Synthetic Lethal Combinations Research Articles

Abstract 1874: Common vulnerabilities of stem cells along the Barrett's-dysplasia-adenocarcinoma sequence

Abstract Barrett's esophagus (BE) is an early precursor of esophageal adenocarcinoma and therefore a relevant target of preemptive therapeutics. Using robust stem cell cloning technologies, we have cloned stem cells from patient-matched biopsies of BE, low-grade dysplasia (LGD), high-grade dysplasia (HGD), and esophageal adenocarcinoma (EAC) from multiple patients. Whole exome sequencing of these stem cells reveals the mutational maturation along the sequence of BE, LGD, HGD, and EAC at unprecedented resolution of both copy number and single nucleotide variation. In vitro differentiation and xenografting of these clones recapitulates their original histology and shows that only the EAC stem cells yield robust tumors in immunodeficient mice. Leveraging the intrinsic immortality of these stem cells, we have performed high-throughput screens for small molecules that selectively kill BE stem cells without harming patient-matched normal esophageal stem cells. These screening efforts evolved into synthetic lethal strategies that identified drug combinations that selectively eliminate, in vitro, BE stem cells from 20 of 23 cases. Remarkably, this same synthetic lethal drug combination also eliminated in vitro stem cells of LGD, HGD, and EAC that were not components of the original screens. We have used synthetic chemistry to generate a series of novel molecules based on the identified drug combination that significantly improved the in vitro efficacy to sub-nanomolar levels. These novel molecules directed to the stem cell component of BE, LGD, HGD, and EAC also show potent effects against tumors generated from EAC stem cells in immunodeficient mice. We anticipate that these synthetically lethal drug combinations will have potential utility against precursor lesions to preempt EAC as well as against EAC itself. Citation Format: Wa Xian, Frank McKeon, Christopher Crum, Jaffer Ajani, Peter Davies, Matthew Vincent, William Bachovchin, Hung-Sen Lai, Shuang Pan, Amber Su, Ashley Hoffman, Raul Caballero Montes, Melina Khorrami, Melika Khorrami, Yusuke Yamamoto. Common vulnerabilities of stem cells along the Barrett's-dysplasia-adenocarcinoma sequence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1874.

Synthetic lethal combination of CHK1 and WEE1 inhibition for treatment of castration-resistant prostate cancer.

WEE1 and CHEK1 (CHK1) kinases are critical regulators of the G2/M cell cycle checkpoint and DNA damage response pathways. The WEE1 inhibitor AZD1775 and the CHK1 inhibitor SRA737 are in clinical trials for various cancers, but have not been thoroughly examined in prostate cancer, particularly castration-resistant (CRPC) and neuroendocrine prostate cancers (NEPC). Our data demonstrated elevated WEE1 and CHK1 expressions in CRPC and NEPC cell lines and patient samples. AZD1775 resulted in rapid and potent cell killing with comparable IC50s across different prostate cancer cell lines, while SRA737 displayed time-dependent progressive cell killing with 10- to 20-fold differences in IC50s. Notably, their combination synergistically reduced the viability of all CRPC cell lines and tumor spheroids in a concentration- and time-dependent manner. Importantly, in a transgenic mouse model of NEPC, both agents alone or in combination suppressed tumor growth, improved overall survival, and reduced the incidence of distant metastases, with SRA737 exhibiting remarkable single agent anticancer activity. Mechanistically, SRA737 synergized with AZD1775 by blocking AZD1775-induced feedback activation of CHK1 in prostate cancer cells, resulting in increased mitotic entry and accumulation of DNA damage. In summary, this preclinical study shows that CHK1 inhibitor SRA737 alone and its combination with AZD1775 offer potential effective treatments for CRPC and NEPC.

An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest

CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.

Targeting CK2-mediated phosphorylation of p53R2 sensitizes BRCA-proficient cancer cells to PARP inhibitors.

