Synthetic Lethal Interactions Research Articles

Abstract P22: C16orf72/HAPSTR1/TAPR1 Interacts with BRCA1 and Senataxin to Regulate R-loops at Stalled Replication Forks and Confer Resistance to PARP Disruption

Abstract Poly (ADP-ribose) polymerase (PARP) proteins play a crucial role in DNA repair, and their inhibition is toxic in cells with defects in homologous recombination (HR). Whilst this interaction is well-established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly explored. Hence, we performed a genome-wide CRISPR screen to identify genes which are synthetic lethal with PARP1/PARP2 disruption. We discovered that the uncharacterized C16orf72/HAPSTR1/TAPR1 gene is essential for the survival of PARP1/PARP2 double knock-out cells but not the parental wildtype cells. We further show that C16orf72 is a novel modulator of R-loop and is crucial in facilitating replication fork restart and suppressing DNA damage in response to replication stress. We show that C16orf72 functions in a parallel pathway to PARP1/PARP2 to suppress DNA:RNA hybrids that accumulate at stalled replication forks, and it does this by interacting with BRCA1 and the DNA/RNA helicase Senataxin. Citation Format: Abhishek Bharadwaj Sharma, Muhammad Khairul Ramlee, Joel Kosmin, Martin R. Higgs, Amy Wolstenholme, George E. Ronson, Dylan Jones, Daniel Ebner, Noor Shamkhi, David Sims, Paul W. G. Wijnhoven, Josep Forment, Ian Gibbs-Seymour, Nicholas D. Lakin. C16orf72/HAPSTR1/TAPR1 Interacts with BRCA1 and Senataxin to Regulate R-loops at Stalled Replication Forks and Confer Resistance to PARP Disruption [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P22.

Abstract 2932: Computational discovery of paralog dependencies drives target identification in lung cancer

Abstract Lung cancer is the leading cause of cancer-related death in the United States, and worldwide, with the 5-year survival rate of non-small cell lung cancer at 24%. Treatment options are limited, and even in cancers that harbor druggable biomarkers, resistance is near-universal. Identifying novel treatment strategies is essential to improve the outcomes of patients with lung cancer and provide secondary strategies for those who acquire resistance to first-line therapeutics. Identifying targeted therapies based on the genotype or expression pattern of cancers is an ongoing endeavor. In lung cancer, treatment of patients that harbor activating mutations in EGFR with specific EGFR inhibitors has improved progression-free survival rates and extended the lifespan of patients diagnosed with this aggressive cancer, demonstrating the utility of targeted therapies based genetic-based vulnerabilities, or synthetic lethal interactions. A rich source of synthetic lethal genetic interactions are paralogous genes. Paralogs result from gene duplication events through evolutionary history, and many paralogs share both sequence homology and functional redundancy. Co-targeting of some paralogous genes induces a fitness defect that is synergistic compared to individual paralog loss alone. Nearly 70% of genes in the human genome are considered paralogs, and until recent efforts from the Berger lab and others, this buffering effect has caused them to be under-represented in lethality studies. Identifying frequently-inactivated paralog genes in lung cancer will provide a platform to identify new vulnerabilities in lung cancer and other cancer types that share mutational backgrounds. To identify patterns of mutation and expression of paralog genes in lung cancer, we have accessed publicly available data sets for both cell line and patient tumor samples. Using TCGA and CCLE data sources, and a set of 1030 paralog pairs identified previously by the Berger lab as potential drug targets, we have identified paralog genes that harbor high mutational frequencies in lung cancer. Using these candidates, we next asked whether genetic inactivation or transcriptional repression of one paralog influences the essentiality of its paired gene using CERES scores generated by the Cancer Dependency Map. Preliminary results identifies several paralog pairs with distinct dependency profiles in cell lines for which expression of one paralog is lost, including MAGOH/MAGOHB and TLK1/TLK2. Further work to characterize these dependencies in relevant cell line models is ongoing. Additionally, the druggability of these targets will be tested using targeted degradation using the dTAG system. This work will uncover a new set of paralog synthetic lethality targets, and further validate the utility of targeting known synthetic lethal paralogs for the treatment of highly lethal lung cancer. Citation Format: Siobhan O'Brien, Alice H. Berger. Computational discovery of paralog dependencies drives target identification in lung 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 2932.

Abstract 5921: PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors

Abstract Background: High microsatellite instability (MSI-H) is highly prevalent across many cancer types including endometrium, gastric, ovary and colorectal cancer. It is well known that there is a clear synthetic lethality (SL) interaction between MSI-H and Werner syndrome protein (WRN) inhibition. WRN is a member of RecQ helicase family and involved in the maintenance of genome integrity. Different from other RecQ family members, WRN has two activity centers responsible for helicase and exonuclease activity, respectively. Further studies confirmed that an inhibition on helicase activity, but not exonuclease activity, is responsible for SL interaction with MSI-H. Also, Recent clinical observations supported that though loss-of-function of WRN could cause an early-aged disease Werner syndrome, selective loss of helicase but not exonuclease leads to only mild to moderate symptoms, suggesting a selective inhibitor of helicase activity has the potential to induce the SL interaction with MSI-H and keep patients safe. Methods: Biochemical assays and cell-based assays were performed to evaluate in vitro activity and selectivity of PH027-1. The in vivo anti-tumor efficacy was tested in two cell-derived xenograft (CDX) mouse models with tumors harboring MSI-H. The in vivo pharmacokinetics (PK) properties were evaluated in mice, rats and dogs, and other in vitro and in vivo PK and safety properties were assessed with corresponding assay methods. Results: Biochemical assays showed that PH027-1 potently inhibited helicase activity of WRN (IC50, 0.8 nM), but had no effect on other RecQ family members even at the highest tested concentration of 10 μM (IC50, all≥10 μM for BLM, RecQ4 and RecQ5). At the same time, PH027-1 did not affect the exonuclease activity of WRN at all tested concentrations. In cell-based assays, PH027-1 potently inhibited cell viability of MSI-H cancer cells SW48 and HCT116 at tens of nM, but had no effect on MSS cells (HT29 cancer cells and CD34+ hematopoietic stem cells) even at 10 μM. The tests were further expanded to 18 cancer cell lines covering uterus, gastric, ovary and colorectal cancer. Nine of twelve MSI-H cell lines were very sensitive to WRN inhibition, and all six MSS cell lines were totally resistant to the treatment. PH027-1 possessed excellent PK profiles as shown by in vivo PK studies, with absolute bioavailability of 58%~102% in mice, rats and dogs. In two CDX models (SW48 and HCT116), oral administration of PH027-1 dose-dependently inhibited tumor growth, and a nearly complete inhibition was observed at a dose of 20 mg/kg QD. Tumor regression was clearly shown at higher doses. All other in vitro and in vivo PK and safety tests showed favorable results and supported for preclinical development of PH027-1. Conclusion: As a highly selective and potent inhibitor on helicase activity of WRN, PH027-1 has the potential to become a novel and safe approach for treatment of MSI-H tumors. Citation Format: Feng Gao, Bin Liu, Liandong Jing, Jing Wang, Yongyong Wu, Jun Wan, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H 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 5921.

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 5802: An orally available small molecule JMBI-001 elicits MYC-synthetic lethality and anti-tumor immunity by disabling MKLP2-mediated cellular processes

Abstract The MYC oncogene, a pivotal regulator of various cellular processes, is deregulated in approximately 70 % of human malignancies. Mitotic Kinesin-like Protein 2 (MKLP2) plays a versatile role in both interphase and mitosis and has emerged as a significant prognostic indicator and therapeutic target in cancer. The undruggability of MYC and the scarcity of MKLP2 inhibitors, however, have impeded clinical translation. Our development of JMBI-001, a potent and orally bioavailable small-molecule compound, overcomes these barriers. JMBI-001 elicits loss of function phenotypes in MKLP2 and a synthetic lethal interaction with MYC overexpression. Extensive kinome and safety profiling have revealed no significant off-target effects, and the compound is well-tolerated in long-term animal studies. In preclinical models, JMBI-001 has demonstrated an average tumor growth inhibition rate of 75 % across more than 20 MYC-overexpressing tumor models, including those in the stomach, lung, colon, liver, breast, kidney, skin, and hematopoietic system. Its anti-tumor activity positively correlates with high MYC abundance, aligning with the selective eradication of cells with abundant MYC both in vitro and in vivo. Notably, JMBI-001 also robustly stimulates systemic anti-tumor immunity, enhancing NK and CD3+ T cell infiltration in tumors of syngeneic cancer models. Moreover, additive or synergistic effects have been observed when combined with anti-PD1 therapy even in tumors refractory to the immune checkpoint blockade (ICB) therapy. The dual therapeutic actions of JMBI-001 stem from its disruption of MKLP2 functionalities, leading to anomalies, such as Golgi fragmentation in interphase and multipolarity in pro-metaphase. These disruptions lead to two key outcomes: apoptosis and immunogenic cell death, marked by secreted ATP, released high mobility group protein B1 (HMGB1), and surface-exposed calreticulin. These abnormalities, primed and amplified by deregulated MYC, are not observed in non-transformed cells, suggesting their potential as pharmacodynamic markers for monitoring JMBI-001's activity in vivo. In conclusion, JMBI-001 represents a novel class of anticancer agents that simultaneously triggers MYC synthetic lethality and anticancer immunity by targeting MKLP2-mediated cellular processes. Its unique mechanisms of action, exceptional bioavailability, potency at low nanomolar concentrations, wide-spectrum efficacy, and favorable safety profile establish JMBI-001 as a promising clinical study-ready drug candidate for treating MYC-driven cancers. Citation Format: Ting Zhang, Qiong Shi, Julia Kalashova, Xumei Liu, Xiaohu Zhou, Chenglu Yang, Yan Long, Hongmei Li, Jinhua Li, Gang Lv, Duo Yu, Xuejiao Jiang, Shenqiu Zhang, Jing Zhang, Hong Liu, Dun Yang. An orally available small molecule JMBI-001 elicits MYC-synthetic lethality and anti-tumor immunity by disabling MKLP2-mediated cellular processes [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 5802.

Abstract 2866: NAPRTsilencing in rhabdomyosarcoma confers therapeutic vulnerabilities to NAD+depletion

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma among children, and new treatments are needed to improve survival. Cells primarily synthesize nicotinamide adenine dinucleotide (NAD) through the Preiss-Handler and Salvage pathways driven by nicotinic acid phosphoribosyltransferase (NAPRT) and nicotinamide phosphoribosyltransferase (NAMPT), respectively. NAMPT inhibitors (NAMPTi) have been tested in clinical trials, but their efficacy has been limited by (a) lack of appropriate biomarkers for patient selection, and (b) significant dose-limiting toxicities (DLTs), including myelosuppression and retinal toxicity, although the latter has been described only in preclinical studies. Previous work from our group indicates that loss of NAPRT expression by tumor-specific promoter CpG island methylation may provide a novel biomarker for sensitivity to NAMPTi in a variety of tumors. Here, we sought to determine if RMS harbors NAPRT silencing that confers a synthetic lethal interaction with NAMPTi. We probed methylation data within the Cancer Dependency Map (DepMap) and found a high frequency of NAPRT gene promoter hypermethylation in RMS models. Predicted NAPRT silencing was confirmed at the protein level by western blot in a panel of RMS cell lines. In vitro growth delay assays demonstrated that NAPRT-silenced cells are exquisitely sensitive to NAMPTi even in the presence of nicotinic acid (NA) supplementation, while NAPRT-expressing cells were completely rescued. This sensitivity to NAMPTi was further mirrored by changes in NAD levels. In vivo, NAMPTi induced significant tumor regression in a NAPRT-silenced RMS xenograft model. To further establish the translational relevance, NAPRT protein expression in a TMA of 51 human RMS tumors was assessed via IHC staining using a newly validated NAPRT monoclonal antibody (4A5D7). Based on a semi-quantitative scoring system accounting for the percentage of positive tumor cells and staining intensity, 28% of RMS samples demonstrated loss of NAPRT protein expression. Overall, these data suggest that NAPRT loss may serve as a therapeutic target in RMS by inducing a synthetic lethal interaction with NAD+ depleting agents. Future studies in patient-derived xenograft (PDX) models will determine the optimal cut-off of NAPRT expression that portends NAMPTi sensitivity, paving the way for the development of biomarker-driven clinical trials in RMS. Ongoing studies will also evaluate the efficacy of combining NAMPTi with existing cytotoxic and DNA damaging chemotherapy in these difficult-to-treat tumors. Citation Format: Sophia J. Zhao, Katelyn J. Noronha, Prateek Bhardwaj, Karlie N. Lucas, Ranjini K. Sundaram, Collin D. Heer, Raffaella Morotti, Josh Spurrier, Mitch Raponi, Ranjit S. Bindra, Juan C. Vasquez. NAPRTsilencing in rhabdomyosarcoma confers therapeutic vulnerabilities to NAD+depletion [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 2866.

Abstract 5902: Targeting MAPK-driven tumors via inhibition of MAP2K4

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination.

Abstract 1080: Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma

Abstract Ewing’s sarcoma (EWS) is an aggressive bone and soft-tissue cancer, characterized by a hallmark fusion oncoprotein comprising the amino terminus of a FET gene and the carboxy terminus of an ETS gene, most commonly (85%), the EWSR1-FLI1 t (11,22) fusion.TK-216 is a first-in-class EWSR1-FLI1 inhibitor currently in phase II clinical trials for EWS. Although TK-216 was designed to target the fusion protein interaction directly, it has demonstrated limited efficacy as a monotherapy. Thus, more effective TK-216 combination therapies are needed. We have used genome wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Knockout screening in EWS cells treated with TK-216 to identify novel synthetic lethality. We have identified 97 genes whose deletion potentially enhances the action of TK-216 in EWS cells. We have also identified several druggable targets, such as USP9X and the oxidative phosphorylation (OXPHOS) pathway as synthetic lethal interactions for TK-216. USP9X is known to deubiquitinate and stabilize the EWS-FLI1 fusion protein. USP9X and EWS-FLI1 protein expression levels were found to be positively correlated in EWS clinical specimens. USP9X siRNA-mediated gene silencing reduced EWS cell viability. Most importantly, USP9X siRNA-mediated gene silencing sensitized EWS cells to TK-216 treatment. In addition, inhibition of USP9X with a small-molecule inhibitor WP1130, led to significant cell death in EWS alone and in combination with TK-216 with wide therapeutic indices. The molecular mechanism of synergy and in vivo efficacy of the novel TK-216 combination will be determined in our study for future clinical translation. Citation Format: Daenikka Ravindrarajah, Sukriti Krishan, Kayleen Simpson, Twishi Gulati, Belamy B. Cheung, Glenn M. Marshall. Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma [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 1080.

Abstract 3364: PELO is a synthetic lethal target in chromosome 9p21-deleted or MSI cancers

Abstract Homozygous deletion of chromosomal region 9p21.3 is one of the most frequently observed somatic copy number alterations in cancers, occurring in approximately 15% of all cancers. Many of the 9p21.3-deleted cancers are further characterized by poor clinical outcomes including subsets of glioblastoma, pancreatic, esophageal, and urothelial cancers. Large deletions of chromosomal regions may also provide unique therapeutic opportunities. Specifically, cancer-specific therapeutic opportunities can arise from co-deletions of passenger genes neighboring tumor suppressors. As such, we sought to identify novel therapeutic targets associated with Ch9p21.3 loss in cancers and identified the pelota mRNA surveillance and ribosome rescue factor (PELO) as the top preferential dependency in 9p21.3-deleted cancers. Intriguingly in another analysis, we identified PELO to be amongst the top preferential dependencies in cancers with microsatellite instability (MSI), which accounts for subsets of colorectal, endometrial, gastric, and ovarian cancers. Given the strength of PELO as a candidate synthetic lethal target in two distinct cancer subsets, we performed focused validation with CRISPR interference across multiple cell lines. Knockdown of PELO preferentially impaired the viability of MSI or 9p21-deleted as compared to control (9p21-proficient and microsatellite stable) cells. Further validation with patient-derived organoid and in vivo xenograft models confirmed these findings. We next sought to refine the predictive biomarkers for PELO dependency and identified loss of FOCAD (a 9p21.3 gene and an interactor of the SKI mRNA surveillance pathway) as necessary and sufficient for PELO dependency in the 9p21.3-deleted context. Our findings implicate splicing alterations involving TTC37, a key protein in the SKI mRNA surveillance complex, as the underlying cause of PELO dependency in MSI cancers. Together, we found that loss of large deletions of 9p21.3 and MSI independently impair the SKI complex, uncovering a dependency upon PELO and highlighting a novel functional interaction between PELO and the SKI complex. Furthermore, the strength of this synthetic lethal interaction in a broad range of cancers with poor outcomes underscores the potential of PELO as a promising drug target for patients with 9p21.3-deleted or MSI cancers. Citation Format: Edmond M. Chan, Patricia Borck, Nolan Bick, Kristina Jankovic, Isabella Boyle, Lucy Parker-Burns, Kyla Foster, Anthony Lau, Ashir Borah, Joshua Dempster, Francisca Vazquez. PELO is a synthetic lethal target in chromosome 9p21-deleted or MSI cancers [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 3364.

Abstract 7148: Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR

Abstract Flap endonuclease 1 (FEN1) is a structure-specific metallonuclease that cleaves 5’ DNA flaps during replication and repair. FEN1 is an attractive target for development of anticancer therapeutics because it is overexpressed in many tumor types and has a large number of synthetic lethality partners including genes in Homologous Recombination (HR) pathway (Mengwasser et al., 2019; Guo et al., 2020). Utilizing fragment-based drug discovery approach we identified a novel metal-binding pharmacophore that binds to the two magnesium ions in the FEN1 active site. Further elaboration using fragment growth strategies resulted in highly potent and selective inhibitors. The current lead (BSM-1516) is ~65-fold more potent against FEN1 than its related enzyme Exonuclease 1 (Exo1) in biochemical assays (IC50 of 7 nM and 460 nM, respectively), an improvement of more than an order of magnitude in selectivity compared to earlier efforts. FEN1 target engagement in live cells was validated by cellular thermal shift assay (CETSA EC50 of 24 nM). Inhibition of FEN1 led to its increased association with chromatin in S-phase cells and recruitment of PARP1 enzyme. In clonogenic assay, BRCA2-deficient DLD1 cells were ~15-fold more sensitive to FEN1 inhibition than their isogenic BRCA2-wild-type counterparts (EC50 of 350 nM and 5 µM, respectively), confirming the increased susceptibility of HR deficient cancer cells to FEN1 inhibition. Treatment of BRCA2-deficient but not wild-type DLD1 cells with BSM-1516 resulted in cell cycle arrest and dose-dependent decrease of S-phase BrdU incorporation into DNA. Cell cycle arrest of BRCA2-deficient DLD1 cells was accompanied by DNA damage signaling and by accumulation of chromatin-bound RPA32, a marker of ssDNA. To explore synthetic lethal interactions of FEN1 with other DNA repair genes we performed FEN1-inhibitor-anchored CRISPR screen. This analysis revealed that in addition to HR pathway inactivation, genomic perturbations in EXO1, USP1 and PARP1 genes sensitized cells to FEN1 inhibition. Synergistic relationships of BSM-1516 and its combination potential were further explored in viability studies with a panel of DDR inhibitors (n=25) in BRCA2-proficient and deficient cell lines. Strong synergy was identified with multiple drug classes that included inhibitors of USP1 (KSQ-4279), PARP (Olaparib, Niraparib, Talazoparib, AZD5305), PARG (PDD 00017273) and ATR (AZD6738, VE-822, Elimusertib). In vitro ADME assays and in vivo PK studies showed that BSM-1516 has properties suitable for in vivo testing, either as a single agent or in combination with synergistic DDR inhibitors, an investigation that is currently underway. Citation Format: Jason Munguia, Sanjay Agarwalla, Elaine Guo, Dave Martin, Junhua Fan, Dave Lonergan, David Puerta, Zachary Zimmerman, Konstantin Taganov. Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR [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 7148.

Abstract 5578: Multi-omics approach reveals a SMAD4-deficiency signature, translational reprogramming and synthetic lethality in esophageal adenocarcinom

Abstract Esophageal adenocarcinoma (EAC) is a cancer of high mutation burden with negligible recurrent and druggable driver genes and, therefore, a paucity of options for targeted therapy. Mutation or loss of the tumor suppressor gene SMAD4 occurs in up to 20% of EAC cases, and we have previously shown that loss of SMAD4 is sufficient to promote EAC tumorigenesis in an in vivo xenograft model. The present study aimed to delineate the role of SMAD4 in EAC tumorigenesis and identify synthetic lethal interactions in SMAD4-deficient cancers as a novel approach for treating EAC. Our multi-omics methodology integrated RNA-sequencing, proteomics, polysome-sequencing, reverse-phase protein arrays, and genome-wide CRISPR-Cas9 screening to unveil a SMAD4-deficiency signature and new therapeutic avenues for EAC. These data reveal a complex interplay between SMAD4 loss and regulation of mTOR signaling, with specific downstream effects to reprogram translation. SMAD4-deficient cells express decreased levels of 4E-BP1, the inhibitory binding partner of EIF4E, which is the rate-limiting component of the cap-dependent translation initiation complex. This was accompanied by increased mTOR activity, including phosphorylation and inactivation of 4E-BP1. We found that SMAD4-deficient cells exhibit vastly different polysome traces compared to their wildtype counterparts and preferentially upregulate cap-dependent translation, acquiring an addiction to translation of oncogenic mRNAs. Furthermore, perturbation of additional negative regulators of mTOR signaling in combination with SMAD4 knockout exacerbated these effects and accelerated tumorigenesis in vivo and growth of patient-derived organoids in vitro. This work advances our knowledge of EAC tumorigenesis, provides new mechanistic insights into SMAD4-driven transformation as well as novel potential therapeutic avenues for SMAD4-deficient EAC. Citation Format: Julia V. Milne, Kenji Fujihara, Eric Kusnadi, Maree Pechlivanis, Niko Thio, Carlos Cabalag, Twishi Gulati, Jovana Gotovac, Kaylene Simpson, Cuong Duong, Luc Furic, Wayne Phillips, Nicholas Clemons. Multi-omics approach reveals a SMAD4-deficiency signature, translational reprogramming and synthetic lethality in esophageal adenocarcinom [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 5578.

Abstract 594: ORIC-613, a potential first- and best-in-class, orally bioavailable, potent and selective PLK4 inhibitor with synthetic lethality in TRIM37 high cancer models

Abstract A synthetic lethal interaction between polo-like kinase 4 (PLK4) and elevated TRIM37 was discovered in breast cancer and neuroblastoma (Meitinger et al. 2020; Yeow et al. 2020). High TRIM37 renders cells dependent on PLK4, a kinase that controls centriole duplication. Amplification and copy number gains of the 17q23 amplicon, where TRIM37 resides, are common in breast cancer and neuroblastoma and associated with early relapse and poor prognosis, making PLK4 a promising new synthetic lethal target for these patients. The development candidate ORIC-613 is an orally bioavailable, potent and exquisitely selective small molecule inhibitor of PLK4, which is synthetic lethal in TRIM37 high cells. ORIC-613 is highly selective against the kinome, including against the closely related aurora and PLK families. ORIC-613 ADME/PK profile includes high cell permeability and low potential for drug-drug interactions based on CYP profiling. ORIC-613 blocked PLK4 trans-autophosphorylation of phosphodegrons, leading to stabilization of PLK4, thus directly demonstrating target engagement in cells. PLK4 protein stabilization correlated with cell viability for ORIC-613, providing a quantifiable pharmacodynamic (PD) association with antitumor activity. Cell viability assessment in cancer cell lines revealed that ORIC-613 showed greater potency in TRIM37 high versus TRIM37 low cell lines. Importantly, ORIC-613 induced significantly greater apoptosis in TRIM37 high versus low cancer cell lines as measured by caspase 3/7 assay. We confirmed that ORIC-613 activity is on-target using an engineered cell line system of PLK4 G95L, in which the leucine mutation maintains enzymatic function but blocks compound binding. In cell viability assays, ORIC-613 was potent in the parental cells and lost activity in G95L cells. Analysis of genomic data from tumors indicates that TRIM37 copy number amplification and gain is prevalent across numerous cancers. Oral dosing of ORIC-613 resulted in tumor growth inhibition and regressions in TRIM37 high xenograft breast tumors with no body weight loss. In summary, ORIC-613 is a potential first- and best-in-class development candidate, with exquisite selectivity for PLK4, which demonstrates synthetic lethality in TRIM37 high tumors and has the potential to benefit these patients. Citation Format: Kyle A. Edgar, Siobhan McRee, Gina Andretta, Chelsea Chen, Christophe Colas, Wei Fang, Wayne Kong, Jason Long, Fang Liu, Jared Moore, Aleskandr Pankov, Dan Shore, Joanne Tan, Robert Warne, Livia Ulicna, Rakesh Vekariya, Anneleen Daemen, F Romero, Melissa R. Junttila, Lori S. Friedman. ORIC-613, a potential first- and best-in-class, orally bioavailable, potent and selective PLK4 inhibitor with synthetic lethality in TRIM37 high cancer models [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 594.

Abstract 4686: Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer

Abstract Breast cancer (BC), the most prevalent malignancy in women, ranks fourth in cancer mortality as of 2022. Mutations in the BRCA genes significantly elevate BC risk and are associated with markedly reduced survival compared to non-carriers. These genes are crucial in homologous recombination, a mechanism essential for error-free DNA repair. Despite advancements, treatment options for BRCA mutation-driven cancers remain limited. Synthetic lethality (SL) has emerged as a promising strategy, with poly(ADP-ribose) polymerase inhibitors (PARPi) being the first developed drugs based on this principle. However, PARPi's effectiveness is limited, as nearly 40% of BRCA-mutated BCs are unresponsive, and resistance often develops in initial responders. Therefore, novel mutation-specific targeted therapies are urgently needed. In this study, we employed the novel computational method 'Mining Synthetic Lethals (MiSL)' to analyze TCGA pan-cancer primary tumor data, identifying ART3 as a potential SL partner of the BRCA1 germline mutation. Our approach focused on the intersection of genetic interactions and cancer vulnerabilities, providing a unique insight into SL relationships. siRNA-mediated silencing of ART3 in BRCA1-mutated cells consistently led to decreased cell viability, apoptosis induction, and G2/M cell cycle arrest. Following stable transfection with an inducible ART3 shRNA construct, we confirmed significant reduction in cell viability in BRCA1-deficient cells through colony formation assays. RNA sequencing of ART3 knockdown cells revealed transcriptional changes in genes involved in cell cycle progression, apoptosis, and growth and proliferation pathways compared with control cells. Immunofluorescent staining revealed increased DNA damage foci with decreased DNA repair in ART3 knockdown BRCA1-deficient cells. In conclusion, our study identifies ART3 as a novel SL partner of the BRCA1 mutation in BC. This discovery paves the way for developing new therapeutic strategies for BRCA1-mutated breast cancers and novel chemoprevention approaches for individuals carrying germline BRCA1 mutations. Future research will focus on further elucidating the molecular mechanisms underlying this SL interaction and exploring its therapeutic potential in clinical settings. Citation Format: Hannah Neiger, Xinyu Pei, Anh Nguyen, Wenjia Wang, Emily L. Siegler, James Zhou, Kristie Lau, Alexandra Tan, Yihui Shi. Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast 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 4686.

Abstract A026: Identifying mechanisms to target in combination with trametinib to improve therapeutic efficacy in low-grade serous ovarian cancer

Abstract A026: Identifying mechanisms to target in combination with trametinib to improve therapeutic efficacy in low-grade serous ovarian cancer

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy-terminal tail in the GPN-loop GTPase Npa3.

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.

Targeting ATR in patients with cancer.

Pharmacological inhibition of the ataxia telangiectasia and Rad3-related protein serine/threonine kinase (ATR; also known as FRAP-related protein (FRP1)) has emerged as a promising strategy for cancer treatment that exploits synthetic lethal interactions with proteins involved in DNA damage repair, overcomes resistance to other therapies and enhances antitumour immunity. Multiple novel, potent ATR inhibitors are being tested in clinical trials using biomarker-directed approaches and involving patients across a broad range of solid cancer types; some of these inhibitors have now entered phase III trials. Further insight into the complex interactions of ATR with other DNA replication stress response pathway components and with the immune system is necessary in order to optimally harness the potential of ATR inhibitors in the clinic and achieve hypomorphic targeting of the various ATR functions. Furthermore, a deeper understanding of the diverse range of predictive biomarkers of response to ATR inhibitors and of the intraclass differences between these agents could help to refine trial design and patient selection strategies. Key challenges that remain in the clinical development of ATR inhibitors include the optimization of their therapeutic index and the development of rational combinations with these agents. In this Review, we detail the molecular mechanisms regulated by ATR and their clinical relevance, and discuss the challenges that must be addressed to extend the benefit of ATR inhibitors to a broad population of patients with cancer.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

Nuclear to cytoplasmic transport is a druggable dependency in MYC-driven hepatocellular carcinoma

The MYC oncogene is often dysregulated in human cancer, including hepatocellular carcinoma (HCC). MYC is considered undruggable to date. Here, we comprehensively identify genes essential for survival of MYChigh but not MYClow cells by a CRISPR/Cas9 genome-wide screen in a MYC-conditional HCC model. Our screen uncovers novel MYC synthetic lethal (MYC-SL) interactions and identifies most MYC-SL genes described previously. In particular, the screen reveals nucleocytoplasmic transport to be a MYC-SL interaction. We show that the majority of MYC-SL nucleocytoplasmic transport genes are upregulated in MYChigh murine HCC and are associated with poor survival in HCC patients. Inhibiting Exportin-1 (XPO1) in vivo induces marked tumor regression in an autochthonous MYC-transgenic HCC model and inhibits tumor growth in HCC patient-derived xenografts. XPO1 expression is associated with poor prognosis only in HCC patients with high MYC activity. We infer that MYC may generally regulate and require altered expression of nucleocytoplasmic transport genes for tumorigenesis.

SL-Miner: a web server for mining evidence and prioritization of cancer-specific synthetic lethality.

Synthetic lethality (SL) refers to a type of genetic interaction in which the simultaneous inactivation of two genes leads to cell death, while the inactivation of a single gene does not affect cell viability. It significantly expands the range of potential therapeutic targets for anti-cancer treatments. SL interactions are primarily identified through experimental screening and computational prediction. Although various computational methods have been proposed, they tend to ignore providing evidence to support their predictions of SL. Besides, they are rarely user-friendly for biologists who likely have limited programming skills. Moreover, the genetic context specificity of SL interactions is often not taken into consideration. Here, we introduce a web server called SL-Miner, which is designed to mine the evidence of SL relationships between a primary gene and a few candidate SL partner genes in a specific type of cancer, and to prioritize these candidate genes by integrating various types of evidence. For intuitive data visualization, SL-Miner provides a range of charts (e.g. volcano plot and box plot) to help users get insights from the data. SL-Miner is available at https://slminer.sist.shanghaitech.edu.cn.

Abstract A117: Molecular stratification of therapeutic targets in pancreatic cancer

Abstract Precision medicine approaches using molecular tumor profiling have resulted in striking clinical benefits in certain patient populations. However, targeted therapy in pancreatic cancer has been unable to displace multi-agent chemotherapy, in part due to lack of actionable biomarkers to anchor stratification. To address this need, we have conducted a biomarker focused analysis of cancer dependencies and cell surface targets to understand specific vulnerabilities present in molecular subgroups. We started by interrogating genome scale CRISPR screening and expression data from The Cancer Dependency Map to identify lineage-enriched, selective dependencies in pancreatic cancer. Our results reveal synthetic lethal interactions such as paralog pairs (e.g., VPS4A/B), dependencies predictable by overexpression (e.g., KLF5), and dependencies predictable by genetic alterations (e.g., RAB10 with SMAD4 deletion). Our results reaffirm the importance of KRAS signaling but also highlight a diversity of downstream essential mechanisms including DOCK5 and RAF1. We also identified vulnerabilities specific to transcriptional subtypes, finding ITGA3 to be a selective dependency of not only the basal-like state in pancreas cancer but also across the spectrum of epithelial malignancies. Given the central role of KRAS mutations in tumorigenesis and the emergence of KRAS inhibitors, we studied predictors of KRAS dependency. GSEA analysis of genes overexpressed in cell lines with lower dependency on KRAS identified gene sets related to focal adhesion, EMT, and neuron development, while lines with high KRAS dependency were enriched in genes related to epithelial differentiation/development and the canonical Hallmark KRAS up signature. These gene signatures also predict resistance and sensitivity to small molecule inhibition of KRAS with a high degree of accuracy (Pearson r = 0.68, 0.94 respectively). In addition to vulnerabilities from intracellular target inhibition, cancer cells may be targeted selectively based on surface proteins using cellular and antibody based therapeutic strategies. We used bulk gene expression data from TCGA and GTEx as well as single cell expression data from The Human Cell Atlas, to calculate enriched genes in pancreas cancer versus critical normal tissues. Our approach captures targets including MSLN, TROP2, MUC1, and CLDN18, which have established tumor specificity, but also identifies PSCA, GPRC5A, and CEACAM5 as additional emerging pancreas tumor specific antigens. We charted interpatient and intratumoral heterogeneity in spatial transcriptomics data using a cohort of 21 treated and untreated pancreatic cancer tissue specimens, identifying MUC1 and GPRC5A as both expressed homogenously across patients and throughout individual tumors while scarcely expressed in normal tissues. Ultimately, these findings may prompt prioritization of new molecularly defined dependencies, and construction of rational combinatorial regimens. Citation Format: Dennis Gong, Jimmy A. Guo, Carina Shiau, Ananya D. Jambhale, Steven Wang, Scott Ginebaugh, Patrick Z. Yu, Kevin S. Kapner, Seema Chugh, Laleh Abbassi, Daniel Zhao, Westley W. Wu, Peter Chen, Harshabad Singh, William L. Hwang, Andrew J. Aguirre. Molecular stratification of therapeutic targets in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A117.