Jeffrey Bacha

Background: Standard-of-care for glioblastoma (GBM) includes surgery, radiation and temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%. Unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance. Second-line treatment with bevacizumab has failed to improve survival and GBMs escape treatment by inducing intratumor hypoxia. VAL-083 is a bi-functional DNA-targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces DNA interstrand crosslinks at N7-guanine, leading to double-strand breaks and cancer cell-death in GBM cells, independent of MGMT. VAL-083 is currently in Phase II clinical trial for the treatment of MGMT promoter unmethylated GBM, both recurrent and treatment-naïve (NCT02717962, NCT03050736), and it remains to be seen if it shows enhanced anti-tumor effect compared to TMZ. Based on its unique monosaccharide backbone structure, VAL-083 may also benefit from bevacizumab-induced GLUT transporter upregulation leading to enhanced uptake and anti-tumor activity. Methods: The cytotoxic effect of VAL-083 and TMZ was verified in 3D GBM organoids derived from 18 patient-derived orthotopic xenograft (PDOX) GBM models of different (epi)genetic background. Cell responses to drugs were calculated as the area under the curve (AUC). We further evaluated VAL-083 ability to decrease tumor growth in vivo in a MGMT-unmethylated, temozolomide-resistant recurrent GBM PDOX model. Mice were grouped into control, bevacizumab, VAL-083, and VAL-083+bevacizmab. Tumor progression was measured by MRI and histopathological assessment. Results: GBM organoids showed only partial response to TMZ. As expected, MGMT- methylated GBMs were less resistant in comparison to MGMT-unmethylated GBMs. VAL-083 was generally more effective than TMZ and response to VAL-083 was not dependent on MGMT promoter methylation status. Responses to TMZ and VAL-083 were comparable between treatment-naïve PDOXs and PDOXs derived from patients previously treated with TMZ and radiation. VAL-083 led to dramatic reduction of tumor growth in vivo (-83% for VAL-083 group, -90% for VAL-083 + bevacizumab). The analysis of tumor growth in time showed further reduction of tumor progression upon combined treatment. Histological assessment showed increased DNA damage (H2AX-P) in tumor cells. H2AX-P was only slightly increased in certain zones of the normal brain, close to meninges and subventricular zone, to a much lower extend in comparison to tumor cell, which was in line with the low toxicity of VAL-083. Citation Format: Anna Golebiewska, Anaïs Oudin, Virginie Baus, Ann-Christin Hau, Eliane Klein, Anne Steino, Jeffrey A. Bacha, Simone P. Niclou, Dennis M. Brown. Dianhydrogalactitol (VAL-083) exhibits strong efficacy in GBM tumors with different (epi)genetic background and treatment history [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5231.

Background: Standard-of-care for glioblastoma (GBM) includes surgery, radiation and temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%. Unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance. Second-line treatment with bevacizumab has not only failed to improve survival, but has also been shown to induce intratumor hypoxia, which is implicated in increased chemoresistance. VAL-083 is a bi-functional DNA-targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces DNA double-strand breaks at N7-guanine and cancer cell-death in GBM cancer stem cells (CSCs) and non-CSCs, independent of MGMT. We have previously shown that bevacizumab treatment upregulates expression of glucose transporters GLUT-1/GLUT-3 on GBM cells. We hypothesized that, based on its unique monosaccharide backbone structure, VAL-083 may benefit from bevacizumab-induced GLUT transporter upregulation leading to enhanced VAL-083 uptake and anti-tumor activity. Methods: To investigate the in vivo anti-tumor effect of VAL-083+bevacizumab, we used a orthotopic patient-derived xenograft GBM model. All mice carried MGMT-unmethylated, temozolomide-resistant recurrent T16 GBM tumors as detected by MRI 35 days post-implantation. Mice were grouped into control, bevacizumab, VAL-083, and VAL-083+bevacizmab. Tumor progression was measured by MRI on days 49 and 56, and tumor growth rate was calculated for the entire study (day 35 vs. 56) and for the last 7 days (day 49 vs. 56). Results: Tumors were significantly smaller in VAL-083-treated mice both compared to control (-83%, p<0.001) and compared to bevacizumab-treated (-75%, p<0.001) mice. Additionally, analysis of tumor growth in-time showed significantly reduced tumor growth rate for VAL-083+bevacizumab compared to VAL-083 alone (p<0.01). Conclusions: These results show strong in vivo anti-tumor efficacy of VAL-083 against MGMT-unmethylated, TMZ-resistant recurrent GBM. This effect was further augmented in combination with bevacizumab, providing rationale of clinical investigation of VAL-083 in combination with bevacizumab in the treatment of GBM. Citation Format: Anna Golebiewska, Anais Oudin, Anne Steino, Simone P. Niclou, Jeffrey Bacha, Dennis M. Brown. Dianhydrogalactitol (VAL-083) reduces glioblastoma tumor growth upon bevacizumab-induced hypoxia, in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1051.

Platinum (Pt)-based chemotherapy plays a key role in ovarian cancer treatment, but patients frequently develop Pt-resistance. Dysfunctional p53 is implicated in Pt-resistance, comprising a therapeutic challenge in high grade serous ovarian carcinoma (HGSOC), where p53 is universally mutated. Attempts to overcome Pt-resistance in HGSOC include agents blocking the DNA repair pathways, most notably PARP inhibitors (PARPi), leading to accumulation of DNA double strand breaks (DSBs) and cancer cell death. Sensitivity to PARPi is correlated with deficiencies in the homologous recombination (HR) DNA repair system, which accurately repairs DSBs. BRCA1 and BRCA2 are key proteins in HR, and mutated BRCA1/2 are well-established biomarkers for PARPi sensitivity. PARPi treatment of Pt-sensitive ovarian cancers have improved progression free survival; however, improvements in overall survival have not been achieved and a 5-year survival rate of 40% remains in ovarian cancer. Additionally, resistance to PARPi has emerged as a significant clinical challenge. VAL-083 is a first-in-class bifunctional DNA damaging agent with demonstrated clinical activity against a range of cancers, including ovarian. VAL-083 rapidly induces interstrand cross-links at guanine-N7 leading to DSBs, activation of the HR repair pathway and cancer cell death. VAL-083 is able to overcome cisplatin-resistance in a panel of ovarian cancer in vitro models. We have also shown that VAL-083 maintains activity independent of prominent DNA repair mechanisms implicated in resistance to numerous chemotherapeutics including cisplatin and PARPi such as MGMT, non-homologous end-joining and mismatch repair. Cancer cells thus rely heavily on a functional HR pathway for repair of VAL-083-induced DSBs. This rationalizes VAL-083 combination therapy with agents inducing DSBs or blocking their repair, including PARPi. Taken together, these data highlight VAL-083's potential for targeting Pt-resistant HGSOC in combination with PARPi. METHODS: The cytotoxicity of VAL-083 in combination with PARPi (olaparib, niraparib, rucaparib, veliparib or talazoparib) against HR-impaired (BRCA siRNA knockdown) and HR-proficient (control siRNA) ovarian cancer cells was studied. RESULTS: We report increased VAL-083 cytotoxicity against HR-impaired A2780 cancer cells. In addition, synergy between VAL-083 and olaparib, talazoparib and niraparib in both HR-proficient and deficient settings. VAL-083 combination with rucaparib produced synergistic cytotoxicity in the HR-deficient setting, while VAL-083 combination with veliparib was additive. These data demonstrate that VAL-083 can synergize with some PARPi in both HR-proficient and HR-deficient settings. CONCLUSION: Our results demonstrate a distinct DNA-damaging mechanism for VAL-083, resulting in the ability to overcome cisplatin-resistance in HR-impaired tumors. In addition, VAL-083 synergized with several PARPi, particularly olaparib, rucaparib and talazoparib, in both HR-proficient and deficient ovarian tumor cells. Citation Format: Anne Steino, Guangan He, Beibei Zhai, Jeffrey Bacha, Dennis M. Brown, Mads Daugaard, Zahid H. Siddik. VAL-083 (dianhydrogalactitol) synergizes with PARP inhibitors in BRCA-proficient and BRCA-deficient ovarian cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P053.

