Synthetic Lethal Approaches Research Articles

The AKT inhibitor AZD5363 elicits synthetic lethality in ARID1A-deficient gastric cancer cells via induction of pyroptosis.

Gastric cancer (GC) is a deadly disease with poor overall survival and limited therapeutic options. Genetic alterations such as mutations and/or deletions in chromatin remodeling gene AT-rich interactive domain 1 A (ARID1A) occur frequently in GC. Although ARID1A mutations/deletions are not a druggable target for conventional treatments, novel therapeutic strategies based on a synthetic lethal approach may be effective for the treatment of ARID1A-deficient cancers. A kinase inhibitor library containing 551 compounds was screened in ARID1A isogenic GC cells for the ability to induce synthetic lethality effect. Selected hits' activity was validated, and the mechanism of the most potent candidate drug, AKT inhibitor AD5363 (capivasertib), on induction of the synthetic lethality with ARID1A deficiency was investigated. After robust vulnerability screening of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A-/- cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the Caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment.

The Pivotal Role of Germline BRCA2 Pathogenic Variants in "Apparently Sporadic" Pancreatic Cancer.

In 1996, Goggins and colleagues demonstrated the importance of germline BRCA2 pathogenic variants in the development of apparently sporadic pancreatic ductal adenocarcinoma (PDAC). Previously, the group identified homozygous deletion of the 13q region in PDACs, enabling the identification of the BRCA2 gene. This 1996 article first revealed loss of BRCA2, both germline and somatic, as a key driver of PDAC at a time when there was still doubt if PDAC even had an inherited component. Contrary to the prevailing wisdom, not all individuals with inherited pathogenic BRCA2 variants had a family history of cancer. The innovative bedside-to-bench nature of this work revealed that individuals with these variants would be missed if genetic testing was limited only to those meeting the family history criteria. Therefore, Goggins and colleagues advocated that universal genetic testing may be indicated for pancreatic cancer at a time when genetic testing was in its infancy. Twenty-three years later, in 2019, universal testing for pancreatic cancer became standard of care in the United States. Additionally, this work and future-related publications by the Kern Laboratory set the stage for targeting BRCA2 and related DNA repair mutations in pancreatic cancer via a synthetic lethal therapeutic approach. The provocative discussion initiated by this team in this publication is still inspiring the field today. In this seminal publication, Goggins and colleagues profoundly impacted the direction of pancreatic cancer research, leading to a more sophisticated approach to designing earlier detection and precision treatment strategies for pancreatic cancer. See related article by Goggins and colleagues, Cancer Res 1996;56:5360-4.

The Pivotal Role of Preclinical Animal Models in Anti-Cancer Drug Discovery and Personalized Cancer Therapy Strategies.

The establishment and utilization of preclinical animal models constitute a pivotal aspect across all facets of cancer research, indispensably contributing to the comprehension of disease initiation and progression mechanisms, as well as facilitating the development of innovative anti-cancer therapeutic approaches. These models have emerged as crucial bridges between basic and clinical research, offering multifaceted support to clinical investigations. This study initially focuses on the importance and benefits of establishing preclinical animal models, discussing the different types of preclinical animal models and recent advancements in cancer research. It then delves into cancer treatment, studying the characteristics of different stages of tumor development and the development of anti-cancer drugs. By integrating tumor hallmarks and preclinical research, we elaborate on the path of anti-cancer drug development and provide guidance on personalized cancer therapy strategies, including synthetic lethality approaches and novel drugs widely adopted in the field. Ultimately, we summarize a strategic framework for selecting preclinical safety experiments, tailored to experimental modalities and preclinical animal species, and present an outlook on the prospects and challenges associated with preclinical animal models. These models undoubtedly offer new avenues for cancer research, encompassing drug development and personalized anti-cancer protocols. Nevertheless, the road ahead continues to be lengthy and fraught with obstacles. Hence, we encourage researchers to persist in harnessing advanced technologies to refine preclinical animal models, thereby empowering these emerging paradigms to positively impact cancer patient outcomes.

Synthetic lethality between toxic amino acids, RTG-target genes and chaperones in Saccharomyces cerevisiae.

