 0.001. Bars represent the mean ± SD. Number of replicates is n > 3.
Figure 2. ActivinB signaling is a potential therapeutic target for patients of group
3 MB
A. Kaplan-Meier representing survival of mice treated with either vehicle or Galunisertib (FY2157299,) or Cisplatin or a combination of Galunisertib and Cisplatin after orthotopic grafting of PDX4 cells into the cerebellum. The rectangle represents Galunisertib treatment duration, while the dotted lines represent the 3 Cisplatin administrations.
B. Boxplot of tumor area after 25 days of treatment. On the right, boxplots represent quantification of P - Smad2 staining on tumor (IHC).
EXAMPLE:
The results reported in the present EXAMPLE were presented in the scientific article: Morabito M, Larcher M, Cavalli FM, Foray C, Forget A, Mirabal-Ortega L, WO 2021/047775 PCT/EP2019/074309
Andrianteranagna M, Druillennec S, Garancher A, Masliah-Planchon J, Leboucher S, Debalkew A, Raso A, Delatre O, Puget S, Doz F, Taylor MD, Ayrault O, Bourdeaut F, Eychene A, Pouponnot C. An autocrine ActivinB mechanism drives TGF b/Activin signaling in Group 3 medulloblastoma. EMBO Mol Med. 2019 Aug;ll(8):e9830.
Accordingly, for the results that are not illustrated in the present specification (i.e. data not shown), one can easily retrieve the corresponding figure from the article as published.
Methods
Bioinformatics analyses
Normalized primary medulloblastoma gene expression data (763 samples) and samples affiliation published in Cavalli et al (2017a) were used to generate scatter plots and gene expression boxplots per subgroup and subtype for the genes of interest. Normalized primary medulloblastoma protein levels data (45 samples) and samples affiliation published in Archer et al (2018a) were used to generate protein levels boxplots per subgroup and subtype for the proteins of interest. Wilcoxon rank-sum tests were performed between subgroups and subtypes. Spearman rank correlation coefficients were computed between the INHBB gene expression values and all other genes for Group 3 samples. The gene pairs were ranked according to the Spearman correlation values.
Patient samples
All MB samples were collected following written informed consent, and study approval was obtained by internal review boards from the following institutions: the Necker Hospital for Sick Children (Paris, France) and the Hospital for Sick Children (Toronto, Canada) (Forget et al, 2018).
Cell culture conditions and treatments
HD-MB03 (named HDMB03) obtained from Dr. Milde (Milde et al, 2012), D458MED (named D458) obtained from Dr. Bigner (He et al, 1991), UW228 (Keles et al, 1995), ONS-76, and DAOY MB cell lines (ATCC) were cultured as described in Garancher et al (2018). 1603MED obtained from Dr. Raso (Raso et al, 2008) and D283MED (ATCC) (named D283) cell lines were maintained in DMEM condition supplemented with 12% fetal bovine serum (GIBCO), 50 units/ml penicillin and streptomycin (Invitrogen) and 0.1 mM non-essential amino WO 2021/047775 PCT/EP2019/074309 acids and sodium pyruvate. 1603MED cell lines were also supplemented with 2 mM L- glutamine. All cells were cultured at 37°C in a humidified atmosphere containing 5% C02. LY363947 and SB431532 resuspended in DMSO (selleckchem) were used at a final concentration of 5 mM for 24 h. Stimulations with TGFB1 and ActivinB were performed for 1 or 24 h at 10 ng/ml. Inhibitions with a recombinant blocking antibody against ActivinB (R&D systems) or recombinant follistatin (R&D systems) were performed for 24 h at 5 and 0.2 pg/ml, respectively.
Growth curves and proliferation assays
For growth curve analyses, 1603MED cells were plated at 8 x 105 cells/ml, and D283 and D458 at 2.5 x 105 cells/ml. Cell were treated once at day 0. Number of viable cells was assessed as indicated in each figure. For D283 and D458, proliferation was monitored using Incucyte Proliferation Assay (Essen bioscience) by analyzing the surface occupied by cells (% confluence).
Conditioned media experiments
Receiving cells (HDMB03) were plated at 1.5 x 105 cells/well in 6-wells plates. 1603MED and HDMB03 conditioned media were obtained by 18 h of incubation at 1 x 106 cells/ml. Non-conditioned media was obtained in the same conditions in absence of cells. Media were collected, filtered, and incubated with PBS as control or blocking antibody against ActivinB (5 pg/ml) for 2 h at 4°C with rotation. Cells were treated with 1 ml of media for 1 h, and cell extracts were collected for WB analysis.
Western Blotting and antibodies
Cell extracts were obtained and WB analyses performed as described in Rocques et al (2007). Membranes were incubated at 4°C overnight with anti-Smad2 (CST, CS86F7, 1/1,000), anti-PhosphoSmad2 (CST, CS138D4, 1/1,000), anti-OTX2 (MerckMillipore, #AB9566, 1/10,000), anti-MYC (CST, CSD3N8F, 1/1,000), anti-PMEPAl (proteintech, 1/500), and anti- b-Actin (Sigma A1978, 1/5,000). Signals were acquired with a CCD camera (G/BOX, Syngene).