Dianhydrogalactitol (VAL-083) is a unique bi-functional alkylating agent causing methylation of N7-guanine and inter-strand DNA crosslinks. VAL-083 is a small water-soluble molecule that readily crosses the blood-brain-barrier. In China, VAL-083 is approved as a chemotherapeutic drug for the treatment of chronic myelogenous leukemia (CML) and lung cancer. In the United States, VAL-083 has been evaluated in more than 40 National Cancer Institute (NCI)-sponsored phase I and phase II clinical trials. Preclinical studies and clinical trial data suggested antineoplastic effects of VAL-083 in a variety of malignancies, including lung cancer, brain tumors, leukemia, cervical cancer, and ovarian cancer. Here we report new insight into VAL-083 mechanism of action by showing that VAL-083 leads to irreversible cell cycle arrest and cell death caused by replication-dependent DNA damage. In lung (H2122, H1792, H23 and A549) and prostate (PC3 and LNCaP) cancer cell lines, VAL-083 treatment caused irreversible cell cycle arrest in late S and G2 phase as measured by propidium iodide (PI) and immunofluorescent (IF) staining in synchronized cultures. Importantly, VAL-083 was cytotoxic to all cell lines tested (IC50 range 3.06 - 25.7 μM). Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. VAL-083 treatment led to phosphorylation of the proximal DNA double-strand break (DSB) sensor Ataxia Telangiectasia Mutated kinase (ATM), the single-strand DNA-binding Replication Protein A (RPA32), and the histone variant H2A.X (γH2A.X). Importantly, the DNA damage was specific to cells in S phase indicating that VAL-083-induced DNA cross-links translates into more severe DNA lesions during replication. Furthermore, S/G2 phase cell cycle arrest and increased phosphorylation of γH2A.X in cancer cells persisted after pulse-treatment with VAL-083, indicating irreversible DNA lesions. Taken together, VAL-083 displayed broad anti-neoplastic activity in lung and prostate cancer cells through the induction of replication-dependent DNA damage. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity in cancer cells will offer help in identifying and predicting efficacy of combination treatments. Citation Format: Beibei Zhai, Anne Steino, Jeffrey Bacha, Dennis Brown, Mads Daugaard. Molecular mechanisms of dianhydrogalactitol (VAL-083) in cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2985.

Dianhydrogalactitol (VAL-083) is a bi-functional DNA-damaging agent that targets N7-guanines and causes DNA inter-strand crosslinks. VAL-083 is a small water-soluble molecule that readily crosses blood-brain-barrier and accumulates in brain tumor tissue, making it a good candidate for targeting brain malignancies, such as glioblastoma multiforme (GBM). VAL-083 has demonstrated anti-tumor activity in prior NCI-sponsored clinical trials in brain tumors and other cancer types. Previous research in our group demonstrates VAL-083-induced DNA inter-strand crosslinks lead to replication-dependent DNA double strand break (DSB) lesions that are preferentially repaired by homologous recombination (HR) in non-small cell lung cancer (NSCLC) cells. To investigate if the observed effects of VAL-083 in NSCLC cells translate to other cancer indications as well, we tested the cytotoxic activity of VAL-083 in a panel of GBM and prostate cancer cell lines. Crystal violet cell proliferation assay showed broad cytotoxicity of VAL-083 in low concentration (µM) range in all the cell lines tested. Immunofluorescent and propidium iodide staining followed by flow cytometric analysis furthermore showed that VAL-083 treatment induced replication-dependent S/G2 cell cycle arrest. DNA repair markers were investigated by western blot and confocal microscopy analyses and confirmed the activation of HR DNA repair pathway after VAL-083 treatment in cancer cells. Using lentiviral transduction of MLH1 and MSH2 expression vectors in HCT116 and LoVo cell lines, we investigated if VAL-083 activity was affected by DNA mismatch repair (MMR), which is the secondary temozolomide (TMZ)-resistance mechanism in GBM patients. Our data showed that VAL-083 cytotoxicity was independent of MMR, suggesting that VAL-083 is able to overcome MMR-mediated TMZ-resistance in GBM patients. Additionally, we demonstrated synergistic effects between VAL-083 and inhibitors of both topoisomerase I and II (camptothecin, irinotecan and etoposide) in GBM and prostate cancer cells. Taken together. our present study validated the broad cytotoxic effect and mechanism-of-action of VAL-083 in a variety of cancer cells and suggested that VAL-083 is able to overcome MMR-mediated TMZ resistance in GBM. We further demonstrated synergy between VAL-083 and topoisomerase inhibitors in GBM, prostate cancer and NSCLC cell lines. This provides good guidance for improved treatment strategies to use VAL-083 either as single agents or as part of combination regimens in the treatment of cancer patients. Citation Format: Beibei Zhai, Sudha Sravanti Kotapalli, Jeffrey A. Bacha, Dennis M. Brown, Anne Steino, Mads Daugaard. Dianhydrogalactitol (VAL-083) synergizes with topoisomerase inhibitors to overcome homologous recombination repair activity in glioblastoma and prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1369.

DNA damage repair (DDR) describes the network of pathways that are responsible for minimizing the effect of daily DNA damage such as mismatched base pairs, single-strand breaks, and double-strand breaks (DSBs). Multiple DNA repair pathways are known, including mismatch repair (MMR), O6-methylguanine DNA methyltransferase (MGMT), non-homologous end joining (NHEJ), and homologous recombination (HR), which act either by repairing the damage, arresting cell growth or, if necessary, promoting cell death. DDR defects are a hallmark of cancer development, rendering the cancer cells highly sensitive to targeted DNA-damaging agents. Dianhydrogalactitol (VAL-083) is a first-in-class DNA-damaging agent that rapidly induces interstrand crosslinks at guanine-N7 causing persistent DNA DSBs that, if left unrepaired, are lethal to the cancer cells. We have previously shown that VAL-083 treatment leads to DNA damage that activates HR and is independent of MGMT DNA repair. Furthermore, we recently showed that VAL-083 induces persistent S/G2 phase cell cycle arrest, proposing potential for combination treatment with S-phase specific DNA-targeting agents such as topoisomerase and PARP inhibitors. In this study, biochemical and microscopic analyses of DNA repair markers were employed to investigate the VAL-083-induced DNA damage response in different MMR- or NHEJ-proficient/deficient cancer cell lines. VAL-083’s activity against HR-impaired cancer cells was also investigated using BRCA1 siRNAs. VAL-083 combination with topoisomerase inhibitors, etoposide (Top2) and camptothecin (Top1), and PARP inhibitor olaparib was investigated in PC3, A549, and A2780 cancer cells. Here, we report VAL-083 is a DNA-targeting agent that induces DNA DSBs, irreversible S/G2-phase cell cycle arrest, activation of the HR repair pathway, and ultimately cell death through mechanisms independent of MGMT, MMR, and NHEJ. We also show increased VAL-083 cytotoxicity against HR-impaired (BRCA1-knockdown) A2780 cancer cells, further supporting HR as the main repair pathway involved in VAL-083-induced cancer cell death, demonstrating the potential of VAL-083 for the treatment of HR-impaired tumors. Additionally, we report synergy between VAL-083 and topoisomerase inhibitors etoposide and camptothecin in A549 and PC3 cancer cell lines, as well as superadditivity with PARP inhibitor olaparib in A2780 cancer cells. Taken together, our results demonstrate a distinct DNA-targeting mechanism of VAL-083, leading to the ability to overcome MGMT- , MMR- , and NHEJ-related chemoresistance to common DNA-targeting agents, including temozolomide and nitrosoureas. In addition, increased VAL-083 cytotoxic effect in cancer cells with impaired HR and synergy/superadditivity with topoisomerase and PARP inhibitors was identified. Citation Format: Beibei Zhai, Guangan He, Anne Steino, Jeffrey A. Bacha, Dennis M. Brown, Zahid Siddik, Mads Daugaard. DNA-damaging agent dianhydrogalactitol (VAL-083) targets HR repair pathway and suggests combination therapy with topoisomerase and PARP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A109.