The toxicity of non-proteinogenic amino acids has been known for decades. Numerous reports describe their antimicrobial/anticancer potential. However, these molecules are often toxic to the host as well; thus, a synthetic lethality approach that reduces the dose of these toxins while maintaining toxicity can be beneficial. Here we investigate synthetic lethality between toxic amino acids, the retrograde pathway, and molecular chaperones. In Saccharomyces cerevisiae, mitochondrial retrograde (RTG) pathway activation induces transcription of RTG-target genes to replenish alpha-ketoglutarate and its downstream product glutamate; both metabolites are required for arginine and lysine biosynthesis. We previously reported that tolerance of canavanine, a toxic arginine derivative, requires an intact RTG pathway, and low-dose canavanine exposure reduces the expression of RTG-target genes. Here we show that only a few of the examined chaperone mutants are sensitive to sublethal doses of canavanine. To predict synthetic lethality potential between RTG-target genes and chaperones, we measured the expression of RTG-target genes in canavanine-sensitive and canavanine-tolerant chaperone mutants. Most RTG-target genes were induced in all chaperone mutants starved for arginine; the same trend was not observed under lysine starvation. Canavanine exposure under arginine starvation attenuated and even reversed RTG-target-gene expression in the tested chaperone mutants. Importantly, under nearly all tested genetic and pharmacological conditions, the expression of IDH1 and/or IDH2 was induced. In agreement, idh1 and idh2 mutants are sensitive to canavanine and thialysine and show synthetic growth inhibition with chaperone mutants. Overall, we show that inhibiting molecular chaperones, RTG-target genes, or both can sensitize cells to low doses of toxic amino acids.

Patents on PARP-1 Inhibitors for the Management of Cancer from 2017-2023.

There are eighteen members of the Poly (ADP-ribose) polymerases (PARPs) family, which oversee various cellular processes such as maintaining the integrity of the genome, regulating transcription, cell cycle progression, initiating the DNA damage response, and apoptosis. PARP1 is an essential member of the PARP family and plays a crucial role in repairing single-strand breaks in eukaryotic cells through a process called BER (base excision repair). It is the most extensively studied and commonly found member of this family. This article discusses the advancements in developing PARP inhibitors for human cancers. It covers the discovery of new PARP1 inhibitors with chemical classification that selectively target multiple areas using cancer models in vitro and in vivo and evaluates them critically. The focus is on patents that have been published from 2017 to 2023, except tankyrase inhibitors. PARP1 inhibitors were developed by various companies and academic groups from the 1990s to enhance the effectiveness of chemo and radiotherapy. However, their progress was hindered due to their severe toxicity when combined with these treatments. Therefore, on finding PARP1 inhibitors that can amplify the ability of chemotherapy agents to kill tumors while causing minimal toxicity, these substances can either be used alone as part of the synthetic lethality approach or in conjunction with radiotherapy or chemotherapy, resulting in a mutually beneficial outcome.

Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

Abstract 3221: Targeting ARID1A-mutated gastric cancer cells with synthetic lethal approaches

Abstract AT-rich interaction domain 1A (ARID1A) functions as a tumor suppressor gene, and its loss or inactivation is a common occurrence in various human malignancies, including around 30% of gastric cancers (GC). This study aimed to identify potential therapeutic targets for GC cells with ARID1A deficiency. After robust screening from a chemical library consisting of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A−/− cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment. Citation Format: Menghan Fang, Youfen Lin, Chaorong Xue, Kaiqin Sheng, Zegeng Guo, Yuting Han, Hanbin Lin, Yuecheng Wu, Stephen B. Howell, Xu Lin, Xinjian Lin. Targeting ARID1A-mutated gastric cancer cells with synthetic lethal approaches [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 3221.