Poly[ADP-ribose] polymerase (PARP) inhibitors, which selectively kills homologous recombination (HR) repair-deficient cancer cells, are widely employed to treat cancer patients harboring BRCA1/2 mutations. However, they display limited efficacy in tumors with wild-type (WT) BRCA1/2. Thus, it is crucial to identify new druggable HR repair regulators and improve the therapeutic efficacy of PARP inhibitors via combination therapies in BRCA1/2-WT tumors. Here, we show that the depletion of ribonucleotide reductase (RNR) subunit p53R2 impairs HR repair and sensitizes BRCA1/2-WT cancer cells to PARP inhibition. We further demonstrate that the loss of p53R2 leads to a decrease of HR repair factor CtIP, as a result of dNTPs shortage-induced ubiquitination of CtIP. Moreover, we identify that casein kinase II (CK2) phosphorylates p53R2 at its ser20, which subsequently activates RNR for dNTPs production. Therefore, pharmacologic inhibition of the CK2-mediated phosphorylation of p53R2 compromises its HR repair capacity in BRCA1/2-WT cancer cells, which renders these cells susceptible to PARP inhibition in vitro and in vivo. Therefore, our study reveals a novel strategy to inhibit HR repair activity and convert BRCA1/2-proficient cancers to be susceptible to PARP inhibitors via synthetic lethal combination.

Inhibition of RRM2 radiosensitizes glioblastoma and uncovers synthetic lethality in combination with targeting CHK1

Glioblastoma (GBM) is an aggressive malignant primary brain tumor. Radioresistance largely contributes to poor clinical outcomes in GBM patients. We targeted ribonucleotide reductase subunit 2 (RRM2) with triapine to radiosensitize GBM. We found RRM2 is associated with increasing tumor grade, is overexpressed in GBM over lower grade gliomas and normal tissue, and is associated with worse survival. We found silencing or inhibition of RRM2 by siRNA or triapine sensitized GBM cells to ionizing radiation (IR) and delayed resolution of IR-induced γ-H2AX nuclear foci. In vivo, triapine and IR reduced tumor growth and increased mouse survival. Intriguingly, triapine led to RRM2 upregulation and CHK1 activation, suggesting a CHK1-dependent RRM2 upregulation following RRM2 inhibition. Consistently, silencing or inhibition of CHK1 with rabusertib abolished the triapine-induced RRM2 upregulation. Accordingly, combining rabusertib and triapine resulted in synthetic lethality in GBM cells. Collectively, our results suggest RRM2 is a promising therapeutic target for GBM, and targeting RRM2 with triapine sensitizes GBM cells to radiation and independently induces synthetic lethality of GBM cells with CHK1 inhibition. Our findings suggest combining triapine with radiation or rabusertib may improve therapeutic outcomes in GBM.

Polymerase θ inhibition steps on the cGAS pedal.

Deficiencies in homologous recombination (HR) repair lead to an accumulation of DNA damage and can predispose individuals to cancer. Polymerase theta (Pol θ, encoded by POLQ) is overexpressed by HR-deficient cancers and promotes cancer cell survival by mediating error-prone double-stranded break (DSB) repair and facilitating resistance against poly-ADP ribose polymerase inhibitor treatment. In this issue of the JCI, Oh, Wang, et al. report on the impact of Pol θ inhibition on activation of antitumor immunity. The authors used pancreatic ductal adenocarcinoma (PDAC) cell and mouse models characterized by HR-associated gene alterations and POLQ overexpression. POLQ knockdown showed synthetic lethality in combination with gene mutations involving DNA repair, including BRCA1, BRCA2, and ATM. Notably, Pol θ deficiency or inhibition suppressed tumor growth, increased the accumulation of unrepaired DNA damage, and enhanced T cell infiltration via the cGAS/STING pathway. These findings suggest a broader scope for Pol θ inhibition in HR-deficient cancers.