PURPOSE: Platinum drug resistance is a major clinical impediment that is normally ascribed to several mechanisms, with dysfunctional p53 playing a critical role, particularly in the highly lethal high-grade ovarian serous carcinoma, where p53 mutations can be >80%. Since restoration of p53 function could restore drug-dependent cytotoxicity, the goal of this study was to examine dianhydrogalactitol (VAL-083) for its potential in an ovarian tumor panel representing different p53 statuses and cisplatin-resistance. VAL-083 has demonstrated clinical activity against a range of tumor types, including ovarian cancer, in historical NCI-sponsored clinical studies, but its activity in a cisplatin-resistance setting is not known. VAL-083 is a bi-functional alkylating agent with a distinct mechanism of action, forming DNA interstrand cross links at the N7 position of guanine, whereas cisplatin and carboplatin predominantly form intrastrand DNA cross-links. METHODS: The tumor panel was composed of cisplatin-sensitive wild type (wt) p53 ovarian cancer cell line A2780, cisplatin-resistant A2780-derived heterozygous p53-V172F mutant 2780CP/Cl-16 cells, and three other cisplatin-resistant models harboring mutant (OVCAR-10) or wt p53 (Hey and OVCA-433). Cells were exposed to cisplatin or VAL-083 and the IC50 cytotoxic parameter was determined by fitting the 5-day MTT cell survival data to a 4-parameter sigmoidal curve. MTT assay was used in conjunction with Bliss Independence Model and combination index approaches to assess VAL-083/cisplatin combinations. Immunoblot analysis assessed p53 response. RESULTS: The IC50 of cisplatin in the cisplatin-sensitive A2780 model was 0.2-0.3 µM. In contrast, the IC50 in the four cisplatin-resistant models (2780CP/Cl-16, OVCAR-10, Hey and OVCA-433) was 3-8 µM; a 10- to 27-fold increase. The corresponding IC50 for VAL-083 was about 0.5 µM in A2780 cells and 2-4 µM in the four cisplatin-resistant models; a 4- to 7-fold increase, suggesting a distinct mode of action of VAL-083 and the ability to circumvent cisplatin resistance. In addition, these results further indicate that VAL-083 activity is less dependent on p53 status. However, immunoblots of 2780CP/Cl-16 cells suggest partial dependency on p53 by demonstrating that VAL-083, unlike cisplatin, can activate mutant p53-V172F through Ser20 phosphorylation, thereby preventing binding to p53 of inhibitory MDM4. VAL-083 thus restores p53 function, which is consistent with its greater potency in p53 mutated cisplatin-resistant ovarian cancer. These results suggest that VAL-083 have two mechanisms: one dependent on p53 and another on a different pathway. The apparent differences in the mechanism between cisplatin and VAL-083 prompted a combination study with the two agents, and consistent synergy was demonstrated. CONCLUSIONS: VAL-083 can circumvent cisplatin-resistance in ovarian tumor models, and its activity is less dependent on p53 status. Together with overt synergy between VAL-083 and cisplatin, our results demonstrate the effectiveness of VAL-083 against cisplatin-resistant ovarian cancer as a single agent or in combination with cisplatin. Citation Format: Anne Steino, Michelle Martinez-Rivera, Guanghan He, Xiaolei Xie, Jeffrey A. Bacha, Dennis M. Brown and Zahid H. Siddik. ACTIVITY OF DIANHYDROGALACTITOL (VAL–083) IN OVARIAN TUMOR MODELS, SENSITIVE OR RESISTANT TO CISPLATIN [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-108.

Cisplatin is an important frontline drug for ovarian carcinoma and non-small cell lung cancer (NSCLC). However, the initial response rates of up to 70% are usually followed by relapse due to the onset of drug resistance. Mechanistically, platinum resistance is multifactorial, with loss of p53 function (by mutation or inhibitory protein binding to wild-type (wt) p53) playing a central role. The appearance of drug resistance is a major clinical barrier and, therefore, new agents are needed to overcome this limitation. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent that induces DNA interstrand cross-links at N7-guanine, a mechanism that is distinct from the intrastrand cross-links by platinum-based drugs. VAL-083 is clinically approved in China for lung cancer, and is undergoing clinical trial in the US for glioma. In the present study, we have examined the in vitro cytotoxicity of VAL-083 as a single agent and in combination with cisplatin or oxaliplatin using the 5-day MTT assay. In the isogenic HCT-116p53+/+ and HCT-116p53-/- colorectal models, loss of p53 increased IC50 of (or resistance to) cisplatin and oxaliplatin by 3- to 6-fold, whereas resistance to VAL-083 was increased only 1.7-fold by loss of p53. These results indicate that the cytotoxicity of VAL-083 is less impacted than the platinum drugs by loss of p53. When tested in cisplatin-sensitive (A2780) vs. cisplatin-resistant wt p53 ovarian tumor models (2780CP-16, OVCAR-10, Hey and OVCA-433) there was a 10- to 40-fold increase in IC50 for cisplatin, while the corresponding increase in IC50 for VAL-083 was only 4- to 7-fold. This indicates only partially cross-resistance between VAL-083 and cisplatin and thus suggests a distinct mode of action for VAL-083 as compared to cisplatin. To further investigate, immunoblots were developed after a 24-h exposure of isogenic A2780 or 2780CP-16 models to cisplatin or VAL-083. The two drugs were equally effective at stabilizing and activating p53 in A2780 cells. However, in cisplatin-resistant 2780CP-16 cells, VAL-083 was more effective than cisplatin at increasing p53 and p21 levels, and at inducing Ser-15 and Ser-20 phosphorylation of p53. This is consistent with the ability of VAL-083 to circumvent cisplatin resistance and demonstrated that this alkylating agent also has the capacity to partially restore wt p53 function in ovarian tumor cells. The independent mode of actions of these drugs suggested the potential for combining VAL-083 with cisplatin or oxaliplatin. These combinations in wt (H460 and A549) and mutant (H1975 and H157) p53 NSCLC models demonstrated significant super-additivity (p<0.05) and/or synergy (CI < 1). Taken together, these results demonstrate the antitumor activity of VAL-083 against both wt and mutant p53 cancers and raise the clinical potential for treatment in a combination setting with platinum drugs. Citation Format: Anne Steino, Guanghan He, Michelle Martinez-Rivera, Jeffrey A. Bacha, Dennis M. Brown, Zahid H. Siddik. Enhanced in vitro activity of dianhydrogalactitol (VAL-083) in combination with platinum drugs: Impact of p53 and platinum-resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2157.

Non-small cell lung cancer (NSCLC) is treated with surgery followed by chemotherapy with either tyrosine kinase inhibitors (TKIs) or platinum-based regimens, but long term prognosis is poor. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent with proven activity against NSCLC in preclinical and clinical studies. VAL-083 is approved for the treatment of lung cancer in the Peoples Republic of China (PRC, Approval No. Guayo Zhunzi H45021133); however, clinical use has been limited by lack of mechanistic data. Here we aimed to investigate in vitro i) the role of p53 status in VAL-083 activity, ii) VAL-083 cytotoxicity in a panel of NSCLC cell lines, iii) the combination of VAL-083 with cisplatin or oxaliplatin in NSCLC cells. We further studied the combination of VAL-083 with cisplatin in NSCLC in vivo. Dependence on p53 status was investigated in isogenic HCT-116p53-/- and HCT-116p53+/+ models. VAL-083, cisplatin and oxaliplatin cytotoxicity was tested in a panel of 9 human NSCLC cell lines: 4 wt, 4 mutant and 1 null for p53. The combination potential for VAL-083 with cisplatin or oxaliplatin was investigated in 3 human NSCLC cell lines; H460 (p53wt), A549 (p53wt) and H1975 (p53mut) by determining superadditivity and synergy using the criteria of combination index (CI)<1. Cytotoxicity was monitored on day 5 with the MTT assay. The in vivo activity of VAL-083 (2, 2.5, or 3 mg/kg i.p.) in combination with cisplatin (2 mg/kg i.v.) was tested in Rag2 mice bearing A549 xenograft tumors. Studies in HCT-116 models showed that loss of p53 increased resistance to cisplatin and oxaliplatin by 3- and 6-fold, respectively, while resistance to VAL-083 was <2-fold, suggesting a more p53-independent mechanism for VAL-083. As single agents, VAL-083, cisplatin and oxaliplatin showed good cytotoxicity in all NSCLC cell lines, with TKI-resistant cell line H460 as the most sensitive (IC50 < 0.5 uM). The combination of VAL-083 with cisplatin or oxaliplatin in H460, A549 and H1975 cells demonstrated significant super-additivity (p<0.05) and synergism (CI < 1) for both combinations in all 3 cell lines. This strongly favors non-overlapping mode of action between the platinum drugs and VAL-083 and demonstrates synergism in TKI-resistant cell lines. In the in vivo model, tumor growth delays of 11, 18 and 25 days were observed for cisplatin combined with 2, 2.5 or 3 mg/kg VAL-083, respectively, while no tumour growth delay was seen between untreated and cisplatin. The median survival time was increased by 2 days for cisplatin alone, while VAL-083 (2, 2.5 and 3 mg/kg) combined with cisplatin increased survival by 17, 17, and 14 days, respectively. In conclusion, when combined with cisplatin or oxaliplatin, VAL-083 demonstrates superadditivity/synergy against NSCLC cells, independent of their p53 status. Further, VAL-083 in combination with cisplatin significantly increased median survival in vivo. These results strongly suggest a potential for VAL-083 as part of combination treatment with platinum drugs for NSCLC, including drug-resistant phenotypes. A clinical trial is planned under the context of the existing PRC approval to investigate these observations in a clinical setting. Results, if favorable, will support expanded clinical use of VAL-083 in PRC and will serve as the basis for global development of VAL-083 as a potentially important chemotherapeutic agent in the treatment of NSCLC. Citation Format: Anne Steino, Jeffrey A. Bacha, Guanghan He, Sarath Kanekal, Nancy Dos Santos, Shun Lu, Dennis M. Brown, Zahid H. Siddik. Dianhydrogalactitol (VAL-083) enhances activity of platinum drugs in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A159.