Real time RT-PCR
All experiments were performed according to the protocols described in Garancher et al
(2018). WO 2021/047775 PCT/EP2019/074309 siRNA and transfection assays
Transfection assays were performed in either 96- or 6-well plates. siRNA transfection was performed according to the manufacturer's instructions (Dharmacon). DharmaFECT 3 transfection reagent was used at 0.15 and 4 m1/100 mΐ of transfection medium for D283 and 1603MED cell lines, respectively. D283 cells were plated at 5 x 105 cells/ml and siRNA were used at a final concentration of 25 nM. 1603MED cells were plated at a concentration of 1 x 106 cells/ml with 10 mM final of siRNA. Transfection assay efficiency was assessed using siGlo (D001630-01-05). siRNA smartpool CTRL (D-001810-00-1005), smartpool GNHBB (L- 011702-00-0010), smartpool PMEPA1 (L-010501-00-0020), ON -TARGETplus individual siRNA PMEPA1#1 (L-010501-05), and PMEPA1#2 (L-010501-08) were purchased from Dharmacon. For rescue experiments, cells were stimulated 10 h after transfection with ActivinB at 10 ng/ml.
Apoptosis and cell cycle analyses by flow cytometry
1603MED and D283 cell lines were plated at 8 x 105 and 2.5 x 105 cell/ml, respectively. Apoptosis was assessed at day 2 using cleaved caspase-3 staining with Apoptosis Kit, APC (BD Bioscience). Cell cycle was analyzed at day 2 using APC BrdU flow Kit (BD Bioscience). Experiments were performed using FACS Kanto (BD Bio science) and analyzed with FlowJo software (Tree Star).
Patient derived xenografts and PDX cultures
PDXs were obtained, maintained, dissociated, and cultured as described in Garancher et al (2018). PDX3, PDX4, and PDX7 correspond to ICN-MB-PDX-3, ICN-MB-PDX-4, and ICN-MB-PDX-7, respectively. All in vitro treatments were performed as described for cell lines.
Animal experimentation
NMRI-nu immunodeficient mice were obtained from Janvier Laboratory. Experiments were performed on 7-8 weeks old female mice after 1 week of acclimation in animal facility of Curie Institute. Mice were housed under a controlled temperature and 12 h/12 h light-dark cycle with access to food and water ad libitum in conventional animal facility. For the animal welfare, mice are maintained in social groups with enrichment. Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved WO 2021/047775 PCT/EP2019/074309 by the ethics committee of the Institut Curie CEEA-IC #118 (Authorization 02383.02 given by National Authority) in compliance with the international guidelines.
Orthotopic transplantation and pharmacological inhibitor treatments
NMRI Nude female mice (Janvier labs) were orthotopically grafted directly in the cerebellum at 7 weeks with 3 x 105 cells/5 mΐ of ICN-MB-PDX-4 cells as described in Garancher et al (2018). After 3 days, mice were administrated 300 mΐ of LY2157299 (Galunisertib, AbMole Bioscience) orally at a dose of 75 mg/kg in 12% DMSO, 30% PEG, and water. Mice were treated 7 days a week twice a day until day 30. Mice were injected with Cisplatin (Sigma) in saline solution at a dose of 2 mg/kg intra-peritoneally at days 4, 8, and 12 post-grafting. Mice were euthanized when scientific and clinical end points were reached and brains were collected and fixed.
Tissue processing and immunohistochemistry (IHC)
After 25 days of treatment, 6 mice per group received ice-cold PBS and 4% formaldehyde/PBS via intracardiac perfusions. Brains were collected and fixed overnight in 4% formaldehyde/PBS at 4°C. IHC was performed on 12-pm-thick sections with the following primary antibodies: anti-PhosphoSmad2 (CST, CS138D4, 1/300), Ki67 (CST, CS9161, 1/500), and cleaved caspase-3 (eBioscience, #14-5698-82, 1/500). Image acquisitions were performed on a Zeiss microscope. Tumor size and IHC staining were assessed using ImageJ software.
Quantification and statistical analyses
Western blot was quantified from digital data acquisition (CCD camera) using ImageJ software. Statistical details can be found in both figures and figure legends. A P < 0.05 is considered as significant. IHC quantifications were assessed using ImageJ software. All experiments were performed, at least, in three independent triplicates.
