Eric Eldering
University of Amsterdam, Experimental Immunology, Faculty Member
Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the... more
Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following C...
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Recurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3’ cryptic transcripts as a result of alternative branchpoint recognition. A large... more
Recurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3’ cryptic transcripts as a result of alternative branchpoint recognition. A large fraction of cryptic transcripts associated with SF3B1 mutations is expected to be sensitive for RNA degradation via nonsense-mediated mRNA decay (NMD). Several studies indicated alterations in various signaling pathways in SF3B1-mutated cells, including an impaired DNA damage response (DDR) in chronic lymphocytic leukemia (CLL). In this study, we investigated isogenic cell lines and treatment naïve primary CLL samples without any TP53 and/or ATM defect, and found no significant effects of SF3B1 mutations on the ATM/p53 response, phosphorylation of H2AX and sensitivity to fludarabine. Cryptic transcripts associated with SF3B1 mutation status were observed at relatively low levels compared to the canonical transcripts and were validated as target for...
Chronic lymphocytic leukemia (CLL) cells cycle between lymphoid tissue sites where they actively proliferate, and the peripheral blood (PB) where they become quiescent. Strong evidence exists for a crucial role of B cell receptor (BCR)... more
Chronic lymphocytic leukemia (CLL) cells cycle between lymphoid tissue sites where they actively proliferate, and the peripheral blood (PB) where they become quiescent. Strong evidence exists for a crucial role of B cell receptor (BCR) triggering, either by (self-)antigen or by receptor auto-engagement in the lymph node (LN) to drive CLL proliferation and provide adhesion. The clinical success of Bruton’s tyrosine kinase (BTK) inhibitors is widely accepted to be based on blockade of the BCR signal. Additional signals in the LN that support CLL survival derive from surrounding cells, such as CD40L-presenting T helper cells, myeloid and stromal cells. It is not quite clear if and to what extent these non-BCR signals contribute to proliferation in situ. In vitro BCR triggering, in contrast, leads to low-level activation and does not result in cell division. Various combinations of non-BCR signals delivered via co-stimulatory receptors, Toll-like receptors (TLRs), and/or soluble cytokines are applied, leading to comparatively modest and short-lived CLL proliferation in vitro. Thus, an unresolved gap exists between the condition in the patient as we now understand it and applicable knowledge that can be harnessed in the laboratory for future therapeutic applications. Even in this era of targeted drugs, CLL remains largely incurable with frequent relapses and emergence of resistance. Therefore, we require better insight into all aspects of CLL growth and potential rewiring of signaling pathways. We aim here to provide an overview of in vivo versus in vitro signals involved in CLL proliferation, point out areas of missing knowledge and suggest future directions for research.
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Cancer cells escape, suppress and exploit the host immune system to sustain themselves, and the tumor microenvironment (TME) actively dampens T cell function by various mechanisms. Over the last years, new immunotherapeutic approaches,... more
Cancer cells escape, suppress and exploit the host immune system to sustain themselves, and the tumor microenvironment (TME) actively dampens T cell function by various mechanisms. Over the last years, new immunotherapeutic approaches, such as adoptive chimeric antigen receptor (CAR) T cell therapy and immune checkpoint inhibitors, have been successfully applied for refractory malignancies that could only be treated in a palliative manner previously. Engaging the anti-tumor activity of the immune system, including CAR T cell therapy to target the CD19 B cell antigen, proved to be effective in acute lymphocytic leukemia. In low-grade hematopoietic B cell malignancies, such as chronic lymphocytic leukemia, clinical outcomes have been tempered by cancer-induced T cell dysfunction characterized in part by a state of metabolic lethargy. In multiple myeloma, novel antigens such as BCMA and CD38 are being explored for CAR T cells. In solid cancers, T cell-based immunotherapies have been ap...