Despite several decades of clinical trial, diffuse intrinsic pontine gliomas (DIPG) continue to have a dismal outcome and survival remains dismal. DIPG is inoperable and standard treatment is radiation alone, as the addition of chemotherapy has not improved survival. Major obstacles to the successful treatment of DIPG include an intact blood-brain barrier impeding drug penetration and inherent tumor-cell resistance mechanisms to chemotherapeutics. Dianhydrogalactitol (VAL-083) is a structurally unique bifunctional DNA targeting agent that readily crosses the blood-brain barrier. VAL-083 forms interstrand DNA crosslinks at the N7 position of guanine, leading to persistent and irreversible DNA double-strand breaks, cell cycle arrest, and ultimately cancer cell death. VAL-083 has cytotoxic activity in several pediatric brain tumors as assessed in historical NCI-sponsored clinical trials, both as a single agent and in combination with other chemotherapeutics. We have previously demonstrated that VAL-083 is able to overcome chemoresistance mediated by DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Expression of MGMT is strongly correlated with resistance to temozolomide (TMZ), which is commonly used in combination with radiation for the treatment of adult brain tumors. VAL-083 activity is also independent of DNA mismatch repair (MMR) system in vitro, a secondary mechanism of resistance to TMZ. VAL-083 potentiates the effect of radiation in TMZ-resistant adult glioblastoma (GBM) cells in vitro and overcomes TMZ resistance in GBM cancer stem cells (CSCs) and non-CSCs. Additionally, VAL-083 demonstrated synergistic efficacy with inhibitors of topoisomerase 1 (camptothecin) and topoisomerase 2 (etoposide) against multiple cancer cell lines. VAL-083’s ability to cross the blood-brain barrier and its ability to overcome common resistance mechanisms, combined with its radiotherapy-potentiating effects, suggest that VAL-083 may provide a new treatment option for DIPG and other pediatric CNS tumors as a single agent, in combination with radiotherapy, or as part of a combination regimen with topoisomerase inhibitors. We recently completed a phase I/II clinical trial in refractory GBM and established a well-tolerated dosing regimen of VAL-083 in adult brain tumor patients. In the present study, we investigated the effects of VAL-083 in combination with radiation or irinotecan (topoisomerase 1 inhibitor) in a panel of DIPG cell lines as well as patient-derived xenografts models. The results will guide a potential clinical trial of VAL-083 in treatment of DIPG, either as part of a chemo-radiation regimen or in combination with topoisomerase inhibitors. Citation Format: Anne Steino, Beibei Zhai, Beibei Zhai, Jeffrey Bacha, Dennis Brown, Shaun Fouse, Joe Costello, Mads Daugaard, Mads Daugaard, Sabine Mueller. Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B30.

Poor outcomes in cancer therapy due to chemo-resistance remain a significant unmet clinical problem for many solid tumors and leukemias. VAL-083 (dianhydrogalactitol) represents a structurally unique first-in-class N7-bifunctional DNA alkylating agent. VAL-083 primarily mediates guanine N7 DNA cross-links, which separates it from other DNA alkylating chemotherapeutics (e.g. temozolomide) that have their primary cytotoxicity by targeting DNA at the O6 position of guanine. As expected from its mechanism, VAL-083 overcomes resistance associated with O6-methylguanine-DNA methyltransferase (MGMT) overexpression in vitro. Furthermore, unlike other N7-acting drugs, such as cisplatin which primarily induces intrastrand crosslinks, VAL-083 mediates interstrand DNA crosslinks. When tested side-by-side in a standard syngeneic mouse fibrosarcoma model (RIF-1 cell-line in C3H mice), VAL-083 demonstrated superiority to cisplatin in tumor growth delay. For mice treated with a single injection of cisplatin of 4 mg/kg, the tumor growth delay was 1.45 days compared to untreated controls. Single IP injection of VAL-083 at a dose of 10 mg/kg delayed tumor growth by 5.6 days. Combination treatment of VAL-083 followed immediately by cisplatin produced a more than additive effect by delaying growth 8.65 days. VAL-083 readily crosses the blood-brain barrier, accumulates in brain tissue and is effective against brain tumor cells both in vitro and in vivo, and against brain cancer stem cells in vitro. VAL-083 is currently in Phase I/II clinical trials for glioblastoma and recurrent metastatic brain tumors. Studies are underway to further characterize the efficacy of VAL-083 in various tumor cell-lines resistant to chemotherapy. Previous clinical studies showing VAL-083 activity in several tumor models, combined with the new data on MGMT-chemo-resistance and synergy with cisplatin makes VAL-083 a promising alternative for both primary and secondary brain tumors as well as cisplatin-resistant tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B252. Citation Format: Anne Steino, Jeffrey A. Bacha, William J. Garner, Sarath Kanekal, Zahid H. Siddik, Dennis M. Brown. The unique mechanism of action of VAL-083 may provide a new treatment option for some chemo-resistant cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B252.

2093 Background: Recurrent glial tumors of the brain continue to be one of the most challenging malignancies to treat, and median survival for patients with recurrent disease is approximately 6 months for glioblastoma multiforme (GBM). The front-line therapy for GBM - temozolomide (TMZ) - is subject to resistance by DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), leading to poor prognoses for patients with recurrent GBM. Dianhydrogalactitol (VAL-083)is a first-in-class bi-functional N7 DNA alkylating agent shown to cross the blood-brain barrier, accumulate in brain tissue, and have activity against GBM. Studies suggest that VAL-083 overcomes MGMT-driven drug resistance in vitro and targets cancer stem cells. The purpose of this study is to determine the maximal tolerated dose (MTD) of VAL-083 in patients with recurrent GBM or progressive secondary brain tumor, and explore the safety, pharmacokinetics and tumor responses to treatment. Methods: Open-label phase I/II dose-escalation study of VAL-083 in patients with histologically confirmed primary WHO grade 4 malignant GBM, now recurrent, previously treated for GBM with surgery and/or radiation, if appropriate, and have failed both bevacizumab and temozolomide; or progressive secondary brain tumor, has failed standard brain radiotherapy, and has brain tumor progression after at least one line of systemic therapy. The study uses a 3 + 3 dose escalation design, until reaching the MTD or maximum specified dose. Patients receive IV VAL-083 on days 1, 2, and 3 of each 21-day treatment cycle. In phase II, additional patients are treated at the MTD (or selected optimum dose) to measure tumor responses. Results: Cohort 1 (3 patients) and cohort 2 (4 patients) were completed without any DLT’s. Adverse events (AEs) have all been grade 1/2, with only 1 grade 3 AE, unrelated to treatment. Cohort 3 currently has 4 patient enrolled, without reaching the MTD. 1/7 (14.3%) patients in cohorts 1and 2 has prolonged stable disease (15+ cycles) on VAL-083 treatment. Conclusions: VAL-083 up to the 2nd dose level was well tolerated without any safety signals. Dose escalation is continuing. Clinical trial information: NCT01478178.