Abstract 4532: Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors

Abstract Cancers exhibiting homologous recombination (HR) repair deficiencies due to mutations in genes like BRCA1 or BRCA2 rely on alternative DNA damage response (DDR) pathways to uphold genomic integrity. This vulnerability allows for the targeted disruption of these pathways through synthetic lethality approaches. DNA polymerase theta (Polθ, encoded by POLQ) assumes a crucial role in mending DNA double-strand breaks (DSB) via microhomology-mediated end-joining (MMEJ), one of the principal pathways for repairing DNA DSB. Notably, Polθ expression is minimal in normal tissues but becomes upregulated in HR-deficient cancer cells, positioning Polθ as a prime target for synthetic lethality in HRD cancers. Reversion mutations in HR-related genes frequently employ micro-homology deletion mechanisms. Consequently, the inhibition of Polθ holds the potential to thwart the emergence of PARPi resistance, primarily driven by BRCA reversion mutations. In this context, we introduce DAT-1000A, a novel small-molecule Polθ inhibitor exhibiting potent activity with a single-digital nanomolar IC50 value. Its efficacy in MMEJ functional assays validates its on-target cellular activity. Assessments of cell viability in HR-deficient BRCA2-KO DLD-1 cells, compared to their parental counterparts, reveal a striking synthetic lethality window exceeding 500-fold. Additionally, DAT-1000A displays synergistic anti-proliferative effects when combined with PARP inhibitors in BRCA1/2-mutated cells. In BRCA1/2-mutated xenograft models, DAT-1000A demonstrates robust and sustained tumor regression, as evidenced by the level of γH2AX, a common marker for double-strand breaks, which closely correlates with anti-tumor efficacy. Importantly, high-dose DAT-1000A treatment in mice elicits no significant abnormalities, underscoring its excellent tolerability. Collectively, DAT-1000A emerges as a novel Polθ inhibitor, offering both potent activity and a favorable safety profile, and holds promise for advancing therapeutic options in the realm of HRD cancer treatment. Citation Format: Dan Yan, Yao Zhang, Yan Zhang, Jingxi Zhang, Bin Wang, Huixian Chen, Jingxue Shi, Xiaoling Lin, Jincong Zhuo, Kevin Zhou. Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors [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 4532.

Abstract 6996: A role for the cell adhesion molecule NCAM1 in regulating activity of the receptor tyrosine kinase RET in cancer

Abstract RET (REarranged during Transfection) is a receptor tyrosine kinase with essential roles in early development; however, genetic alterations lead to aberrant RET activity that contributes to oncogenic processes in several cancers. While some of the mechanisms by which RET mediates these processes are known, the network of proteins facilitating its activity remains largely undiscovered. To address this, we performed a genome-wide synthetic dosage lethal screen which identified NCAM1 (Neural Cell Adhesion Molecule 1) as one potential candidate modulating RET activity. Using an NCAM1 knockdown (KD) in SH-SY5Y neuroblastoma cells, we show that loss of NCAM1 reduces total protein levels of RET with no effect on RET transcription. Despite reduced abundance of RET, we show that the mature form of RET has a prolonged half-life in the absence of NCAM1. Assessing the functional outcomes of reduced RET protein levels, we observe reduced activation of RET by its extracellular ligand GDNF (Glial cell line-derived neurotrophic factor) and a subsequent reduction in activation of proliferative signaling pathways downstream of RET. As a result, RET-mediated proliferation is diminished in the absence of NCAM1, with reduced viability of NCAM1 KD SH-SY5Y observed in response to GDNF. Together, our data suggest that the synthetic lethal relationship between RET and NCAM1 may arise from a role for NCAM1 in regulating total RET protein available for activation, thereby reducing RET-mediated cell viability and proliferation. Our work has uncovered a role for NCAM1 in modulating RET activity in cancer and validates the use of our synthetic dosage lethal approach in uncovering proteins facilitating oncogenic RET activity. Citation Format: Montdher H. Hussain, Brandy D. Hyndman, Frederick S. Vizeacoumar, Mathieu J. Crupi, Franco J. Vizeacoumar, Lois M. Mulligan. A role for the cell adhesion molecule NCAM1 in regulating activity of the receptor tyrosine kinase RET in cancer [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 6996.

Abstract 5882: Targeting vulnerabilities of drug-tolerant persister cells in gastric cancer