Abstract 1637: MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations

Abstract Methylthioadenosine phosphorylase (MTAP) is co-deleted with the tumor suppressor CDKN2A in approximately 15% of solid tumors and can elicit dependency on methionine adenosyltransferase 2A (MAT2A) production of S-adenosyl methionine (SAM), the major methyl donor for cellular methyltransferase reactions. IDE397, a potent small molecule inhibitor of MAT2A, was developed to exploit this synthetic lethal relationship for therapeutic benefit in patients harboring MTAP-/- cancers and demonstrates selective anti-tumor activity in MTAP-/- models. Evaluation of IDE397 efficacy across a panel of 48 MTAP-/- PDX models revealed consistent tumor growth inhibition (TGI>60%) across diverse lineages, with distinct enrichment of tumor regressions in squamous lung cancers. To further enhance the efficacy of IDE397 within and among tumor lineages, we sought to identify rational combination partners. A three-pronged approach was employed that dovetailed molecular profiling of IDE397 drug effects in vivo, chemogenomic evaluation of selective drug sensitivities in MTAP-/- cell lines across the CCLE, and high-throughput in vitro drug combination screens. Unbiased pathway analysis of IDE397-dependent changes in MTAP-/- PDX transcriptomes revealed perturbations in pre-mRNA splicing, DNA damage repair, and mitotic spindle assembly as commonly enriched across diverse models. Evaluation of altered pre-mRNA splicing patterns suggested that many of these changes may be a consequence of selective impairment of PRMT5-dependent support of spliceosome activity. Notably, sensitivities to drugs that inhibit the fidelity of pre-mRNA splicing, genome stability, and microtubule stability were enriched in MTAP-/- cell lines, suggesting that the biological processes selectively altered by IDE397 in tumors are already partially impaired by MTAP deletion. Finally, an IDE397 in vitro drug combination viability screen of over 400 drugs returned taxanes, platins, topoisomerase inhibitors, splicing inhibitors, and anti-folates as selectively synergistic with IDE397 in MTAP-/- models. Synergy between IDE397 and pemetrexed was of particular interest given mechanistic convergence of the methionine salvage and folate cycle pathways on nucleotide synthesis in the context of MTAP-/-. Accordingly, this combination provided enhanced tumor growth inhibition in vivo in MTAP-/- NSCLC and bladder models and is currently under evaluation in phase 1. Collectively, these observations indicate that MAT2A inhibition can generate cell states in MTAP-/- tumor cells that are selectively vulnerable to approved chemotherapies and targeted therapies. These synergistic relationships may provide a predictive biomarker strategy for multiple IDE397 synthetic lethal combination therapies. Citation Format: Kimberline Y. Gerrick, Jenny Laraio, Kelsey Annen, Hoang Tran, Marcus M. Fischer, Yevgeniy Freyman, Geeta Sharma, Isaac Bishof, Stephen Federowicz, Divya Pankajakshan, Claire L. Neilan, Biju Mangatt, Anthony Mazurek, Mark R. Lackner, Michael White, Zineb Mounir. MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1637.

Abstract 5015: Wee1 inhibition and its synthetic lethal combination with Chk1 inhibition in mouse model of neuroendocrine prostate cancer

Abstract The mitotic regulator Wee1 is a critical regulator of G2/M cell cycle checkpoint and DNA damage response pathways. Wee1-targeted agents are under active clinical investigation in multiple cancer types. However, Wee1 and its inhibitors have not been well studied in prostate cancer, particularly neuroendocrine prostate cancer (NEPC). In this study, we investigated the therapeutic potential of Wee1 inhibitor AZD1775 and its synthetic lethal combination with Chk1 inhibitor in the context of NEPC. Our data demonstrated increased Wee1 expression in NEPC cells and tumors. Treatment of NEPC cells with AZD1775 inhibited NEPC tumor cell survival with greater potency as compared to other prostate cancer cells, demonstrating increased vulnerability of NEPC cells to Wee1 inhibition. We further identified synthetic lethality between the inhibition of Wee1 and several protein kinases, particularly Chk1, another key G2/M checkpoint kinase. Using an autochthonous adenocarcinoma mouse prostate (TRAMP) model that gives rise to NEPC tumors, we assessed the efficacy of AZD1775 and a highly selective Chk1 inhibitor SRA737 as single agents and in combination on the progression, survival, and metastatic spread of NEPC tumors in vivo. AZD1775 and SRA737 when used as single agents significantly suppressed tumor growth, improved overall survival, and incidence of distant metastases of mice with NEPC tumors. The combination of AZD1775 and SRA737 on a sequential 2-wk treatment schedule was more efficacious in suppressing tumor growth and metastasis than each drug alone in castrated TRAMP NEPC tumors. Mechanistically, the combination of Chk1 and Wee1 inhibitors synergized to reduce the inhibitory phosphorylation of CDK1 at Y15 and Cdc25c at S216, resulting in enhanced activation of CDK1/cyclin B complex, abrogation of G2/M checkpoint, and consequent premature mitotic entry, which ultimately led to mitotic catastrophe and apoptosis. Our findings provide promising preclinical evidence supporting the therapeutic values of Wee1 and Chk1 inhibitors as single agents and in combination for the treatment of NEPC. Citation Format: Yapeng Chao, Yuzhou Chen, Yu Liu, Jaclyn A. Connelly, Hong Wang, Qiming Jane Wang. Wee1 inhibition and its synthetic lethal combination with Chk1 inhibition in mouse model of neuroendocrine prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5015.