Results
TGFp/ActivinB signaling pathway is active in Group 3 MB
Since different genomic alterations in the TGFp/Activin pathway have been previously described in Group 3 MB (Northcott et al, 2012b, 2017; Lin et al, 2016), we first investigated whether the pathway is activated in patient samples. We performed WB analysis on 38 medulloblastomas: 7 WNT, 12 SHH, 10 Group 3, and 9 Group 4 tumors. Activation of the WO 2021/047775 PCT/EP2019/074309 pathway, monitored by the level of Smad2 phosphorylation (P-Smad2), was observed in some patient samples from all MB groups (data not shown). An inter-tumor heterogeneity was observed in each group, with some samples with high P-Smad2. However, an overall higher level of Smad2 phosphorylation was observed in Group 3 when normalized to b-actin (data not shown). This was not evidenced when normalized to total Smad2 (data not shown) since an important variation of Smad2 level was observed (data not shown). This is in line with the modification of Smad2 stability by auto -regulatory mechanisms (Yan et al, 2018). Thus, the overall level of P-Smad2/p-actin, which formally reflects the level of nuclear and active Smad2, led us to conclude that TGFp/Activin pathway is activated in some Group 3 patients.
Considering that amplifications of receptors of the pathway have been described in less than 10% of Group 3 tumors (Northcott et al, 2012b), we hypothesized that other mechanisms may account for pathway activation in several G3 samples. Activation of the Smad2/3 pathway in cancer is frequently due to autocrine/paracrine activation by TGFP ligands (Rodon et al, 2014). Therefore, we analyzed the expression of major mediators of the TGFp/Activin pathway, including ligands and receptors in previously published MB dataset at the mRNA (Data ref: Cavalli et al, 2017b) and protein (Data ref: Archer et al, 2018b) levels. No major difference in the expression of the different receptors was observed between the different groups (data not shown). In contrast, striking differences were observed for the ligands. For example, TGFB2 was found highly expressed in SHH tumors (data not shown). We observed higher expression levels of TGFB1, TGFB3, and GNHBB (encoding ActivinB) in Group 3 in comparison with the other ones although expression of TGFB3 is similar between Group 3 and Group 4 (data not shown). These results were confirmed at the protein level (data not shown). These data were compatible with an autocrine activation of the pathway by one of those ligands listed above in Group 3 MB.
We next investigated the activation of TGFp/Activin pathway in MB cell lines. We analyzed the level of P-Smad2 in four well-established Group 3 MB cell lines (HDMB03, D458, 1603MED, and D283) as well as in three cell lines classified as non-Group 3 (DAOY, ONS76, and UW228). Western Blot (WB) analyses showed higher basal intensity of P-Smad2 signal in Group 3 cell lines (data not shown), confirming that the pathway is activated in this group. As in patient samples, we observed heterogeneity in the activation of the pathway, with a very WO 2021/047775 PCT/EP2019/074309 strong basal level of pathway activation being observed in the 1603MED cell line while in some cell lines its level was modest.
To understand what drives the basal activation of the pathway in Group 3 cell lines, we investigated the expression level of different ligands and receptors of the pathway by RT-qPCR (data not shown). No marked difference in the expression of the receptors was found between Group 3 and non-Group 3 cell lines, except a higher expression of ACVR1B, ACVR2A, and ACVR2B (data not shown) and a lower expression of TGFBR2, an obligatory partner for TGFBR1, in Group 3 cell lines. We did not observe any direct correspondence between the expression of the different receptors and the level of activation of the pathway in the different Group 3 cell lines (data not shown), suggesting that pathway activation is not directly linked to the deregulation of receptors expression. We investigated the expression of different ligands (data not shown) and found a higher expression of INHBB in the 1603MED and D283 Group 3 cell lines as compared to the others (data not shown). Interestingly, this level of expression directly corresponded to that of P-Smad2 levels, strong in 1603MED to intermediate in D283. This suggested that the ActivinB, encoded by INHBB, could be the major driver of Smad2/Smad3 phosphorylation in this group. The same observation could be drawn for TGFB3 in 1603MED and to a lesser extent in D283, while genes encoding the other ligands were not overexpressed in the cell lines showing a high level of P-Smad2 (data not shown). Taken together, these results suggested the potential existence of an autocrine mechanism involving either TGFB3 or INHBB that could be responsible for TGFp/ Activin signaling activation in Group 3 MB.