Abstract Cytotoxic drugs often fail to eradicate cancers due to the presence of treatment-persistent tumor cells that represent a reservoir for relapse. These “residual” cancer cells escape from chemotherapy- induced death by entering a reversible slow proliferation state, known as drug tolerant persister (DTP) state. Although improvement of treatment options achieved a survival benefit, Gastric Cancer (GC) is still endowed with poor prognosis. Surgery and neo/adjuvant chemotherapy remain the keystone of treatment. However, most patients with advanced cancer relapse even after complete surgical resection, suggesting a critical role for DTP cells. We obtained GC DTP cells from primary cells upon first- and second-line chemotherapy regimens (FLOT and FOLFIRI). Cells were treated with doses corresponding to the drug maximal plasmatic concentration. At the end of the treatment, cells were left without drugs (washout). During this window of time, when cells had regrown, they were re-challenged with chemotherapy and resulted still sensitive to the treatments, thus fulfilling the criteria for bona fide DTP cells. GC DTPs showed increased expression of markers associated with gastric stem cells (i.e. LGR5), downregulation of proliferative markers (i.e. Ki67) and decreased levels of PS6, evaluated as a readout of mTOR activation. Interestingly, DTPs resulted positive to X-gal-based β-galactosidase staining, suggesting a chemotherapy-induced senescence-like status. RNA-seq analysis and gene expression arrays allowed the identification of genes specifically down/upregulated in DTP cells. Using the intuitive enrichment analysis web-based tool Enrichr, we discovered that 54/173 of the downregulated genes characterizing the persister state were targets of a single miRNA. By quantitative real-time PCR, we validated the expression of this miR in all the models of FOLFIRI- and FLOT-obtained persister cells and we found it strongly upregulated (up to 8-fold), highlighting its possible contribute in the establishment of the persister state. Since among the target genes of this miR there are genes involved in the Homolougus Recombination (HR) system, we performed TaqMan™ Array assay for Human DNA repair gene expression on FOLFIRI-obtained DTPs. We identified a significant downregulation of genes strictly correlated to the HR machinery, suggesting that the FOLFIRI-induced persister cells can temporarily enter in a “BRCAness” status that may be used for a synthetic lethality approach using PARP inhibitors. Targeting the drug tolerant persister state is an essential approach to diminish cancer cells when they are potentially in their most vulnerable state. Citation Format: Elisabetta Puliga, Lisa Negro, Martina Olivero, Claudia Orrù, Fabrizio Maina, Daniela Conticelli, Simona Corso, Silvia Giordano. Targeting vulnerabilities of drug-tolerant persister cells in gastric cancer [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 5882.

A novel targeted NGS panel identifies numerous homologous recombination deficiency (HRD)-associated gene mutations in addition to known BRCA mutations

Deleterious mutations in the BRCA1 and BRCA2 genes have significant therapeutic relevance in clinical settings regarding personalized therapy approaches. BRCA1 and BRCA2 play a pivotal role in homologous recombination (HR) and thus are sensitive for PARP inhibitors (PARPi). Beyond the narrow scope of evaluating only the BRCA mutation status, PARPi can be beneficial for HR deficient (HRD) patients, who harbor mutations in other HR-associated genes. In the present retrospective study, a novel targeted HRD gene panel was validated and implemented for use with FFPE tissue. Samples of patients with ovarian, breast, pancreatic and prostate cancer were included. Variants were robustly detected with various DNA input amounts and the use of test samples showed complete concordance between previously known mutations and HRD panel results. From all the 90 samples included in this cohort, TP53 was the most frequently altered gene (73%). Deleterious BRCA1/2 mutations were found in 20 (22%) of all samples. New pathogenic or likely pathogenic mutations in additional HR-associated genes were identified in 22 (24%) patients. Taken together, the present study proves the feasibility of a new HRD gene panel with reliable panel performance and offers the possibility to easily screen for resistance mutations acquired over treatment time.Mutations in HR-associated genes, besides BRCA1/2, might represent promising potential targets for synthetic lethality approaches. Thus, a substantial number of patients may benefit from expanding the scope of therapeutic agents like PARPi.

Poly (ADP-ribose) polymerase inhibitor therapy and mechanisms of resistance in epithelial ovarian cancer.

Advanced epithelial ovarian cancer is the commonest cause of gynaecological cancer deaths. First-line treatment for advanced disease includes a combination of platinum-taxane chemotherapy (post-operatively or peri-operatively) and maximal debulking surgery whenever feasible. Initial response rate to chemotherapy is high (up to 80%) but most patients will develop recurrence (approximately 70-90%) and succumb to the disease. Recently, poly-ADP-ribose polymerase (PARP) inhibition (by drugs such as Olaparib, Niraparib or Rucaparib) directed synthetic lethality approach in BRCA germline mutant or platinum sensitive disease has generated real hope for patients. PARP inhibitor (PARPi) maintenance therapy can prolong survival but therapeutic response is not sustained due to intrinsic or acquired secondary resistance to PARPi therapy. Reversion of BRCA1/2 mutation can lead to clinical PARPi resistance in BRCA-germline mutated ovarian cancer. However, in the more common platinum sensitive sporadic HGSOC, the clinical mechanisms of development of PARPi resistance remains to be defined. Here we provide a comprehensive review of the current status of PARPi and the mechanisms of resistance to therapy.