Abstract 2670: Drug combinations against Barrett's stem cells show efficacy across advanced lesions in esophageal cancer

Abstract We have applied a single cell cloning technology to patient-matched endoscopic biopsies of Barrett's esophagus and co-existing lesions of low-grade dysplasia (LGD), high-grade dysplasia (HGD), and esophageal adenocarcinoma (EAC). In vitro differentiation and xenografting of these clones yields epithelia with histology corresponding to the origin of these clones, and the EAC clones yield aggressive tumors in vivo. In addition, genomic analyses of these clones reveals their phylogenetic relationships based on common and accumulating mutations. In efforts to identify drugs that would target the Barrett's stem cells for preemptive therapeutics, we performed parallel screens of small molecule libraries against Barrett's stem cells and patient-matched normal esophageal stem cells. Synthetic lethal strategies ultimately yielded drug combinations that showed low nanomolar efficacy against Barrett's stem cells that spared normal esophageal stem cells even at micromolar concentrations. The synthesis of new compositions of matter corresponding to these leads has identified powerful analogs that not only selectively eliminate stem cells of Barrett's esophagus but show similar efficacy in vitro against stem cells of LGD, HGD, and EAC. In vitro, these drug combinations show broad efficacy against Barrett's and EAC, with nanomolar elimination of 20 of 23 Barrett's cases and 6 of 7 EAC cases. In tumors derived from xenografting EAC clones, these new drug combinations show robust activity marked by tumor shrinkage and a profound loss of tumor cell clonogenicity. We foresee the potential of these synthetic lethal drug combination both as topical delivery for premetastatic lesions in the distal esophagus and perhaps as systemic applications for advanced EAC cases. Citation Format: Wa Xian, Jennifer Lin, Mona Oraei, Frank McKeon, Shan Wang, Jaffer Ajani, Christopher Crum, William Bachovchin, Melina Khorrami, Amber Su, Melika Khorrami, Shumei Song, Jeremy Siegelman, Matt Vincent, Khek Yu Ho, Yusuke Yamamoto. Drug combinations against Barrett's stem cells show efficacy across advanced lesions in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2670.

Abstract SY32-03: Overcoming PARP inhibitor resistance through rational combinations in the clinic

Abstract Genomic instability is a key hallmark of cancer that arises owing to defects in the DNA damage response (DDR) and/or increased replication stress. The therapeutic landscape of antitumor agents targeting the DDR has rapidly expanded to include inhibitors of key mediators of DNA repair and replication beyond PARP, such as ATR and WEE1. Dr Yap will cover the latest advances in biomarker-driven synthetic lethal combination treatment strategies for different cancers. He will discuss efforts to optimize combination therapies that are ongoing across a range of cancers, involving the development of predictive biomarkers of response, assessment of the mechanisms underlying resistance, and the evaluation of rational, tolerable combinations with novel molecularly targeted agents, immune-checkpoint inhibitors and antibody drug conjugates. Citation Format: Timothy A. Yap. Overcoming PARP inhibitor resistance through rational combinations in the clinic. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY32-03.

Abstract P2-17-01: Targeted CRISPR screen to identify synthetic lethal combinations between APOBEC3B and DNA repair