Background: Recurrent glial tumors of the brain remain one of the most challenging malignancies to treat. Median survival for patients with recurrent disease is approximately 6 months for glioblastoma multiforme (GBM), as front-line therapy temozolomide (TMZ) is subject to resistance by DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). VAL-083 represents a first-in-class bi-functional N7 DNA alkylating agent that readily crosses the blood-brain barrier and accumulates in brain tissue. Previously published pre-clinical and clinical studies suggest that VAL-083 has activity against a range of tumor types, including GBM. Furthermore, research shows that VAL-083 overcomes MGMT-driven drug resistance in vitro and has activity against cancer stem cells. Hence, the purpose of this Phase I/II study is to determine the safety and the maximal tolerated dose (MTD) of VAL-083 in patients with recurrent GBM or progressive secondary brain tumor, and to explore the pharmacokinetic properties and tumor responses to treatment. Objectives Part 1: 1) Establish the dose-limiting toxicities (DLT) of VAL-083 and identify appropriate dose and dosing regimen of VAL-083 2) Assess the safety and characterize the toxicities associated with VAL-083 3) Collect information about anti-tumor activity of VAL-083 Objectives Part 2: 4) Confirm the safety and tolerability of the chosen dose/dosing regimen in a larger number of patients 5) Obtain preliminary evidence of anti-tumor activity in GBM, as measured by response rate and progression free survival Methods: An open-label, single arm Phase I/II dose-escalation study designed to evaluate the safety, tolerability, pharmacokinetics and anti-tumor activity of VAL-083 in patients with i) histologically confirmed initial diagnosis of primary WHO Grade IV malignant GBM, now recurrent, or ii) progressive secondary brain tumor, having failed standard brain radiotherapy, and with brain tumor progression after at least one line of systemic therapy. The study utilizes a 3 + 3 dose escalation design, until the MTD or the maximum specified dose is reached. Patients receive VAL-083 intravenously at the assigned dose on days 1, 2, and 3 of each 21-day treatment cycle. In Phase II, additional patients will be treated at the MTD (or other selected optimum Phase II dose) to measure tumor responses. All patients enrolled have previously been treated with surgery and/or radiation, if appropriate, and must have failed both bevacizumab and TMZ, unless contraindicated. Results (study ongoing): Cohort 1 (3 patients) and cohort 2 (4 patients) were completed without reaching DLT and no drug-related adverse effects (AEs) were detected. 28.5% (2/7 patients) show stable disease or tumor regression in response to VAL-083 treatment as assessed by regular MRI scans and check-ups. Cohort 3 currently has 1 patient enrolled without reaching DLT. ClinicalTrials.gov Identifier: NCT01478178 Citation Format: Dennis M. Brown, Jeffrey A. Bacha, William J. Garner, Kent C. Shih, Howard A. Burris, Richard Schwartz. Phase I/II study of val-083 in patients with recurrent malignant glioma or secondary brain tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4672. doi:10.1158/1538-7445.AM2013-4672

Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard of care includes surgery, radiation and treatment with temozolomide (TMZ), however nearly all tumors recur and the prognosis for recurrent GBM is dismal. Most GBM tumors have unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT); a validated biomarker for TMZ-resistance. Second-line treatment with anti-angiogenic agent bevacizumab has not improved overall survival (OS) and 5-year survival is less than 3%. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent targeting N7-Guanine and inducing interstrand cross-links, DNA double-strand breaks and cell-death in GBM cell-lines and GBM cancer stem cells. VAL-083’s cytotoxicity is independent of MGMT status and VAL-083 overcomes TMZ-resistance in vitro. Our recent phase I/II clinical trial in recurrent GBM patients failing both TMZ and bevacizumab, suggested VAL-083 offers clinically meaningful survival benefits for patients with recurrent GBM and pinpointing a new dosing regimen (40 mg/m2/d on days 1,2,3 of a 21-day cycle). A pivotal Phase 3 study in recurrent GBM after failing both TMZ and bevacizumab is being planned. If successful, this study will serve as the basis for a New Drug Application (NDA) submission for VAL-083. In addition, a single-arm Phase 2 study to confirm the tolerability of the new dosing regimen in combination with radiotherapy and to explore the activity of VAL-083 in newly diagnosed MGMT-unmethylated GBM patients whose tumors are known to express high MGMT levels is proceeding. In the present Phase 2 clinical trial, the main goal is to assess the overall survival (OS) in MGMT-unmethylated, recurrent, bevacizumab-naive GBM. RATIONALE: The vast majority of GBM patients experience recurrent/progressive disease within a year from initial diagnosis and median survival after recurrence is 3-9 months. Chemotherapy regimens for these patients are lacking and there is a significant unmet medical need. Given VAL-083’s novel alkylating mechanism, promising clinical benefit, and favorable safety profile, a trial studying VAL-083 in MGMT-unmethylated recurrent GBM is warranted. METHOD: Open label, single-arm, biomarker-driven Phase 2 clinical trial in MGMT-unmethylated adult GBM patients at first recurrence/progression, prior to bevacizumab. 48 patients will be enrolled to determine if treatment with VAL-083 will improve OS at 9-months compared to historical control with lomustine. The patients will receive VAL-083 40mg/m2/day on days 1,2,3 of a 21-day cycle. Patients will be followed until death or for at least 9 months from enrollment, whichever occurs earlier. Survival will be compared to recently published EORTC26101 for recurrent MGMT-unmethylated GBM patients treated with lomustine. Secondary outcome measures include progression-free survival and overall response rate. Clinicaltrials.gov identifier: NCT02717962. Citation Format: Barbara J. O9Brien, Jeffrey A. Bacha, Dennis M. Brown, Anne Steino, Richard Schwartz, Sarath Kanekal, Lorena Lopez, Marta Penas-Prado. Phase II study of dianhydrogalactitol in patients with MGMT-unmethylated bevacizumab-naive recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT054. doi:10.1158/1538-7445.AM2017-CT054

2063Background: Glioblastoma (GBM) is the most common CNS tumor. Patients with recurrent GBM have few treatment options and dismal prognosis. O6-methylguanine-DNA-methyltransferase (MGMT) is correlated with resistance to front-line systemic therapy with temozolomide (TMZ) and poor patient outcomes. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent that readily crosses the blood-brain barrier and has demonstrated activity independent of MGMT in multiple GBM cell lines and cancer stem cells in vitro. VAL-083 showed promise against CNS tumors in prior NCI-sponsored clinical trials. The goal of this clinical trial is to determine the appropriate dose for VAL-083 for advancement to Phase III trials as a new treatment for recurrent GBM. Methods: Study Population: Patients must have recurrent GBM following surgery, radiation, TMZ and bevacizumab. Phase I: Open-label, single-arm dose-escalation study (3+3 design). Patients received VAL-083 on days 1, 2, 3 of a 21-day cycle, until MTD was reached. ...