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Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and... more
Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and pro-survival signals from surrounding cells, and become metabolically activated. However, detailed insight into the altered metabolism of LN CLL and how this may be related to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we have applied a two-tiered strategy. By using PB samples taken from patients before/after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR), which drives CLL cells out of the LN, combined with in vitro re-stimulation of TME signals, we indirectly mapped the metabolism of CLL in their TME, as well as the effects of IBR treatment. We hypothesized that the overlapping/distinct metabolites affected by IBR and in vitro stimulations would reflect th...
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Alterations in expression of specifically BCL-XL and MCL-1 dictate sensitivity of CLL cells to the Bcl-2 specific inhibitor venetoclax (VEN). We and others have shown upregulation of these anti-apoptotic proteins by interaction of CLL... more
Alterations in expression of specifically BCL-XL and MCL-1 dictate sensitivity of CLL cells to the Bcl-2 specific inhibitor venetoclax (VEN). We and others have shown upregulation of these anti-apoptotic proteins by interaction of CLL cells with CD4+ T helper cells within their lymph node microenvironment (LN-ME) mediated by CD40 signalling. We also reported significant metabolic changes of LN-ME activated CLL cells but whether metabolic alterations can be linked to VEN resistance remains unclear. As VEN is increasingly used in early stages of CLL, better understanding and tools to circumvent VEN resistance are highly needed. We aim to reveal the metabolic adaption of CLL to CD40 signalling in connection with VEN resistance. After in vitro CD40 signalling stimulation of peripheral blood (PB) CLL cells, mitochondrial mass and glucose uptake were measured by flow cytometry, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on Seahorse XF Analyser....
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The classic view of CLL as a static disease has recently been challenged by the first in vivo CLL cell kinetics study (Messmer BT, et al. J Clin Invest. 2005). In a clinical heterogeneous CLL patient cohort not only substantial... more
The classic view of CLL as a static disease has recently been challenged by the first in vivo CLL cell kinetics study (Messmer BT, et al. J Clin Invest. 2005). In a clinical heterogeneous CLL patient cohort not only substantial proliferation rates but also increased death rates were observed, supporting the concept of CLL as a dynamic disease. Whether this dynamic state also applies to patients with untreated clinically stable disease (and lymphocyte numbers) is currently not known. Proliferation of tumor cells is assumed to take place in proliferation centers in lymph nodes (LN) and bone marrow (BM), where survival largely depends on micro-environmental stimuli within these tissues. CLL cell death is thought to occur preferentially in the peripheral blood (PB). Although involvement of lymph nodes in both proliferation and cell survival has been studied well the precise role of the bone marrow is much less clear. The aim of the present study was to measure and model in vivo CLL kine...
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The treatment of chronic lymphocytic leukemia (CLL) with inhibitors targeting B cell receptor signaling and other survival mechanisms holds great promise. Especially the early clinical success of Ibrutinib, an irreversible inhibitor of... more
The treatment of chronic lymphocytic leukemia (CLL) with inhibitors targeting B cell receptor signaling and other survival mechanisms holds great promise. Especially the early clinical success of Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), has received widespread attention. In this review we will focus on the fundamental and clinical aspects of BTK inhibitors in CLL, with emphasis on Ibrutinib as the best studied of this class of drugs. Furthermore, we summarize recent laboratory as well as clinical findings relating to the first cases of Ibrutinib resistance. Finally, we address combination strategies with Ibrutinib, and attempt to extrapolate its current status to the near future in the clinic.Oncogene advance online publication, 23 June 2014; doi:10.1038/onc.2014.181.