Abstract APOBEC-catalyzed deamination of cytosine bases is the largest enzymatic and second largest overall source of mutation in cancer. One member from the APOBEC family of enzymes, APOBEC3B (A3B) is overexpressed and dysregulated in many different cancer types. In addition to hallmark C-to-T transitions and C-to-G transversions, APOBEC-catalyzed uracil lesions can be processed into single- and double-strand DNA breaks. Therefore, A3B-positive tumors are under continual stress to repair DNA breaks and may be vulnerable to DNA repair inhibition. Previous results from the Harris lab have identified UNG2, DNA uracil glycosylase 2 (UNG2), initiator of the base excision repair pathway, as synthetic lethal pair with A3B. The genetic disruption of UNG2 combined with high expression of A3B, causes cell death. This provides the rational to hypothesize that also other DNA repair proteins could be putative synthetic lethal pairs with A3B, when its expression and activity are high. To test this hypothesis, CRISPR guide RNA library targeting 237 DNA damage repair and response genes was used in doxycycline-inducible TREX-293-A3Bi-eGFP cell line. Cells expressing or not A3B were harvested for DNA extraction and sequencing at different time points. Comparison of guide RNA abundance between doxycycline and H2O treated cells revealed the dropout guides that disrupt genes and create putative synthetic lethal combinations with A3B. The screen design and overall results will be presented Citation Format: Bojana Stefanovska, Kevin Lin, Benjamin Troness, Chad Myers, Reuben Harris. Targeted CRISPR screen to identify synthetic lethal combinations between APOBEC3B and DNA repair [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-01.

SPRTN patient variants cause global-genome DNA-protein crosslink repair defects

DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.

PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors.

Alterations in the ATM gene are among the most common somatic and hereditary cancer mutations, and ATM-deficient tumors are hypersensitive to DNA-damaging agents. A synthetic lethal combination of DNA-damaging agents and DNA repair inhibitors could have widespread utility in ATM-deficient cancers. However, overlapping normal tissue toxicities from these drug classes have precluded their clinical translation. We investigated PLX038, a releasable polyethylene glycol-conjugate of the topoisomerase I inhibitor SN-38, in ATM wild-type and null isogenic xenografts and in a BRCA1-deficient xenograft. PLX038 monotherapy and combination with PARP inhibition potently inhibited the growth of both BRCA1- and ATM-deficient tumors. A patient with an ATM-mutated breast cancer treated with PLX038 and the PARP inhibitor rucaparib achieved rapid, symptomatic, and radiographic complete response lasting 12 months. Single-agent PLX038 or PLX038 in combination with DNA damage response inhibitors are novel therapeutic paradigms for patients with ATM-loss cancers.

Abstract 2597: Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a grim prognosis. Though the overall 5-year survival rates have recently improved to 10%, there is still an unmet need for better therapies. PARP inhibitors (PARPi)/platinum therapies have demonstrated clinical efficacy in BRCA1/2 mutated PDAC and has recently led to the FDA approval of PARPi (olaparib) in the maintenance setting, offering the best personalized treatment approach for such patients. Although promising, the benefits of these therapeutic strategies are currently limited to only a few patients. We have recently established that BARD1 (BRCA1- Associated-Ring-Domain-1), the main binding partner of BRCA1 in regulating homologous recombination repair is upregulated in PDAC cells exposed to PARPi/platinum compounds, by a post-transcriptional RNA binding mechanism. We therefore hypothesized that inhibition of BARD1 will sensitize cells to these agents. We found that inhibition of BARD1 (both by siRNA/shRNA and CRISPR-KO) not only sensitized the cells to olaparib or oxaliplatin but enhanced DNA damage in cells, as analyzed by comet assays and γ-H2AX immunofluorescence staining. BARD1 inhibition led to a decrease in homologous repair efficiency of cells as analyzed by the well established pDRGFP assay. To further understand BARD1 dependent downstream signaling, we performed comparative transcriptomic analysis of BARD1 silenced cells compared to scramble control. RNA seq analysis revealed modulation of several cell cycle checkpoints and DNA damage response pathways. Notably, through gene set enrichment analysis (GSEA), we found that silencing BARD1 causes de-enrichment of pathway regulating Polo-Like-Kinase-1 (PLK1, a serine/threonine kinase) activity (p-val=0.05, FDR=0.07). PLK1 is frequently upregulated in PDAC cells and promotes progression of cells through mitosis. Based on these results, we found that a PLK1 specific inhibitor (onvansertib) synergized with olaparib to reduce cell survival of homologous repair proficient (HR-P) PDAC cells. Importantly, the mechanism of drug synergy involved induction of apoptosis and DNA damage, as assessed by cleaved caspase-3 and γ-H2AX protein analysis. These results are suggestive of targeting the BARD1-PLK1 axis in order to enhance sensitivity of PARPi/platinums in HR proficient cells. Our ongoing and future studies are aimed at understanding the interaction of BARD1 and PLK1 in PDAC cells and analyze the effects of onvansertib and olaparib on tumor growth in vivo. Accomplishment of these studies will establish novel therapeutic targets and synthetic lethal combinations that have not been explored yet for deadly pancreatic cancers, ultimately benefiting patient survival in the long run. Citation Format: Carolyn Coats, Anoohya Arkala, Eric Londin, Avinoam Nevler, Jonathan R. Brody, Charles J. Yeo, Aditi Jain. Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2597.