An autocrine stimulation involving ActivinB
To further investigate the presence of a potential autocrine mechanism, we first analyzed the ability of cell lines to respond to exogenous stimulation by either TGFP or Activin ligands, each requiring different sets of receptors. Non-Group 3 cell lines showed an increase in P- Smad2 signals in response to TGFP stimulation, while no modulation was observed upon Activin stimulation (data not shown). Strikingly, Group 3 MB cell lines showed the complete opposite profile: P-Smad2 signal was increased upon Activin stimulation, while it remained unchanged upon TGFP stimulation (data not shown). Noteworthily, 1603MED displayed a very high basal level of P-Smad2 which is constitutive. The reason for which G3 cell lines respond to Activin but not to TGFP is currently unknown. However, we noticed a lower level of TGFBR2 in these cells, a receptor required for TGFP response (data not shown). This was WO 2021/047775 PCT/EP2019/074309 also observed in G3 tumor samples at the RNA and protein level (data not shown). These opposite responses suggested a ligand-specific response between MB subgroups with Group 3 MB cell lines being able to respond to Activin but not to TGFp, thereby excluding TGFP ligands as a potential autocrine source for Smad2 activation. Since Group 3 cell lines displayed concomitant pathway activation and INHBB expression, these results strongly suggested that an ActivinB (encoded by INHBB) autocrine stimulation could be responsible for the activation of the pathway in the 1603MED and D283 Group 3 cell lines. To further investigate the potential role of ActivinB in the basal Smad2 activation in these cell lines, we focused on the 1603MED cell line, which shows the strongest basal activation. Treatment of 1603MED cells with an ActivinB blocking antibody induced a decrease in P-Smad2 level (data not shown). Importantly, the specificity of this antibody toward ActivinB was verified by showing that it does not block TGFP stimulation (data not shown). These experiments supported that an autocrine ActivinB production induced, at least partially, a strong activation of the pathway in 1603MED. This was further supported by P-Smad2 inhibition upon treatment with follistatin, a ligand trap for Activins (data not shown). We next sought to directly demonstrate that 1603MED cells secrete ActivinB. HDMB03 cells were used as receiving cells to conditioned media, since they showed the lowest basal activation of the pathway among G3 cell lines (data not shown) but efficiently responded to exogenous ActivinB and not to TGFP stimulation (data not shown). Three culture media were tested as follows: a non-conditioned media that had never been in contact with any cells, an HDMB03 -conditioned media, both of them being used as negative controls, and a 1603MED conditioned media. HDMB03 cells were treated with these different media for 1 h, and the effect on the Smad2 pathway was tested by WB (data not shown). 1603MED conditioned media induced a strong P-Smad2 signal as compared to the two control media. This induction was prevented by incubation with an ActivinB blocking antibody (data not shown), strongly supporting that 1603MED secreted active ActivinB ligand. To further substantiate this hypothesis, we targeted INHBB expression by siRNA in 1603MED. Although expression of INHBB was reduced to only 40% (data not shown), we nonetheless observed a decrease in P-Smad2 level (data not shown) resulting in decreased cell growth (data not shown). All these effects were rescued by exogenous addition of ActivinB (data not shown). Altogether, these results strongly support an autocrine secretion of ActivinB by 1603MED cells leading to P-Smad2 activation and promoting 1603MED cell proliferation.
ActivinB stimulation promotes proliferation WO 2021/047775 PCT/EP2019/074309
We next investigated the role of Activin pathway activation in Group 3 MB cell lines. D458 (data not shown) and D283 (data not shown) cells, which showed intermediate basal activation of the pathway (data not shown), were stimulated with ActivinB (data not shown). Activation of the pathway was validated by monitoring P-Smad2 levels (data not shown). Incucyte Proliferation Assay revealed an increase in cell proliferation upon ActivinB stimulation in both cell lines (data not shown). It remains to be determined why ActivinB did not promote cell growth while activating the pathway in HDMB03 (data not shown). An increase in cell proliferation can result from faster cell cycle progression, a reduction in cell death, or both. We analyzed the cell cycle profile by BrdU incorporation and 7AAD labeling and observed an increase in the number of cells in S phase following ActivinB stimulation, concomitant with a decrease in G0/G1 (data not shown). Apoptosis was monitored by FACS analysis of cleaved caspase-3 staining. We did not detect consistent effects on apoptosis, with a slight decrease in D458 cell line following stimulation after 2 days (data not shown), while no changes were detected in D283 (data not shown). These results indicated that ActivinB stimulates cell proliferation in Group 3 cell lines mainly by promoting cell cycle progression.
Inhibition of the pathway decreases proliferation
We next investigated the consequences of pharmacological inhibition of the pathway in Group 3 MB cell lines (Fig 1). One Group 3 cell line that exhibits a very high basal activation of the pathway (1603MED, Fig 1A-D) and one with an intermediate level (D283, Fig 1E-H) were treated with LY364947 or SB431542. These compounds prevent the phosphorylation of Smad2/3 by the TGFP and Activin type I receptors. Indeed, we verified that they prevent TGFp- as well as ActivinB -induced P-Smad2 (data not shown). After 24 h of treatment, the level of P-Smad2 was decreased in 1603MED and D283 cell lines (Fig 1A and E, respectively). This pathway inactivation was accompanied by a decrease in cell proliferation (Fig IB and F). FACS analyses were performed to measure BrdU incorporation and 7AAD labeling. Treatment with inhibitors induced a decrease in the percentage of cells in S phase concomitant with an increase in G0/G1 (Fig 1C and G). A very slight increase in the percentage of cells positive for cleaved caspase-3 staining was also observed (Fig ID and H), showing that the inhibition of the pathway mainly impacted on cell cycle and to a much lesser extent on apoptosis.