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Research Interests: Immunology, Food, Biology, Cytokines, Apoptosis, and 6 moreMedicine, Signal Transduction, Humans, Animals, Lymphocytes, and Protein Binding
CD70 and CD27 are costimulatory molecules that provide essential signals for the expansion and differentiation of CD8(+) T cells. Here, we show that CD27-driven costimulation lowered the threshold of T cell receptor activation on CD8(+)... more
CD70 and CD27 are costimulatory molecules that provide essential signals for the expansion and differentiation of CD8(+) T cells. Here, we show that CD27-driven costimulation lowered the threshold of T cell receptor activation on CD8(+) T cells and enabled responses against low-affinity antigens. Using influenza infection to study in vivo consequences, we found that CD27-driven costimulation promoted a CD8(+) T cell response of overall low affinity. These qualitative effects of CD27 on T cell responses were maintained into the memory phase. On a clonal level, CD27-driven costimulation established a higher degree of variety in memory CD8(+) T cells. The benefit became apparent when mice were reinfected, given that CD27 improved CD8(+) T cell responses against reinfection with viral variants, but not with identical virus. We propose that CD27-driven costimulation is a strategy to generate memory clones that have potential reactivity to a wide array of mutable pathogens.
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Research Interests: Immunology, Immune response, Flow Cytometry, Immunohistochemistry, Apoptosis, and 15 moreGene expression, Cell separation, Protein Stability, Signal Transduction, Mice, Immunity, Adaptive Immunity, Animals, Immunoprecipitation, Immune system, T cell activation, Cell Expansion, Gene expression profiling, Immunoblotting, and reverse transcriptase polymerase chain reaction
The efficiency of humoral immune responses depends on the selective outgrowth of B cells and plasmacells that produce high affinity antibodies. The factors responsible for affinity maturation of B cell clones in the germinal center (GC)... more
The efficiency of humoral immune responses depends on the selective outgrowth of B cells and plasmacells that produce high affinity antibodies. The factors responsible for affinity maturation of B cell clones in the germinal center (GC) have been well established but selection mechanisms that allow clones to enter the GC are largely unknown. Here we identify apoptosis, regulated by the proapoptotic BH3-only member Noxa (Pmaip1), as a critical factor for the selection of high-affinity clones during B cell expansion after antigen triggering. Noxa is induced in activated B cells, and its ablation provides a survival advantage both in vitro and in vivo. After immunization or influenza infection, Noxa−/− mice display enlarged GCs, in which B cells with reduced antigen affinity accumulate. As a consequence, Noxa−/− mice mount low affinity antibody responses compared with wild-type animals. Importantly, the low affinity responses correlate with increased immunoglobulin diversity, and canno...
Research Interests: Flow Cytometry, Apoptosis, Gene expression, Mice, Female, and 14 moreAnimals, Blood, Male, Immunization, Clinical Sciences, B Lymphocytes, Cell Survival, immunoglobulin G, Germinal Center, Hemocyanin, reverse transcriptase polymerase chain reaction, Plasma cells, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
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Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious... more
Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple clones with slightly different affinities, thereby creating T cell memory with a certain degree of diversity. Currently, the mechanisms that control size, diversity, and cross-reactivity of the memory T cell pool are incompletely defined. Previously, we established a role for apoptosis, mediated by the BH3-only protein Noxa, in controlling diversity of the effector T cell population. This function might positively or negatively impact T cell memory in terms of function, pool size, and cross-reactivity during recall responses. Therefore, we investigated the role of Noxa in T cell memory during acute and chronic infections. Upon influenza infection, Noxa(-/-) mice generate a memory compartm...
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In chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We... more
In chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boos...
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Background. In healthy individuals it has been shown that regulatory T cells (Treg) differentiate from rapidly proliferating memory T cells. Rapid proliferation in combination with high susceptibility to apoptosis, make the Treg... more
Background. In healthy individuals it has been shown that regulatory T cells (Treg) differentiate from rapidly proliferating memory T cells. Rapid proliferation in combination with high susceptibility to apoptosis, make the Treg population highly dynamic. Tregs are thought to play an important role in immune evasion by malignancies. High Treg numbers in patients with malignancies are often associated with poor prognosis. An important question to be answered is whether tumor cells can promote the expansion and activation of Tregs. Recently it has been described that chronic lymphocytic leukemia (CLL) patients have increased numbers of Treg, with highest Treg frequencies in progressive patients1. The mechanism of this expansion however remains unknown. In the present study, we analysed the mechanism behind Treg expansion in CLL. Results. Neither analysis of the T cell receptor (TCR) repertoire nor CD45 isoform expression of Treg from CLL patients provided evidence for chronic (tumor) ...