DNA damage response (DDR) is a network of intracellular pathways designed to minimize the impact of DNA damage. Multiple DNA repair pathways are known, including mismatch repair (MMR), O6-methylguanine DNA methyltransferase (MGMT), non-homologous end joining (NHEJ), and homologous recombination (HR), which act either by repairing the damage, arresting cell growth or, if necessary, promoting cell death. DDR defects are a hallmark of cancer development, rendering the cancer cells highly sensitive to targeted DNA-damaging agents. Ovarian cancer is normally treated with platinum (Pt)-based chemotherapy, which forms predominantly intrastrand DNA crosslinks; however, most advanced ovarian cancer patients develop episodes of recurrent disease with progressively shorter disease-free intervals. Dysfunctional p53 diminishes the therapeutic cytotoxicity of many DNA-targeting drugs, including Pt-based agents, comprising a therapeutic challenge in high-grade serous ovarian cancer (HGSOC), where p53 is almost universally mutated (~96%). Attempts to overcome Pt-resistance in HGSOC include PARP inhibitors (PARPi), which target tumors with deficiencies in the HR pathway (50% of HGSOC), responsible for the repair of DNA double-strand breaks (DSBs). Unfortunately, PARPi resistance frequently arises, leading to a 5-year survival rate of only 40% in this cancer. VAL-083 is a first-in-class DNA-damaging agent that has demonstrated clinical activity against a range of tumor types, including ovarian cancer, in historical NCI-sponsored clinical studies. VAL-083 rapidly induces interstrand crosslinks at guanine-N7, causing DNA DSBs that, if left unrepaired, are lethal to the cancer cell. We have shown that VAL-083 treatment leads to DNA damage that activates HR and is independent of common DNA repair pathways, including MGMT, MMR, and NHEJ. Notably, VAL-083 induces persistent S/G2 phase cell cycle arrest through two parallel pathways--one p53-independent and one p53-dependent. Taken together, these data propose the potential for targeting HR-impaired tumors and for combination treatment with S-phase specific DNA-targeting agents, including PARPi. We have previously seen VAL-083 cytotoxicity against a panel of ovarian cancer cells with differing p53 status and superadditivity between VAL-083 and PARPi olaparib, in vitro. The purpose of this study was to expand on these studies by examining the cytotoxicity of VAL-083 against HR-impaired ovarian cancer cells, the impact of p53 status, and the potential for VAL-083 combination with PARPi as part of a treatment strategy for HR-impaired, p53 dysfunctional HGSOC. VAL-083 cytotoxicity was investigated using the 5-day MTT assay in A2780 ovarian cancer cells. HR-impaired A2780 were examined using BRCA1 siRNA oligos prior to VAL-083 exposure. Combinations with PARPi veliparib (catalytic) and talazoparib (trapping) were examined in HR-proficient and HR–impaired A2780. The impact of p53 status was investigated by CRISPR/cas 9 knockout of p53 in the wildtype (wt) A2780 cells and in p53-mutated OVCAR3 cancer cells. We report increased VAL-083 cytotoxicity against HR-impaired A2780 cancer cells, further supporting HR as the main repair pathway for VAL-083-induced DNA damage, and suggesting HR-impaired HGSOC as a target for VAL-083 treatment. We further report superadditivity between VAL-083 and PARPi veliparib and talazoparib. Significantly, VAL-083 cytotoxicity against wt and knockout p53 A2780 cells and against p53-mutated OVCAR3 cells differed only by a factor of ~2, supporting a p53-independent mechanism of action for VAL-083. In conclusion, our results demonstrate a distinct DNA-damaging mechanism for VAL-083, resulting in the ability to target HR-impaired tumors and overcome MGMT- , MMR- , and NHEJ-related chemoresistance. VAL-083 activity was independent of p53 status, and superadditivity with PARPi was identified in HR-proficient A2780. Studies in HR-impaired A2780 are ongoing and will also be presented. Citation Format: Jeffrey A. Bacha, Guangan He, Xiaolei Xie, Anne Steino, Dennis M. Brown, Zahid H. Siddik. Distinct mechanism of action of DNA-damaging agent dianhydrogalactitol (VAL-083) suggests combination therapy with PARP inhibitors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A01.

Ovarian cancer is usually treated with platinum (Pt)-based chemotherapy, but patients frequently develop Pt-resistance. Dysfunctional p53 is implicated in Pt-resistance, comprising a therapeutic challenge in high grade serous ovarian cancer (HGSOC), where p53 is 96% mutated. Attempts to overcome Pt-resistance in HGSOC include agents blocking the DNA repair pathways, most notably the PARP inhibitors (PARPi), leading to accumulation of DNA double strand breaks (DSBs) and cancer cell death. However, PARPi resistance often arises, leading to a 5-year survival rate of 40% in HGSOC. VAL-083 is a first-in-class DNA damaging agent with demonstrated clinical activity against a range of tumors, including ovarian. VAL-083 rapidly induces interstrand cross-links at guanine-N7 leading to DSBs, activation of the homologous recombination (HR) DNA repair pathway, S/G2 cell cycle arrest and cancer cell death. Notably, VAL-083 induces cell death through two parallel pathways; one p53-independent and one p53-dependent. We have shown that VAL-083 is able to overcome cisplatin resistance in a panel of ovarian cancer cells, independent of p53 status. We have also shown that VAL-083 maintains activity independent of DNA repair mechanisms such as O6-methylguanine DNA methyltransferase (MGMT), non-homologous end-joining (NHEJ) and mismatch repair (MMR), implicated in resistance to chemotherapeutics, including cisplatin and PARPi. Cancer cells thus rely heavily on functional HR for repair of VAL-083-induced DSBs, proposing combination therapy with agents further inducing DSBs or blocking their repair, including PARPi. Taken together, these data propose VAL-083 for treatment of Pt-resistant HGSOC and for combination therapy with PARPi. Here, we study the cytotoxicity of VAL-083 in combination with PARPi (olaparib, niraparib, rucaparib, veliparib or talazoparib) against HR-proficient and HR-impaired ovarian cancer cells, the impact of p53 status and Pt-resistance. VAL-083 cytotoxicity alone and in combination with PARPi was investigated using the MTT assay in HR-proficient and HR-impaired A2780 ovarian cancer cells. The impact of p53 status was studied by CRISPR/cas 9 knockout of p53 in wildtype A2780 cells and in a panel of p53-mutated ovarian cancer cells. We report increased VAL-083 cytotoxicity against HR-impaired A2780 cells. We further report superadditivity in both HR-proficient and HR-deficient A2780 cells between VAL-083 and olaparib, niraparib, talazoparib or rucaparib. In conclusion, our results demonstrated a distinct DNA damaging mechanism for VAL-083, resulting in the ability to overcome Pt-resistance, target HR-impaired tumors and overcome MGMT, MMR and NHEJ-related chemoresistance. In addition, VAL-083 activity was independent of p53 status and superadditive with PARPi in HR-proficient and HR-deficient A2780 tumor cells. Citation Format: Jeffrey A. Bacha, Guangan He, Anne Steino, Dennis M. Brown, Zahid H. Siddik. Dianhydrogalactitol (VAL-083) has a distinct mechanism of action that suggests combination with PARP inhibitors as an effective therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1962.

Standard treatments for glioblastoma (GBM) include surgery, radiation and chemotherapy with temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%, largely due to chemoresistance. Evidence shows that cancer cells utilize DNA damage repair pathways to overcome cytotoxic effects of chemotherapy. GBM tumors expressing O6-methylguanine-DNA-methyltransferase (MGMT) display intrinsic chemoresistance to TMZ and nitrosoureas, while a deficient DNA mismatch repair system (MMR) confers chemoresistance to TMZ and platinum agents. Alterations in p53, particularly gain-of-function mutations, are correlated with increased MGMT-expression and poor prognoses in GBM. Second line anti-angiogenic treatment with bevacizumab has not improved overall survival and has been shown to induce intratumor hypoxia and increased chemoresistance. VAL-083 is a bi-functional alkylating agent that readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has demonstrated activity against GBM in prior NCI-sponsored clinical trials. VAL-083 induces interstrand cross-links at guanine-N7 causing DNA double-strand breaks and cancer cell death. VAL-083 is equiactive against GBM cancer stem cells (CSCs) and non-CSCs independent of MGMT and p53 status, in vitro. We recently showed that VAL-083 leads to irreversible S/G2-phase cell cycle arrest, proposing synergy with S-phase specific chemotherapeutics, including topoisomerase and PARP inhibitors. VAL-083 further showed persistent activation of the homologous recombination (HR) DNA repair pathway and its potency was increased when HR was impaired, demonstrating that VAL-083-induced lesions are repaired via HR suggesting increased VAL-083 potency in HR-impaired tumors. Further, hypoxic cancer cells are known to downregulate their HR pathway, proposing increased VAL-083 potency in hypoxic tumors. Bevacizumab treatment increases hypoxia in tumor cells, presumably impairing HR, proposing VAL-083 as a treatment option in HR-deficient or hypoxic cancers following, or as part of a combination treatment with, bevacizumab. Here, VAL-083 cytotoxicity and DNA damage response was evaluated by crystal violet assays, western blot and flow cytometry. VAL-083’s ability to overcome MMR-related chemoresistance was investigated using lentiviral MLH1 and MSH2 vectors in HCT116 and LoVo cancer cells. We report synergy between VAL-083 and etoposide or camptothecin in A549 and PC3 cancer cell lines. We also investigated the potency of VAL-083 in GBM under hypoxia either in vitro or in vivo as part of a combination treatment with bevacizumab. Our results demonstrate a distinct anti-cancer mechanism for VAL-083, resulting in the ability to overcome resistance to TMZ and nitrosoureas, increased activity in cancers with impaired HR and synergy with etoposide or camptothecin. Citation Format: Beibei Zhai, Anna Gobielewska, Anne Steino, Jeffrey A. Bacha, Dennis M. Brown, Simone Niclou, Mads Daugaard. Molecular mechanisms of dianhydrogalactitol (VAL-083) in overcoming chemoresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2483. doi:10.1158/1538-7445.AM2017-2483