Synthetic Lethality Approaches in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL), a remarkable cancer that mainly affects children, has seen commendable advances in its treatment. However, the occurrence of relapses after initial treatments poses a major threat and is one of the leading causes of cancer-related mortality in pediatric patients. To address this problem, innovative therapeutic approaches for ALL need to be continuously developed and refined. Synthetic lethality, an interaction between genes in which alteration of only one allows survival, but simultaneous alteration of both leads to inviability, is emerging as a promising therapeutic approach against ALL and other cancers. In this regard, the review aims to examine the documented cases of synthetic lethality in ALL reported to date (2023) and to elucidate the molecular mechanisms underlying this phenomenon. Furthermore, this review explores possible targets that have so far gone unnoticed, justifying their importance in this context.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

Preomic profile of BxPC-3 cells after treatment with BRC4

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SignificanceRAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

The Development of p53-Targeted Therapies for Human Cancers.

p53 plays a critical role in tumor suppression and is the most frequently mutated gene in human cancers. Most p53 mutants (mutp53) are missense mutations and are thus expressed in human cancers. In human cancers that retain wtp53, the wtp53 activities are downregulated through multiple mechanisms. For example, the overexpression of the negative regulators of p53, MDM2/MDMX, can also efficiently destabilize and inactivate wtp53. Therefore, both wtp53 and mutp53 have become promising and intensively explored therapeutic targets for cancer treatment. Current efforts include the development of small molecule compounds to disrupt the interaction between wtp53 and MDM2/MDMX in human cancers expressing wtp53 and to restore wtp53-like activity to p53 mutants in human cancers expressing mutp53. In addition, a synthetic lethality approach has been applied to identify signaling pathways affected by p53 dysfunction, which, when targeted, can lead to cell death. While an intensive search for p53-targeted cancer therapy has produced potential candidates with encouraging preclinical efficacy data, it remains challenging to develop such drugs with good efficacy and safety profiles. A more in-depth understanding of the mechanisms of action of these p53-targeting drugs will help to overcome these challenges.

CDK12 alterations and ARID1A mutations are predictors of poor prognosis and therapeutic targets in high-grade salivary gland carcinoma: analysis of the National Genomic Profiling Database.

Due to the diversity of histopathologic types in salivary gland carcinoma, genomic analysis of large cohorts with next-generation sequencing by histologic type has not been adequately performed. We analysed data from 93 patients with salivary duct carcinoma and 243 patients with adenoid cystic carcinoma who underwent comprehensive genomic profiling testing in the Center for Cancer Genomics and Advanced Therapeutics database, a Japanese national genome profiling database. We visualised gene mutation profiles using the OncoPrinter platform. Fisher's exact test, Kaplan-Meier analysis, log-rank test and Cox regression models were used for statistical analysis. In salivary duct carcinoma, a population with CDK12 and ERBB2 co-amplification was detected in 20 of 37 (54.1%) patients with ERBB2 amplification. We identified five loss-of-function variants in genes related to homologous recombination deficiency, such as BRCA2 and CDK12. Cox survival analysis showed that CDK12 and ERBB2 co-amplification is associated with overall survival (hazard ratio, 3.597; P=0.045). In salivary duct carcinoma, NOTCH1 mutations were the most common, followed by mutations in chromatin modification genes such as KMT2D, BCOR, KDM6A, ARID1A, EP300 and CREBBP. In the multivariate Cox analysis, activating NOTCH1 mutations (hazard ratio, 3.569; P=0.009) and ARID1A mutations (hazard ratio, 4.029; P=0.034) were significantly associated with overall survival. CDK12 and ERBB2 co-amplification is associated with a poor prognosis in salivary duct carcinoma. Chromatin remodelling genes are deeply involved in tumour progression in adenoid cystic carcinoma. One such gene, ARID1A, was an independent prognostic factor. In salivary duct carcinoma and adenoid cystic carcinoma, there might be minor populations with mutations that could be targeted for treatment with the synthetic lethality approach.