PMEPA1 is implicated in ActivinB promotion of cell growth
To identify relevant genes downstream of Activin signaling in Group 3 MB, we sorted the top 10 genes, whose expression was correlated with GNHBB in Group 3 patient samples WO 2021/047775 PCT/EP2019/074309
(data not shown). PMEPA1, which scored as the top gene, is a well-established Smad2/3 target gene in different cell types including P19 cells stimulated by Activin (Coda et al, 2017). Accordingly, we found that PMEPA1 expression level was enriched in Group 3 MB (data not shown) and correlated with INHBB expression in MB (data not shown). This correlation is highest in G3 as compared to the other groups (data not shown). Accordingly, we observed a good correspondence between P-Smad2 overall level and PMEPA1 protein expression in patient samples by Western blot analysis (data not shown). We next tested whether PMEPA1 is also a target of the Smad2 signaling in MB by modulating pathway activation (data not shown). Activation of the pathway by ActivinB induced an increase in PMEPA1 mRNA and protein levels, while its inhibition by LY364947, SB431542, blocking ActivinB antibody, or follistatin had the opposite effect in G3 cell lines (data not shown). MYC and OTX2 are key players in Group 3 MB and are also known as Smad2/3 target genes in other cell types (Jia et al, 2009; Brown et al, 2011; Coda et al, 2017). Therefore, we investigated whether their expression could be modulated by this pathway in Group 3 MB cell lines. In contrast to PMEPA1, no major change was observed at the mRNA (data not shown) and protein (data not shown) levels upon pathway inhibition regarding OTX2, while a slight decrease could be observed for MYC. However, no significant increase in MYC expression was observed upon ActivinB treatment (data not shown). Interestingly siRNA-mediated INHBB knockdown decreased PMEPA1 expression that could be rescued upon ActivinB treatment (data not shown). These results suggested that PMEPA1 is a target gene of the Activin pathway in Group 3 MB but that neither MYC nor OTX2, two important players of this group, appears to be consistently regulated by this signaling pathway although minor effects are observed on MYC. The role of PMEPA1 in cancer remains unclear and is likely to be cell type specific. It has been shown to either promote or restrain cancer progression (Liu et al, 2011; Fournier et al, 2015; Nie et al, 2016). Therefore, we investigated its role in Group 3 MB. siRNA-mediated PMEPA1 knockdown resulted in cell growth inhibition in both 1603MED and D283 cell lines (data not shown), suggesting that PMEPA1 is an important mediator of Activin signaling-mediated proliferation in Group 3 MB.
TGFp/ActivinB signaling pathway in Group 3 MB Patient Derived Xenografts (PDXs)
We further validated the importance of the pathway in patient derived xenograft (PDX) models, known to remain close to the original tumor (data not shown). As observed in Group 3 patient samples and cell lines, we found heterogeneous levels of P-Smad2, from high to WO 2021/047775 PCT/EP2019/074309 moderate, in the three Group 3 PDXs tested (data not shown). PDX4 displayed a very strong activation of the pathway, similar to that observed in the 1603MED cell line. We investigated the expression level of different mediators of the pathway by RT-qPCR (data not shown). This analysis showed heterogeneous expression levels of INHBB in the 3 PDXs (data not shown), which tightly corresponded to the level of P-Smad2. PDX4, which showed the highest level of expression of INHBB, also displayed the highest P-Smad2 signal (data not shown). As in cell lines, Group 3 PDXs responded to Activin but not to TGFP stimulation (data not shown). This result supported the observations in MB cell lines, suggesting a ligand specificity toward Activin in Group 3 MB. To further investigate the possibility of an autocrine mechanism involving ActivinB, we performed conditioned media experiments. Conditioned media from PDX4, which displays a strong activation of the pathway, markedly increased P-Smad2 phosphorylation in the receiving HDMB03 cells (data not shown). This induction could be partially prevented when the media was pre-incubated with an ActivinB blocking antibody (data not shown). Moreover, PDX4 treated with the same antibody also showed a decrease in P-Smad2 (data not shown). P-Smad2 signal could also be inhibited following treatment with inhibitors of type I receptors and follistatin (data not shown). We next assessed if this signaling pathway controls PMEPA1 expression. As in cell lines, a decrease in PMEPA1 expression was observed in PDXs after treatment with inhibitors and increased by ActivinB treatment. The expression of MYC and OTX2 remained mostly unchanged (data not shown). Altogether, these results confirmed those obtained in cell lines, highlighting the presence of an autocrine stimulation involving ActivinB in Group 3 MB and identified PMEPA1 as a gene, whose expression is controlled by this signaling pathway. We next investigated if inhibition of this pathway could be of therapeutic interest in vivo. The human PDX4, which displays a very high level of activation of the pathway, was orthotopically grafted into the cerebellum of nude mice. Animals were then treated 7 days per week twice a day with Galunisertib, a pharmacological inhibitor currently in clinical trial for Glioblastoma, Cisplatin as described in Niklison-Chirou et al (2017), or a combination. Galunisertib is described as a TGFP type I inhibitor but, since TGFP and Activin type I receptors are very similar, it also inhibits very efficiently ActivinB- induced Smad2 activation (data not shown). Accordingly, we verified that Galunisertib recapitulated the main in vitro data obtained with LY364947 and SB431542 (data not shown). Galunisertib -treated mice survived longer as compared to controls (Fig 2A), demonstrating the benefit of such treatment in tumors displaying high level of activation of the pathway. Accordingly, Galunisertib -treated mice displayed smaller tumors with less P-Smad2 (Fig 2B). WO 2021/047775 PCT/EP2019/074309
No major difference was observed for Ki67 and cleaved caspase-3 staining (data not shown). Although we did not observe any benefit from the combination of Galunisertib with Cisplatin (Figs 2A-B and data not shown), we cannot not exclude that different treatment kinetics could be more efficient. In this respect, other combinations with different drugs or radiotherapy remain to be evaluated.