Therapeutic Targeting of Tumor Cells and Tumor Immune Microenvironment Vulnerabilities.

Therapeutic targeting of tumor vulnerabilities is emerging as a key area of research. This review is focused on exploiting the vulnerabilities of tumor cells and the immune cells in the tumor immune microenvironment (TIME), including tumor hypoxia, tumor acidity, the bidirectional proton-coupled monocarboxylate transporters (MCTs) of lactate, mitochondrial oxidative phosphorylation (OXPHOS), and redox enzymes in the tricarboxylic acid cycle. Cancer cells use glucose for energy even under normoxic conditions. Although cancer cells predominantly rely on glycolysis, many have fully functional mitochondria, suggesting that mitochondria are a vulnerable target organelle in cancer cells. Thus, one key distinction between cancer and normal cell metabolism is metabolic reprogramming. Mitochondria-targeted small molecule inhibitors of OXPHOS inhibit tumor proliferation and growth. Another hallmark of cancer is extracellular acidification due lactate accumulation. Emerging results show that lactate acts as a fuel for mitochondrial metabolism and supports tumor proliferation and growth. Metabolic reprogramming occurs in glycolysis-deficient tumor phenotypes and in kinase-targeted, drug-resistant cancers overexpressing OXPHOS genes. Glycolytic cancer cells located away from the vasculature overexpress MCT4 transporter to prevent overacidification by exporting lactate, and the oxidative cancer cells located near the vasculature express MCT1 transporter to provide energy through incorporation of lactate into the tricarboxylic acid cycle. MCTs are, therefore, a vulnerable target in cancer metabolism. MCT inhibitors exert synthetic lethality in combination with metformin, a weak inhibitor of OXPHOS, in cancer cells. Simultaneously targeting multiple vulnerabilities within mitochondria shows synergistic antiproliferative and antitumor effects. Developing tumor-selective, small molecule inhibitors of OXPHOS with a high therapeutic index is critical to fully exploiting the mitochondrial vulnerabilities. We and others developed small-molecule inhibitors containing triphenylphosphonium cation that potently inhibit OXPHOS in tumor cells and tissues. Factors affecting tumor cell vulnerabilities also impact immune cells in the TIME. Glycolytic tumor cells supply lactate to the tumor-suppressing regulatory T cells overexpressing MCTs. Therapeutic opportunities for targeting vulnerabilities in tumor cells and the TIME, as well as the implications on cancer health disparities and cancer treatment, are addressed.

Synthetic Lethality in Ovarian Cancer.

Ovarian cancers include several distinct malignancies which differ with respect to clinicopathologic features and prognosis. High-grade serous cancer is the most common histologic subtype and accounts for most ovarian cancer-related deaths. High-grade serous ovarian cancer (HGSOC) is treated with surgery and platinum-based chemotherapy, but most patients relapse and succumb to chemoresistant disease. The genetic concept of synthetic lethality, in which the synergy of mutations in multiple genes results in cell death, provides a framework to design novel therapeutic approaches to overcome chemoresistance in ovarian cancer. Recent progress in understanding the genomic architecture and hereditary drivers of ovarian cancer has shown potential for synthetic lethality strategies designed around homologous DNA repair. Clinical trials have validated high response rates for PARP inhibitors in patients with BRCA1 or BRCA2 mutations. Here we discuss the biological rationale behind targeting BRCA-PARP synthetic lethality based on genetic context in ovarian cancer and how this approach is being assessed in the clinic. Applying the concept of synthetic lethality to target non-BRCA-mutant cancers is an ongoing challenge, and we discuss novel approaches to target ovarian cancer using synthetic lethality in combination with and beyond PARP inhibitors. This review will also describe obstacles for synthetic lethality in ovarian cancer and new opportunities to develop potent targeted drugs for patients with ovarian cancer.

Pharmaco-transcriptomic correlation analysis reveals novel responsive signatures to HDAC inhibitors and identifies Dasatinib as a synergistic interactor in small-cell lung cancer.