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Chronic lymphocytic leukemia (CLL) cells can lose function of P53 and acquire resistance to standard chemotherapy. We found that expression P73, a member of the P53 family, could be induced in CLL cells following CD40-ligation via a c-abl... more
Chronic lymphocytic leukemia (CLL) cells can lose function of P53 and acquire resistance to standard chemotherapy. We found that expression P73, a member of the P53 family, could be induced in CLL cells following CD40-ligation via a c-abl dependent pathway. Induced expression of P73 was associated with re-acquired sensitivity to drugs commonly used treat this disease, such as Fludarabine monophosphate. Similar effects also were noted for CLL cells transduced with an adenovirus vector encoding P731. Prior studies found that P73 also can be induced in breast and colon cancer cell lines following treatment with platinum-based compounds, potentially accounting for the activity of this class of drugs. We hypothesized that treatment of CLL cells with such compound also could induce P73 and that such induced expression also might be associated with re-acquired sensitivity of P53-deficient CLL cells to standard anti-cancer drugs. We studied the mechanisms and effects of platinum-based compo...
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Seliciclib (cyc202, R-roscovitine) is a novel cyclin dependent kinase inhibitor currently in pre-clinical testing. It indirectly inhibits RNA polymerase II and has been shown to be effective against B-cell malignancies by inducing... more
Seliciclib (cyc202, R-roscovitine) is a novel cyclin dependent kinase inhibitor currently in pre-clinical testing. It indirectly inhibits RNA polymerase II and has been shown to be effective against B-cell malignancies by inducing apoptosis and Mcl-1 degradation. The aim of this study was to dissect the underlying apoptotic pathway(s) of seliciclib in chronic lymphocytic leukemia (B-CLL). Treatment of CLL cells with >10 μM seliciclib in vitro induced apoptosis within 24 hours, irrespective of IgVH mutation status (n=20). Gene profiling by means of RT-MLPA, did not reveal involvement of p53 as indicated by the absence of Puma upregulation. None of the >30 other apoptosis genes tested were induced; instead signals for certain labile mRNAs (Mcl-1, A1/Bfl-1, PI-9) were clearly decreased upon seliciclib. Detailed investigation of B cell lines overexpressing various anti-apoptotic proteins (Bcl-2, caspase-9-DN, Flip, FADD-DN), indicated that seliciclib activated the mitochondrial bu...
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The clinical success of PI3K and Btk inhibitors that block upstream BCR signals, has been primarily attributed to inhibition of adhesion and migration of CLL cells. However, the effects of these inhibitors on the proliferative capacity of... more
The clinical success of PI3K and Btk inhibitors that block upstream BCR signals, has been primarily attributed to inhibition of adhesion and migration of CLL cells. However, the effects of these inhibitors on the proliferative capacity of CLL cells is largely unknown. Interestingly, in vitro BCR triggering does not induce proliferation of CLL cells. Instead, we have found that activated T cell-derived signals, specifically CD40L and IL-21, initiate CLL proliferation in vitro and can be traced in CLL lymph node samples from patients [Pascutti et al., Blood 2013]. Targeting the underlying signaling pathways may be clinically relevant, and therefore we aimed to characterize the molecular mechanisms underlying antigen-independent proliferation. In the present study we applied CD40L/IL-21 stimulation to induce proliferation, and tested various specific kinase inhibitors for blocking potential. Surprisingly, the Btk inhibitor ibrutinib significantly suppressed CD40L/IL-21-induced prolifer...