TGFp/ActivinB signaling pathway in Group 3a subtype of MB
As mentioned above, tumor samples, PDXs, and cell lines from Group 3 displayed an inter-tumoral heterogeneity regarding the level of pathway activation, some of them showing a very strong P-Smad2 basal level. Recently, intragroup heterogeneity has been described in MB (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017) with the definition of new subtypes within Group 3 tumors. We wondered if this intragroup heterogeneity could explain our results. Since we showed that this strong activation was linked to an autocrine mechanism involving ActivinB, we investigated INHBB expression in these newly described subtypes of Group 3 tumors (data not shown). We found that INHBB displayed a significantly higher expression level in the Group 3a subtype as compared to Group 3b and Group 3g according to Cavalli et al (2017a) subtyping. Interestingly, PMEPA1 displayed the same profile, and consequently, INHBB and PMEPA1 expression was tightly correlated in Group 3 (data not shown). In contrast, MYC expression showed an opposite expression pattern as compared to INHBB (Fig 7D): Group 3g subtype, which is characterized by an enrichment of MYC amplifications, displayed the highest MYC expression levels, whereas the a subtype showed the lowest (Cavalli et al, 2017a). We recently reported that NRL and CRX control photoreceptor genes expression and define a subset of Group 3 tumors (Garancher et al, 2018). We found that alike INHBB, NRL is highly expressed in the G3a subtype (data not shown). This identifies Group 3a as the subtype that expresses high level of INHBB and high photoreceptor genes.
Discussion:
Group 3 is the most aggressive MB group with patients showing the poorest prognosis. Several genomic alterations have been identified, including those targeting the TGFp/Activin pathway at very low frequency. Indeed, SCNA analyses have identified uncommon gains and/or amplifications of genes encoding receptors of the TGFp/Activin pathway. Activation of the cognate Smad2/3 pathway in Group 3 tumors has never been investigated, neither its potential biological consequences nor its potential therapeutic targeting. Using patient samples, PDXs, and cell lines, we showed that, beside these infrequent genomic alterations, the WO 2021/047775 PCT/EP2019/074309
TGFp/Activin pathway is also activated in a specific subtype of Group 3, through an autocrine mechanism involving ActivinB. This pathway is involved in MB growth and represents an interesting therapeutic target.
ActivinB mediates Smad2/3 signaling in Group 3 MB
While activation of the TGFp/Activin pathway has been described in SHH group, no data are currently available regarding its activation in Group 3. A recent report showed that Prune- 1 may activate the TGFP pathway in Group 3 MB but the level of pathway activation in Group 3 was not investigated nor its functional relevance (Ferrucci et al, 2018). It has also been suggested that TGFP ligands determine the promigratory potential of bFGF signaling in MB but this study was performed in non-Group 3 cell lines and in atypical MB-PDX (Santhana Kumar et al, 2018). Using patient samples, we showed here that the TGFp/Activin pathway is activated in a subset of Group 3. We confirmed these results using PDXs as well as MB cell lines. In many different cancers, TGFP pathway activation involves autocrine loops, due to the high expression of genes encoding the different TGFP ligands (Rodon et al, 2014). We investigated the potential mechanism of activation of the pathway in Group 3. As in other cancers, we observed high expression of TGFB1 and TGFB3 in Group 3 MB. In addition, we also observed very high expression of INHBB, which encodes ActivinB, suggesting that TGFP 1 , TGFP3, and ActivinB ligands could be potentially responsible for pathway activation. Unexpectedly, our data clearly showed that Group 3 cells do not respond to TGFP stimulation, while they are highly sensitive to Activin, excluding de facto TGFpi and TGFP3 as potential ligands that would activate the pathway in an autocrine manner. The mechanism underlying the lack of TGFP responsiveness in G3 models is currently unknown. However, we noticed a significant decrease in RNA and protein TGFBR2 levels in G3 samples. Since TGFBR2 is absolutely required for signal transduction by TGFP ligands, this observation may provide a plausible explanation to this lack of response. In any case, our experiments based on conditioned medium, blocking antibody, follistatin treatment, and siRNA on cell lines clearly pointed out on ActivinB as an important determinant of pathway activation in Group 3. Importantly, these observations were confirmed on PDXs. According to transcriptomic data showing that INHBB expression is found in a large number of Group 3 MB, this autocrine mechanism is very likely the main mechanism leading to pathway activation in this group. Additional mechanisms, such as amplifications of receptors or Prune- 1 expression (see above), could also contribute to this activation, either by cooperating with ActivinB or by being involved in a more restricted number of Group 3 MBs that do not exhibit this autocrine WO 2021/047775 PCT/EP2019/074309 mechanism. Interestingly, while TGFPs and Activins activate the same Smad pathway (Smad2/3), TGFPs autocrine mechanisms have been much more frequently described to be implicated in cancer progression than Activins (Chen et al, 2002; Wakefield & Hill, 2013), highlighting a singularity of Group 3 MBs. Since Activin is involved in developmental processes (Wu & Hill, 2009), its implication in Group 3 MB instead of TGFP may relate to the pediatric nature of these tumors or to their cell of origin. In support of the latter and according to brain atlas data, INHBB displays a very cell-specific and dynamic profile during cerebellar development.