Non-small cell lung cancer (NSCLC) treatment usually involves surgery and chemotherapy with tyrosine kinase inhibitors (TKI) in patients with EGFR mutations (10-15% in Western population, 40% in Asian populations) or with platinum-based regimens. Response to TKI treatment is short lived, and tumors recur with new mutations, primarily T790M. Recurrent NSCLC with T790M is sensitive to third generation TKIs, but resistance usually emerges through new mutations, including KRAS. Resistance to cisplatin and carboplatin, partly due to p53 mutation, is also a major clinical limitation and long-term prognosis in NSCLC is poor. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent with demonstrated clinical activity against NSCLC in historical NCI-sponsored trials and VAL-083 is approved for lung cancer treatment in China. However, the mechanism-of-action of how DNA damage signals are propagated and their effects on NSCLC cells are not fully understood. Therefore, we examined VAL-083 in a panel of 11 human NSCLC cell lines harboring wild-type p53 (H460, A549, H226), mutant p53 (H1975, SkLU1, H2122, H157, H1792, H23) or null p53 (H838, H1299). Importantly, as determined by the 5-day MTT assay, VAL-083 was cytotoxic against all 11 cell lines at low μM concentrations and cytotoxicity was independent of p53 status. We chose 3 TKI-resistant cell lines with different mutation profiles for cell cycle kinetics studies: i) H1975 (p53 mut, EGFR-T790M mut, KRAS wt) with IC50 0.9μM, ii) A549 (p53 wt, EGFR wt, KRAS mut) with IC50 1.8μM, and iii) H157 (p53 mut, EGFR wt, KRAS mut) with IC50 4.5μM. In all 3 cell lines, early response at 18 hr showed dose-dependent increase in cells in S-phase, with continued slow cell cycle progression resulting in accumulation of cells in G2/M phase by 36 hr, suggesting persistent cell cycle arrest. DNA damage signaling was examined by immunoblot analysis in A549 and H1975 cells. In p53-wt A549, VAL-083 induced the phospho-Ser15 form of p53, total p53 and total p21, thus indicating that VAL-083 treatment activated p53 function. On the other hand, in p53-mutant H1975, VAL-083 treatment did not result in consistent p53 or p21 increases, but did readily induce phospho-Ser15 p53. This is consistent with a lack of p53 function, as anticipated for mutant p53 cells. Interestingly, H1975 was 2-fold more sensitive to VAL-083 than A549, suggesting, that in p53-mutant cells, VAL-083 acts through a p53-independent mechanism. Examination of ATR, ATM, Chk1 and Chk2 indicated that DNA damage by VAL-083 prompted phosphorylation of these kinases. Notably, the total anti-apoptotic Chk1 was more prominently reduced in H1975 than A549, which may partly explain the stronger cytotoxicity of VAL-083 in p53-mutant H1975. These preclinical data strongly support VAL-083 as a potential treatment of mutant p53 and TKI-resistant NSCLC, and indicate DNA damage signaling is mediated via ATM, ATR, Chk1 and Chk2. Citation Format: Anne Steino, Guangan He, Jeffrey A. Bacha, Dennis M. Brown, Zahid Siddik. DNA damage response to dianhydrogalactitol (VAL-083) in p53-deficient non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1429. doi:10.1158/1538-7445.AM2017-1429

Introduction: Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. In China, VAL-083 is approved to use in the chemotherapeutic treatment of lung cancer and chronic myelogenous leukemia. In the United States, VAL-083 is currently undergoing investigation as a new therapy in the treatment of temozolomide refractory glioblastoma (GBM). VAL-083 is a small water-soluble molecule that readily crosses blood-brain-barrier and accumulates in the tumor tissues in brain, making it a good candidate for targeting brain malignancies, such as GBM and medulloblastoma. Historical data from preclinical studies and clinical trials sponsored by the US National Cancer Institute (NCI) support anti-neoplastic effects of VAL-083 in a variety of cancer types in addition to GBM, including lung cancer, leukemia, cervical cancer, and ovarian cancer. However, the detailed molecular mechanisms mediating VAL-083 sensitivity or resistance in cancer cells is still unclear. Therefore, we investigated the distinct mechanism of action of VAL-083 in different cancer cell lines. Methods: VAL-083 cytotoxicity was evaluated in a panel of human non-small cell lung cancer (NSCLC) cell lines (A549, H2122, H1792, and H23) and prostate cancer cell lines (PC3 and LNCaP) by crystal violet assays. Cell cycle analysis and DNA damage response were investigated by propidium iodide (PI) and immunofluorescent (IF) staining. Western blot and IF staining analyses were employed to elucidate the DNA repair mechanism involved in VAL-083-treated cancer cells. Results: In this study, we report new insights into VAL-083's mechanisms of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA double-strand breaks (DSBs). In all the cancer cells tested, VAL-083 showed broad cytotoxicity with an IC50 range of 3.1 - 25.7 μM. In lung cancer (H2122, H1792, and A549) and prostate cancer (PC3 and LNCaP) cell lines, VAL-083 treatment caused irreversible cell cycle arrest at S/G2 phase as measured by PI and IF staining in synchronized cells, indicating that VAL-083-induced inter-strand crosslinks result in more difficult to repair DNA lesions during replication, including DSBs. Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. The S/G2 phase cell cycle arrest and the increased γH2A.X (an indication of DSB lesions) expression persisted for 48-72 h after treatment with VAL-083, indicating prolonged unrepaired DNA lesions caused by VAL-083. VAL-083 pulse-treatment led to persistent phosphorylation of DSB sensors ataxia telangiectasia mutated (ATM), single-strand DNA-binding replication protein A (RPA32), and H2A.X. Furthermore, Western blot analyses also demonstrated activation of the downstream effectors of ATM and ataxia telangiectasia and Rad3-related protein (ATR) kinases, Chk2 (T68) and Chk1 (S317 and S345). These results suggest that VAL-083-induced persistent and irreversible DNA damage activated the homologous recombination DNA repair signaling pathway in the panel of cancer cells studied. Conclusions: VAL-083 displayed broad anti-neoplastic activity in different lung and prostate cancer cells through the replication-dependent DSBs. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity provides guidance for improved treatment strategies for cancer patients with VAL-083 in either single or combination regimens. Citation Format: Beibei Zhai, Anne Steino, Jeffrey Bacha, Dennis Brown, Mads Daugaard. Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B15.