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Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been... more
Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been described. T cell mediated lysis is another tool currently exploited in hematologic malignancies. In contrast to acute lymphoblastic leukemia (ALL) however, efficacy of autologous based T cell therapy, such as CAR T cells, in CLL has been low. This is linked to a CLL mediated acquired T cell dysfunction. Bispecific T cell engagers targeting CD19 are successfully applied in ALL, but whether it overcomes the acquired T cell dysfunction in CLL is unknown. We therefore tested efficacy of a CD3xCD19 Dual Affinity Re-Targeting molecule (DART) in CLL. Since it has been observed that bispecific antibodies can overcome deficient synapse formation in CLL (Robinson et al, 2018) and based on our assumption that T cell mediated lysis differs from venetoclax-medi...
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Introduction Genomic studies have revealed that individual tumours are genetically heterogeneous containing genetic alterations which can evolve over time. The recently discovered novel recurrent mutations in CLL, i.e. SF3B1, NOTCH1, but... more
Introduction Genomic studies have revealed that individual tumours are genetically heterogeneous containing genetic alterations which can evolve over time. The recently discovered novel recurrent mutations in CLL, i.e. SF3B1, NOTCH1, but also TP53 and ATM, were more often subclonal, indicating that they tend to arise later in leukemic development and contribute to disease progression. Although, it has been suggested that the presence of a subclonal driver mutation is an independent risk factor for disease progression in CLL, this has not yet been tested in a prospective trial with treatment naive patients. Aim To study the incidence and clinical impact of clonality of novel recurrent mutations in a prospective trial in high risk treatment naive CLL patients treated with fludarabine and cyclophosphamide with (FCA) or without (FC) alemtuzumab. Methods In the HOVON68 trial, treatment naïve CLL patients with a high risk profile defined as either 17p deletion, 11q deletion, trisomy 12, u...
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Mainly based on the observation that overexpression of CD23 in B-CLL cells is regulated by Notch2, deregulation of the Notch pathway has been suggested to contribute to the pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). The... more
Mainly based on the observation that overexpression of CD23 in B-CLL cells is regulated by Notch2, deregulation of the Notch pathway has been suggested to contribute to the pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). The aim of the present study was to assess a possible functional role of the Notch pathway in CLL. To this end we performed two kind of experiments. First we co-cultured primary CLL cells with either L cells or OP9 cells expressing the Notch ligands DeltaLike1 (DL1) and Jagged1 (Jag1). This did not affect cell survival in CLL. Next we evaluated the effect of the g-secretase inhibitors GSI-1 and GSI-9 (DAPT) which have been shown to block intracellular processing of all four Notch receptors. Here we encountered a surprising dichotomy: whereas DAPT was unable to induce apoptosis in CLL, it did inhibit lineage commitment of early thymic precursors by means of DL-1 triggering of the Notch1 receptor (Dontje et al, Blood 15 March 2006, Vol. 107, No. 6). In co...
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Background : Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived anti-apoptotic Mcl-1 protein, but it is unclear if and... more
Background : Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived anti-apoptotic Mcl-1 protein, but it is unclear if and how this leads to Bax or Bak activation that is required for most forms of cell death. Aim: To study the effects of seliciclib on apoptosis gene expression and Mcl-1 and Bcl-2 protein interactions in B cell chronic lymphocytic leukemia (B-CLL), a malignancy with known aberrant apoptosis regulation. Methods: Purified B-CLL cells (PBMC consisting of >90% B-CLL cells; n=20) and Ramos cell lines overexpressing different apoptosis regulators were used in this study. The effect of seliciclib on viability, apoptosis gene expression pattern, and protein associations was investigated via RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western blotting and co-immunoprecipitation assays. Ramos cells were transduced with retroviral vectors expre...