ActivinB induces PMEPA1 expression and promotes cell cycle progression
The TGFp/ Activin pathway is highly pleiotropic and sometimes displays antagonistic functions during carcinogenic processes. For example, it can promote either cell cycle arrest or proliferation, depending on the context. This opposite role has been well illustrated in Glioblastoma in which the epigenetic status of the cells, in particular its DNA methylation profile, is responsible for this duality (Bruna et al, 2007). In agreement with this pro-mitogenic activity, we found that pathway inhibition decreased cell proliferation in Group 3 MB, while ActivinB stimulation increased it by consistently promoting cell cycle progression. MYC and OTX2, two genes known to promote cell proliferation in Group 3 MB, are target genes of the Smad2/3 pathway in other contexts (Jia et al, 2009; Brown et al, 2011; Coda et al, 2017). In general, this signaling pathway reduces MYC expression (Warner et al, 1999; Seoane et al, 2001), although it can be induced in human embryonic stem cells (Brown et al, 2011). Since OTX2 has been demonstrated to be a major Smad2/3 target gene in the nervous system (Jia et al, 2009), it has been proposed to be a Smad2/3 inducible gene in Group 3 MB (Ferrucci et al, 2018) and considered as part of this signaling pathway in MB (Northcott et al, 2012b). We did not detect any consistent changes in MYC and OTX2 expression upon modulation of the Activin pathway, suggesting that this signaling pathway does not regulate these two genes in Group 3 tumors and promotes tumor growth through other mechanisms. In contrast, we showed that PMEPA1, whose expression is induced by TGFP or Activin signaling in many different contexts (Coda et al, 2017), is also an Activin-regulated gene in Group 3 MB. Indeed, inhibition or activation of the Activin signaling pathway modulated PMEPA1 expression accordingly. This regulation is likely to be relevant in patients since INHBB and PMEPA1 expression is correlated in human MB samples. PMEPA1 is the top correlated gene with INHBB within Group 3 MB, showing that their expression is strongly linked in this group. In all MB samples, the correlation is lower than within Group 3 samples. Indeed, PMEPA1 expression is higher in WO 2021/047775 PCT/EP2019/074309
Group 3 but reaches an intermediate level in WNT and SHH groups that do not express INHBB. In WNT and SHH groups, PMEPA1 expression is likely due to TGFp/Activin pathway activation, as highlighted by the high level of P-Smad2 found in patient samples in those two groups, although pathway activation is independent on ActivinB autocrine stimulation. Thus, PMEPA1 expression likely constitutes a relevant and general readout of Smad2/3 activation, which is due to an ActivinB autocrine stimulation in Group 3 and to other mechanisms in SHH and WNT groups. The role of PMEPA1 in cancer appears to be quite complex. It has been shown to act as negative auto -regulatory loop by limiting Smad2/3 activation (Watanabe et al, 2010) although this appears to be isoform dependent (Fournier et al, 2015). Other reports suggested that PMEPA1 could promote cell proliferation in cancer cells (Vo Nguyen et al, 2014; Nie et al, 2016) and convert TGFp/Activin signaling from a tumor suppressor to tumor promoting pathway (Singha et al, 2010). Although not excluding that PMEPA1 may limit Smad2/3 activation in Group 3 MB without abolishing it, our results are in line with those latter reports. Indeed, siRNA-mediated PMEPA1 downregulation decreased Group 3 cell proliferation showing that it is an important mediator of ActivinB promoting Group 3 MB growth.