The median overall survival time for patients with stage IV non-small cell lung cancer (NSCLC) is 4 months, and 1- and 5-year survival is less than 16% and 2%, respectively. NSCLC is usually treated with surgery followed by treatment with either Tyrosine Kinase Inhibitors (TKIs) or platinum-based regimens. Unfortunately, TKI resistance has emerged as a significant unmet medical need, and long-term prognosis with platinum-based therapies is poor. Dianhydrogalactitol (VAL-083) is a structurally unique bi-functional alkylating agent mediating interstrand DNA crosslinks at N7 of guanine. It has previously demonstrated activity against NSCLC in preclinical and clinical trials, and is approved for treatment of lung cancer in China, suggesting that it may be a therapeutic option for drug-resistant NSCLC. However, the underlying basis for its activity remains unclear. We thus aimed to investigate in vitro i) the role of p53 status in the activity of VAL-083, ii) VAL-083 activity in comparison to cisplatin and oxaliplatin, and iii) the combination of VAL-083 with cisplatin or oxaliplatin. The dependence on p53 status was investigated in isogenic models with (HCT-116p53-/-) or without (HCT-116p53+/+) p53 knockout. The cytotoxic activity of VAL-083 was tested in a panel of 9 human NSCLC cell lines, of which 4 were wild-type (wt) p53, 4 were mutant p53 and 1 was null for p53. The potential for combination was investigated by determining superadditivity and assessing synergy using the criteria of combination index (CI) of <1, obtained by following the Compusyn constant-dose ratio protocol. Cytotoxicty in all cases was monitored on day 5 with the MTT assay. Studies in HCT-116 models demonstrated that loss of p53 increased resistance to cisplatin and oxaliplatin by 3- and 6-fold, respectively, whereas resistance to VAL-083 was <2-fold. As single agents, VAL-083, cisplatin and oxaliplatin displayed cytotoxic activity in all 9 NSCLC cell lines to varying degrees, with H460 being the most sensitive to the three agents (IC50 < 0.5 uM). The IC50 in the other cell lines ranged from 0.9 to 6.1 μM, 0.5 to 2.2 μM and 0.6 to 2.6 μM for VAL-083, cisplatin and oxaliplatin, respectively, and there was no overt difference in drug sensitivity between the wt and mutant/null p53 group. This suggests that either wt p53 is not activated and/or other genetic alterations attenuate cytotoxic activities. If the agents have similar mode of action, then combinations may only demonstrate cytotoxic additivity. However, the combination of VAL-083 with cisplatin or oxaliplatin in the A549 NSCLC model, demonstrated significant superadditivity (p<0.05) and synergism (CI < 1) for both combinations. This strongly favors non-overlapping mechanism of action between the platinum drugs and VAL-083. In conclusion, VAL-083 is less dependent on p53 for its activity, and demonstrates superadditivity/synergy against NSCLC cells when combined with either cisplatin or oxaliplatin. Citation Format: Anne Steino, Guangan He, Jeffrey A. Bacha, Sarath Kanekal, Dennis M. Brown, Zahid H. Siddik. In vitro activity of dianhydrogalactitol alone or with platinum drugs in the treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 751. doi:10.1158/1538-7445.AM2015-751

TPS10063 Background: Intravenous irinotecan hydrochloride (IRN-IV) is approved for the treatment of adult colorectal cancer. IRN-IV is also widely used off-label for a range of adult and pediatric solid tumors including recurrent Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, hepatoblastoma, Wilms tumor, gynecologic cancers, lung cancer and medulloblastoma. Previously, a regimen of IRN-IV administered as a 60-min i.v. infusion daily for 5 days, every 21 days has been recommended use in treating children with solid tumors (Blaney. ClinCanRes, 2001 ). Protracted administration schedule of intravenous irinotecan is inconvenient for patients, so oral regimens utilizing IRN-IV have been developed (Wagner. ClinSarcRes, 2015 ). Unfortunately, the palatability of the intravenous preparation is poor, leading to reduced compliance especially in younger pediatric patients. Development of an advanced formulation to improve tolerability and patient compliance is an important unmet clinical need...

Intravenous irinotecan hydrochloride (IRN-IV) is approved for the treatment of adult colorectal cancer. IRN-IV is also widely used off-label for a range of adult and pediatric solid tumors including recurrent Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, hepatoblastoma, Wilms tumor, gynecologic cancers, lung cancer and medulloblastoma. Recently, commercially available intravenous irinotecan has been administered orally (IRN-IVPO) in pediatric patients to improve convenience, reduce in-clinic time and overall cost of treatment. Unfortunately, the palatability of the intravenous preparation is poor, leading to reduced compliance especially in younger pediatric patients. Development of an advanced formulation to improve tolerability and patient compliance is an important unmet need. VAL-413 is a novel formulation developed to improve palatability of oral irinotecan. We will report on an ongoing Phase 1-2 first-in-human clinical trial of VAL-413, designed to test the hypothesis that V...

Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard-of-care for glioblastoma (GBM) includes surgery followed by concurrent therapy with radiation and temozolomide (TMZ) followed by adjuvant TMZ (days 1-5 every 28 days). Almost all GBM patients experience recurrent/progressive disease, with a median survival of 3-9 months after recurrence. Second-line treatment for recurrent GBM with bevacizumab (BEV) has not improved survival, and effective therapies for GBM are lacking. Unmethylated promoter status for O6-methylguanine DNA methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is correlated with poor prognosis. VAL-083 is a bi-functional DNA-targeting agent rapidly inducing interstrand cross-links at N7-guanine, leading to DNA double-strand breaks and cell death. VAL-083's cytotoxicity is independent of MGMT status, and VAL-083 overcomes TMZ-resistance in GBM cell lines, GBM cancer stem cells, and in vivo GBM models. The trial described here is an open-label two-arm biomarker-driven phase 2 clinical trial in MGMT-unmethylated GBM patients with either recurrent disease (Group 1) or newly diagnosed GBM patients requiring adjuvant therapy after chemoirradiation with temozolomide (Group 2). Patients receive VAL-083 IV at 30 or 40 mg/m2/day on days 1, 2, and 3 of a 21-day cycle. The primary objective of this study is to determine the effect of VAL-083 on median overall survival (mOS) in MGMT-unmethylated recurrent GBM patients (Group 1) compared to historical control, and progression-free survival (PFS) in newly diagnosed GBM patients requiring adjuvant therapy after chemoirradiation with temozolomide (Group 2), compared to historical control. Secondary efficacy endpoints include progression-free survival (PFS) (Group 1), overall response rate (ORR), duration of response (DOR), and quality-of-life (QoL). Tumor response will be assessed by MRI approximately every 42 days, as per RANO criteria. The initial starting dose in this study was 40 mg/m2/day, which was subsequently reduced to 30 mg/m2/day to improve tolerance due to myelosuppression. As of 21st January 2020, thirty-five (35) subjects had enrolled at a starting dose of 40 mg/m2/day, and 31 subjects had enrolled at a starting dose of 30 mg/m2/day in Group 1, and 9 subjects enrolled at a starting dose of 30 mg/m2/day in Group 2. As anticipated from prior studies with VAL-083, myelosuppression (thrombocytopenia and neutropenia) has been the most common adverse event observed. Enrollment and safety data update will be provided at the meeting. Clinicaltrials.gov identifier: NCT02717962 Citation Format: Barbara J. O'Brien, Carlos Kamiya-Matsuoka, Shiao-Pei Weathers, Alfred Yung, Monica Loghin, Rebecca Harrison, Nazanin Majd, Jeffrey A. Bacha, Dennis M. Brown, Anne Steino, Gregory Johnson, Sarath Kanekal, John Langlands, Lorena M. Lopez, Richard M. Schwartz, Marta Penas-Prado, John F. de Groot. Phase 2 study of dianhydrogalactitol (VAL-083) in patients with MGMT-unmethylated, bevacizumab-naïve glioblastoma in the recurrent or adjuvant setting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT272.

Purpose:Histone deacetylase (HDAC) inhibition has been shown to induce pharmacologic “BRCAness” in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors (PARPi). Here, we report the concept and characterization of a novel bifunctional PARPi (kt-3283) with dual activity toward PARP1/2 and HDAC enzymes in Ewing sarcoma cells.Experimental Design:Inhibition of PARP1/2 and HDAC was measured using PARP1/2, HDAC activity, and PAR formation assays. Cytotoxicity was assessed by IncuCyte live cell imaging, CellTiter-Glo, and spheroid assays. Cell-cycle profiles were determined using propidium iodide staining and flow cytometry. DNA damage was examined by γH2AX expression and comet assay. Inhibition of metastatic potential by kt-3283 was evaluated via ex vivo pulmonary metastasis assay (PuMA).Results:Compared with FDA-approved PARP (olapa...