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1798 Introduction Development of chemoresistance in chronic lymphocytic leukemia (CLL) is at least partly mediated by protective stimuli within the lymph node (LN) microenvironment. Dasatinib has activity against multiple kinases which... more
1798 Introduction Development of chemoresistance in chronic lymphocytic leukemia (CLL) is at least partly mediated by protective stimuli within the lymph node (LN) microenvironment. Dasatinib has activity against multiple kinases which have been reported to be activated by the microenvironment, including SRC, c-Abl and BTK. We have recently shown that Dasatinib can effectively inhibit the anti-apoptotic program and as a consequence, restore fludarabine sensitivity in vitro. Patients and methods We conducted an open-label phase 2 trial of Dasatinib combined with fludarabine in twenty refractory CLL patients. Patients were treated with Dasatinib 100 mg once daily for 28 days. In patients who did not reach at least a PR, fludarabine was added (40 mg/m2 for 3 consecutive days q28) for at least 2 and a maximum of 6 cycles. Response assessments included CT-scans at baseline, following 4 weeks of Dasatinib monotherapy, after 2 cycles of combination therapy and at the end of protocol. In a ...
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The new BH3-mimetic ABT-199 antagonizes Bcl-2 and avoids the thrombocytopenia associated with clinical application of its predecessor ABT-263 (navitoclax). Chronic lymphocytic leukemia (CLL) cells are highly sensitive to ABT-199 and the... more
The new BH3-mimetic ABT-199 antagonizes Bcl-2 and avoids the thrombocytopenia associated with clinical application of its predecessor ABT-263 (navitoclax). Chronic lymphocytic leukemia (CLL) cells are highly sensitive to ABT-199 and the first clinical results show clear reductions in peripheral and bone marrow CLL cells and in lymph node size. In the lymph node, CLL cells receive pro-survival signals that upregulate Bcl-XL, Mcl-1 and Bfl-11. These Bcl-2 family members are not targeted by ABT-199, which poses the potential risk of remaining clones with residual viability. Here, we aimed to define the signals that determine sensitivity for ABT-199 and ABT-737 in an in vitro lymph node model of CLL. We applied CD40 and cytokine stimulation in combination with kinase inhibitors that are known to change microenviroenmental signals and increase drug resistance in CLL. Stimulation via CD40 plus IL-4 or IL-21 differentially affected the expression of Mcl-1, Bcl-XL, Bfl-1 and Noxa and this c...
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CLL progression and chemoresistance can result from signals from the lymph node (LN) microenvironment and from acquired aberrations in the DNA damage repair (DDR) pathway. Clinical targeting of kinases upstream in the B cell receptor... more
CLL progression and chemoresistance can result from signals from the lymph node (LN) microenvironment and from acquired aberrations in the DNA damage repair (DDR) pathway. Clinical targeting of kinases upstream in the B cell receptor (BCR) activation pathway, such as Btk or PI3Kδ results in egress of cells from the LN microenvironment [1,2]. Such prolonged lymphocytosis during kinase-inhibitor treatment appears to pose no clinical disadvantage [3]. However, it enhances the chance of accumulating resistance-inducing mutations, and therefore drugs that combine LN egress with direct cytoxicity could provide an improved therapeutic strategy for CLL. The mTOR complex, consisting of mTOR1 and 2, is the main downstream kinase of the PI3K/Akt pathway and contributes to proliferation and survival. DNA-PK is a kinase required for non-homologous end joining (NHEJ) of the DNA repair pathway. Inhibitors of crucial components of the DDR pathway might be active in CLL, especially in patients harbo...