BackgroundHistone acetylation/deacetylase process is one of the most studied epigenetic modifications. Histone deacetylase inhibitors (HDACis) have shown clinical benefits in haematological malignancies but failed in solid tumours due to the lack of biomarker-driven stratification.MethodsWe perform integrative pharmaco-transcriptomic analysis by correlating drug response profiles of five pan-HDACis with transcriptomes of solid cancer cell lines (n=659) to systematically identify generalizable gene signatures associated with HDACis sensitivity and resistance. The established signatures are then applied to identify cancer subtypes that are potentially sensitive or resistant to HDACis, and drugs that enhance the efficacy of HDACis. Finally, the reproductivity of the established HDACis signatures is evaluated by multiple independent drug response datasets and experimental assays.FindingsWe successfully delineate generalizable gene signatures predicting sensitivity (containing 46 genes) and resistance (containing 53 genes) to all five HDACis, with their reproductivity confirmed by multiple external sources and independent internal assays. Using the gene signatures, we identify low-grade glioma harbouring isocitrate dehydrogenase 1/2 (IDH1/2) mutation and non-YAP1-driven subsets of small-cell lung cancer (SCLC) that particularly benefit from HDACis monotherapy. Further, based on the resistance gene signature, we identify clinically-approved Dasatinib as a synthetic lethal drug with HDACi, synergizing in inducing apoptosis and reactive oxygen species on a panel of SCLC. Finally, Dasatinib significantly enhances the therapeutic efficacy of Vorinostat in SCLC xenografts.InterpretationOur work establishes robust gene signatures predicting HDACis sensitivity/resistance in solid cancer and uncovers combined Dasatinib/HDACi as a synthetic lethal combination therapy for SCLC.

Abstract 1053: A large cellular screen charting the landscape of synergistic drug combinations in lung cancer

Abstract Introduction Non-small-cell lung cancer (NSCLC) is among the most common cancers, accounting for 85% of all lung cancer cases. Drug combinations are known to more effective than single drugs and can delay drug resistance. NSCLC has a poor prognosis and there are no approved targeted cancer drug combinations. Here we have conducted the first large-scale screen of drug combinations in NSCLC cell lines to identify new candidate synergistic combinations and map the landscape of synthetic lethal and synergistic pathways underlying them. Results We conducted the largest of its kind in vitro drug combination screening of 81 NSCLC cell-lines for 21 x 242 cancer drug combinations at 5 different doses. Our collection of approved and investigational drugs included mainly targeted drugs as well as some chemotherapy drugs. For each drug combination, we computed the synergy score using the Bliss model. Our key findings are as follows: 1. We identify drug combinations (like CHEK and WEE1 inhibitors) that are highly synergistic in many NSCLC cell lines. 2. Combinations of targeted therapies are overall more synergistic than chemotherapeutic drug combinations. 3. Synergistic combinations occur more within pathways. 4. We see that most drug combinations do not make cell lines more sensitive than the most sensitive cell line to the corresponding single agents across all the cell lines tested. 5. We identify a new class of drug combinations - called super-sensitizers - whose combined effects are larger than the effects observed for the individual drugs composing across all of the cohort cell-lines. 6. Combinations of multi-target drugs tend to be more synergistic than single-target drugs. 7. We identify and characterize a set of context-specific drug combinations that are differentially synergistic or differentially effective (i.e. viability after treatment) between wild-type vs mutant cell lines of various cancer drivers (KRAS, TP53, STK11, PIK3CA, EGFR). 8. Studying potential mechanistic underpinnings of drug synergism in NSCLC, we find that: (a) synergistic combinations target genes that have more proximal physical interactions, (b) some synergistic combinations target synthetic lethal combinations, (c) a subset of synergistic combinations have further support by NSCLC patients survival analysis. Conclusions We performed the first large in vitro drug combination screen in NSCLC. We identify top drug combinations that are highly synergistic and effective in many cell lines and characterize key biological properties and mechanisms underlying them. The screen results are provided in a user-friendly public data resource that can help facilitate the development of targeted drug combinations in lung cancer therapy. Citation Format: Nishanth Ulhas Nair, Adam Friedman, Patricia Greninger, Avinash D. Sahu, Ellen Murchie, Giovanna Stein Crowther, Joe McClanaghan, Joo Sang Lee, Daniel A. Haber, Jeffrey Engelman, Eytan Ruppin, Cyril Benes. A large cellular screen charting the landscape of synergistic drug combinations in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1053.