Targeting the TGFp/Activin pathway in Group 3 as a therapeutic perspective
We observed an activation of the Smad2 pathway in Group 3 cell lines, PDXs, and patient samples. However, this activation appears to be heterogeneous. For example, some cell lines and PDXs displayed a very high basal level of Smad2 activation, while others a much more moderate and this held true on patient samples. Since different Group 3 subtypes have been described recently (Cavalli et al, 2017a; Northcott et al, 2017; Schwalbe et al, 2017), we investigated whether INHBB expression could be enriched in a given subtype. We observed that INHBB expression is higher in subtype Group 3a according to the classification of Cavalli et al (2017a). This subtype is characterized by the lack of MYC amplification and, as shown in this study, an overall moderate to low MYC expression level. This subtype displays high photoreceptor gene expression (Cavalli et al, 2017a), including those of the two master regulators of this program, NRF and CRX. Accordingly, we recently showed that their expression defines a specific subtype within Group 3 (Garancher et al, 2018). Our data may suggest that the expression of INHBB could lead to Smad2/3 activation in this subtype. Indeed, we found that PMEPA1, whose expression can be considered as a readout of Smad2/3 activation (see above), is significantly higher in Group 3a subtype as compared to other Group 3 subtypes. Moreover, its expression is tightly correlated to that of INHBB in the Group 3 WO 2021/047775 PCT/EP2019/074309 tumors, suggesting that INHBB expression leads to productive pathway activation. In support to this, PDX4, which expresses very high level of INHBB, also displays very strong Smad2 activation. This PDX is not MYC amplified and highly expresses the photoreceptor genes (Garancher et al, 2018). It should be nevertheless mentioned that the 1603MED cell line is also characterized by high INHBB expression and high Smad2 activation but is MYC amplified and does not express high level of photoreceptor genes (Raso et al, 2008). Thus, we proposed that activation of the Smad2/3 pathway involving an Activin B autocrine stimulation is enriched in subtype Group 3a, although not limited to this subtype. Interestingly, treatment with Galunisertib, whose toxicity and efficacy is currently tested in clinical trials for Glioblastoma patients, increased the survival of mice orthotopically grafted with PDX4. This suggests that Group 3a patients may be particularly sensitive to pathway inhibition.
Conclusion:
In conclusion, the TGFp/ Activin signaling pathway is activated through an ActivinB autocrine mechanism in a subset of Group 3 MB subtype. Not only this pathway is activated, but it also plays a growth -promoting role and constitutes an important driver of therapeutic interest in these tumors. We propose that high levels of INHBB, PMEPA1 expression, and Smad2 phosphorylation might constitute biomarkers for potential Group 3 patients to be eligible to treatment with inhibitors of the TGFp/Activin signaling pathway, in particularly Galunisertib.
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Claims

WO 2021/047775 PCT/EP2019/074309 CLAIMS:
1. A method of treating a patient suffering from medulloblastoma group 3 comprising administering to the patient a therapeutically effective amount of an inhibitor of the TGFp/ActivinB signaling pathway.
2. The method of claim 1 wherein the patient belongs to the subtype Group 3a.
3. The method of claim 1 wherein the inhibitor of the TGFp/ActivinB signaling pathway is selected from the group consisting of LY-580276, LY-364947, LY-2109761, LY- 2157299, LY-573636, SB- 505124, SB-431542, SB-525234, SD-208, SD-093, Ki- 26894, NPC-30345, SX-007, IN- 1130, EW-7203, EW-7195, EW-7197 and GW6604.
4. The method of claim 1 wherein the inhibitor of the TGFp/ActivinB signaling pathway is Galunisertib.
5. The method of claim 1 wherein the inhibitor of the TGFp/ActivinB signaling pathway is an antibody having specificity for Activin B.
6. The method of claim 1 wherein the inhibitor of the TGFp/ActivinB signaling pathway is an inhibitor of PMEPA1 expression.
7. The method of claim 1 wherein the patient is characterized by high level of Smad2 phosphorylation, high expression level of INHBB, and high expression level of PMEPA1.
8. The method of claim 1 comprising the step of determining the level of Smad2 phosphorylation, the level of INHBB expression and the level PMEPA1 expression in a tumor sample obtained from the patient.
9. The method of claim 8 wherein each determined level is compared to a predetermined reference value wherein when the three determined levels are higher than their respective predetermined reference value, the patient is thus eligible to a treatment with the inhibitor of the TGFp/ActivinB signaling pathway.
10. The method of claim 1 wherein the inhibitor of the TGFp/ActivinB signaling pathway is administered to the patient in combination with chemotherapy. WO 2021/047775 PCT/EP2019/074309
11. The method claim 10 wherein the chemotherapeutic agent is cisplatin.
12. The method of claim 1 wherein the inhibitor of the TGFp/ActivinB signaling pathway is administered to the patient in combination with radiotherapy.
13. A method for determining whether a patient suffering from a medulloblastoma will achieve a response with an inhibitor of the TGFp/ActivinB signaling pathway comprising determining the expression level of PMEPA1, wherein said expression level correlates with the response of the patient to the treatment.
14. The method of claim 13 comprising the steps of i) determining the expression level of PMEPA1, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will achieve a response when the level determined at step i) is higher than the predetermined reference value.
PCT/EP2019/074309 2019-09-12 2019-09-12 Use of inhibitors of tgfb/activinb signaling pathway for the treatment of patients suffering from medulloblastoma group 3 WO2021047775A1 (en)

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