A synthetic lethal approach to drug targeting of G-quadruplexes based on CX-5461

DNA G-quadruplex (G4) structures are enriched at human genome loci critical for cancer development, such as in oncogene promoters, telomeres, and rDNA. Medicinal chemistry approaches to developing drugs that target G4 structures date back to over 20 years ago. Small-molecule drugs were designed to target and stabilize G4 structures, thereby blocking replication and transcription, resulting in cancer cell death. CX-3543 (Quarfloxin) was the first G4-targeting drug to enter clinical trials in 2005; however, because of the lack of efficacy, it was withdrawn from Phase 2 clinical trials. Efficacy problems also occurred in the clinical trial of patients with advanced hematologic malignancies using CX-5461 (Pidnarulex), another G4-stabilizing drug. Only after the discovery of synthetic lethal (SL) interactions between Pidnarulex and the BRCA1/2-mediated homologous recombination (HR) pathway in 2017, promising clinical efficacy was achieved. In this case, Pidnarulex was used in a clinical trial to treat solid tumors deficient in BRCA2 and PALB2. The history of the development of Pidnarulex highlights the importance of SL in identifying cancer patients responsive to G4-targeting drugs. In order to identify additional cancer patients responsive to Pidnarulex, several genetic interaction screens have been performed with Pidnarulex and other G4-targeting drugs using human cancer cell lines or C. elegans. Screening results confirmed the synthetic lethal interaction between G4 stabilizers and HR genes and also uncovered other novel genetic interactions, including genes in other DNA damage repair pathways and genes in transcription, epigenetic, and RNA processing deficiencies. In addition to patient identification, synthetic lethality is also important for the design of drug combination therapy for G4-targeting drugs in order to achieve better clinical outcomes.

Abstract B019: A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer

Abstract Despite substantial improvements in the treatment landscape of prostate cancer, the evolution of hormone therapy-resistant and metastatic prostate cancer remains a major cause of cancer-related death globally. The mainstay of treatment for advanced prostate cancer is targeting of androgen receptor signaling, including androgen deprivation therapy plus second-generation androgen receptor blockade (e.g., enzalutamide, apalutamide, darolutamide), and/or androgen synthesis inhibition (abiraterone). While these agents have significantly prolonged the lives of patients with advanced prostate cancer, the evolution of resistance to these treatments in nearly universal. This therapy resistance is mediated by diverse mechanisms, including both androgen receptor-dependent mechanisms, such as androgen receptor mutations, amplifications, alternatively spliced isoforms, and structural rearrangements, as well as non-androgen receptor-mediated mechanisms, such as lineage plasticity toward neuroendocrine-like or epithelial-mesenchymal transition (EMT)-like lineages. Our prior work identified the EMT transcriptional regulator Snail as critical to hormonal therapy resistance and commonly detected in human metastatic prostate cancer. In the current study, we sought to interrogate the actionable landscape of EMT-mediated hormone therapy-resistant prostate cancer to identify synthetic lethality and collateral sensitivity approaches to treating this aggressive disease state. Using a combination of high-throughput drug screens and multi-parameter phenotyping by confluence imaging, ATP production, and phenotypic plasticity reporters of EMT, we identified candidate synthetic lethalities to Snail-mediated EMT in prostate cancer. These analyses identified multiple actionable targets, such as XPO1, PI3K/mTOR, aurora kinases, c-MET, polo-like kinases, and JAK/STAT as synthetic lethalities in Snail+ prostate cancer. We validated these targets in a subsequent validation screen in an LNCaP-derived model of resistance to sequential androgen deprivation and enzalutamide. This follow-up screen provided validation of inhibitors of JAK/STAT and PI3K/mTOR as therapeutic vulnerabilities for Snail+ and enzalutamide-resistant prostate cancer. Citation Format: Kathryn E. Ware, Beatrice C. Thomas, Pelumi Olawuni, Maya U. Sheth, Nathan Hawkey, Yeshwanth M, Brian C. Miller, Katherine J. Vietor, Mohit Kumar Jolly, So Young Kim, Andrew J. Armstrong, Jason A. Somarelli. A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B019.

POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.