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711 Mutations or deletions of the tumor suppressor p53 or its upstream kinase ATM are well-known determinants of poor prognosis in Chronic Lymphocytic Leukemia (CLL). In recent years, genome wide sequencing has uncovered novel gene... more
711 Mutations or deletions of the tumor suppressor p53 or its upstream kinase ATM are well-known determinants of poor prognosis in Chronic Lymphocytic Leukemia (CLL). In recent years, genome wide sequencing has uncovered novel gene mutations that correspond with poor prognosis. Specifically, recurrent mutations in the splicing factor SF3B1 and the Notch and NRAS/KRAS oncogenes have been found. These mutations were (in part) mutually exclusive with p53 and/or ATM mutations, which suggested overlap in biological function. Here, we report results of a comparative analysis of p53 target genes and in vitro drug responses in CLL samples with either p53 (n=9), ATM (n=10), SF3B1 (n=11), Notch (n=6), or NRAS/KRAS (n=4) gene deletions/mutations. We found that upon irradiation, mRNA induction of all tested p53 targets genes (p21, Puma, CD95, Bax, PCNA, FXDR) was clearly decreased in all SF3B1 mutated CLL samples (overall p<0.001). SF3B1 samples resembled ATM mutated/11q− CLL in displaying a...
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Key Points CD8+ T cells from CLL patients display aberrations in mitochondrial and glucose metabolism prior to and after stimulation. CD8+ CD19-CAR T cells have enhanced mitochondrial biogenesis in complete responding CLL patients... more
Key Points CD8+ T cells from CLL patients display aberrations in mitochondrial and glucose metabolism prior to and after stimulation. CD8+ CD19-CAR T cells have enhanced mitochondrial biogenesis in complete responding CLL patients correlating to expansion and persistence.
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Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We... more
Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increase...
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Stimulation of CD27, a member of the tumour necrosis factor receptor family, by its ligand CD70 induces expansion of IFNg secreting CD41 and CD81 T cells in vivo. We here analysed the
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C1-inhibitor(Mo), a dysfunctional C1-inhibitor molecule produced in two kindred with type II hereditary angioedema, has a mutation at the P10 position (Ala436 to Thr). Like most serpins with hinge region mutations (P14, P12, P10),... more
C1-inhibitor(Mo), a dysfunctional C1-inhibitor molecule produced in two kindred with type II hereditary angioedema, has a mutation at the P10 position (Ala436 to Thr). Like most serpins with hinge region mutations (P14, P12, P10), C1-inhibitor(Mo) loses its inhibitory activity. However, unlike the other hinge region mutations, this mutant is not converted to a substrate. As shown by nondenaturing gel electrophoresis, gel filtration, sucrose density gradient ultracentrifugation, and electron microscopy, C1-inhibitor(Mo) exists in both monomeric and multimeric forms. Polymerization probably results from reactive center loop insertion into the A sheet of an adjacent molecule. Native C1-inhibitor(Mo) was shown to have a thermal stability profile intermediate to those of intact and of cleaved normal C1-inhibitor. Native C1-inhibitor(Mo) did not bind to monoclonal antibody KII, which binds only to reactive center-cleaved normal C1-inhibitor. It did, however, react with monoclonal antibody...
Research Interests: Thermodynamics, Transmission Electron Microscopy, Biological Chemistry, Biological Sciences, Cell line, and 13 moreHumans, Animals, Biological, Threonine, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Molecular cloning, Transfection, Alanine, Amino Acid Sequence, Drug Stability, Protein Denaturation, and Medical and Health Sciences
Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties. The ability to bind to target proteases, as well as... more
Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties. The ability to bind to target proteases, as well as potential substrate-like behavior, was investigated with radioimmunoassays. The P1-Lys variant retained binding capacity toward C1s, plasmin, and kallikrein. In addition, complex formation with C1s was detected for P1-Asn and P1-His. All other P1 substitutions resulted in C1 inhibitor variants that neither complexed with nor were inactivated by C1s, kallikrein, beta-factor XIIa, or plasmin. Electrophoretic studies confirmed that P1-Lys and P1-His can form sodium dodecyl sulfate-resistant complexes with C1s. In contrast, the C1s-P1-Asn complex dissociated upon addition of sodium dodecyl sulfate. Kinetic experiments by the method of progress curves generated association rate constants (kon) with C1s of 4.2 x 10(4) M-1 s-1 for recombinant wild-type C1 ...