Targeting DNA Repair Mechanisms to Overcome Drug Resistance in Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of adult non-Hodgkin lymphomas. Most DLBCL patients achieve long-term remission after treatment, but a third relapse after conventional Rituximab (R)-based chemotherapy regimens, such as CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) (Siegel et al, 2012).Cancer cells are exposed to chronic replication stress, which impedes the duplication of their genome and induces mitotic catastrophe (Shaheen et al, 2011). Functional DNA repair pathways are therefore important for the survival of cancer cells. This dependence can be exploited therapeutically to hamper repair of the intrinsic DNA damage occurring during replication or to exacerbate DNA damage induced by chemotherapy (Shaheen et al, Blood 2011). Furthermore, high-risk DLBCL patients overexpress genes potentially involved in resistance to CHOP-based regimens, such as genes of the nucleotide excision repair (NER) pathway (Bret et al, Oncotarget 2012, Cell Cycle 2013).We recently developed GEP-based DNA repair scores that allow to identify high-risk DLBCL patients that could benefit from treatment with DNA repair inhibitors (Bret at al, BJH 2015).DLBCL treatments include cyclophosphamide, a nitrogen mustard derivate that induces interstrand crosslinks (ICLs), and doxorubicin, a DNA topoisomerase inhibitor that induces DNA double-strand breaks, DNA adducts and formaldehyde-dependent ICL formation. Inhibiting DNA repair is a promising strategy to improve the efficacy of genotoxic drugs and overcome drug resistance. Our data support the view that inhibitors of DNA damage signaling and DNA repair have potential therapeutic interest in DLBCL.We characterized the drug-response of 16 DLBCL cell lines to 9 DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1/2 inhibitor), AZD6738 (ATR inhibitor), MK8776 (CHK1 inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor), Lomeguatrib (MGMT) and genotoxic agents used in DLBCL treatment (Cyclophosphamide, Gemcitabine, Doxorubicin and Etoposide) (Table 1). All drugs induced significant apoptosis (Annexin V staining and PARP cleavage) and significant inhibition of proliferation (BrdU incorporation) in the different DLBCL cell lines tested (P <0.05). CHK1/2 inhibitor, Wee1 inhibitor, Cyclophosphamide, Gemcitabine and Doxorubicin induced DNA damages monitored by H2AX phosphorylation. Correlating drug response of each compounds with our GEP-based DNA repair scores (Bret et al, BJH 2015), we identified a significant correlation between FANC score and response to ATR inhibitor (AZD6738) and HRR score/BER score and response to Etoposide (P<0.05). High-risk DLBCL patients identified with GEP-based FANC, HRR and BER scores may benefit from treatment by ATR inhibitors or Etoposide respectively.Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 of DNA repair targeted treatments with conventional genotoxic agents in DLBCL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone, a total of 3 synthetic lethal combinations have been identified including cyclophosphamide with CHK1/2 inhibitor (PF477736) (IC50 reduced from 2.63 µM to 1.20 µM), cyclophosphamide and ATR inhibitor (AZD6738) (IC50 reduced from 2.63 µM to 0.74 µM) and doxorubicin with DNAPK inhibitor (NU7441) (IC50 reduced from 57 nM to 14 nM).Furthermore we identified new potent synergistic combinations (combination index < 1) including CHK1/2 inhibitor (PF477736) with etoposide (IC50 reduced from 560 nM to 437 nM), ATR inhibitor (AZD6738) (IC50 reduced from 560 nM to 364 nM) with etoposide and ATM inhibitor (KU55933) with etoposide (IC50 reduced from 560 nM to 269 nM).Despite overall improvements in the treatment of DLBCL, including the use of rituximab, approximately one-third of patients fail to achieve complete remission or experience relapse. This remains a major cause of morbidity and mortality. The DNA repair scores could be useful to identify high-risk DLBCL patients and define the best synthetic lethal approach combining DNA repair inhibitors with conventional chemotherapy. These results open new perspectives to improve the treatment of DLBCL patients and provide new strategies to overcome drug resistance. [Display omitted] DisclosuresCartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding.