Isogenic Cell Research Articles

Real-time monitoring of replication errors’ fate reveals the origin and dynamics of spontaneous mutations

The efficiency of replication error repair is a critical factor governing the emergence of mutations. However, it has so far been impossible to study this efficiency at the level of individual cells and to investigate if it varies within isogenic cell populations. In addition, why some errors escape repair remains unknown. Here we apply a combination of fluorescent labelling of the Escherichia coli Mismatch Repair (MMR) complex, microfluidics, and time-lapse microscopy, to monitor in real-time the fate of >20000 replication errors. We show that i) many mutations result from errors that are detected by MMR but inefficiently repaired ii) this limited repair efficiency is due to a temporal constraint imposed by the transient nature of the DNA strand discrimination signal, a constraint that is likely conserved across organisms, and iii) repair capacity varies from cell to cell, resulting in a subpopulation of cells with higher mutation rate. Such variations could influence the fitness and adaptability of populations, accelerating for instance the emergence of antibiotic resistance.

Abstract 7123: Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML)

Abstract Isocitrate dehydrogenase 1 and 2 (IDH1/2) convert isocitrate to α-ketoglutarate in the tricarboxylic acid (TCA) cycle. A missense mutation in IDH1/2 leads to accelerated conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent dioxygenases including Ten-Eleven Translocation (TET) enzymes. TET enzymes are DNA demethylases that convert 5-methylcytosine to 5-carboxycytosine and regulate CpG island promoter methylation signatures. In IDH1/2 mutant glioma, inhibition of TET2 enzyme by 2-HG can lead to promoter hypermethylation of O6-methylguanine methyltransferase (MGMT) and reduce its expression. Approximately 10-15% of de novo or treatment-related AML harbor mutually exclusive mutations in IDH1/2 or TET2, and emerging resistance towards clinical IDH1/2 inhibitors, therefore warrants novel therapeutic options. We performed a pan-cancer TCGA analysis and observed MGMT promoter hypermethylation and reduced expression in IDH1/2 AML. Our independent study on 10 AML patients (5 IDH1/2 and 5 TET2 mutant) corroborated the TCGA data and showed a significantly reduced expression of MGMT in comparison to 7 wild-type AML samples. As MGMT resolves O6-alkylguanine lesions during DNA damage, we performed an exhaustive alkylator screen, where temozolomide (TMZ) and our novel compound KL-50 emerged as the top hits that imparted a maximum therapeutic index in multiple MGMT (MGMT+/–) isogenic AML cell line models. Both TMZ and KL-50 induced MGMT-dependent DNA strand breaks and replication stress and activated ATM and ATR DNA damage response pathways in a time-dependent manner. Inhibition of ATR activation with Ceralasertib, an ATR inhibitor (ATRi) currently in clinical trials for chronic myelomonocytic leukemia and myelodysplastic syndromes, imparted a profound MGMT-dependent synergy with TMZ and KL-50. Researchers have established in glioma that mismatch repair (MMR) loss confers resistance to TMZ, and reported studies also show evidence of MMR loss in refractory or relapsed AML patients. Thus, we developed numerous MMR-deficient isogenic AML cell lines in an MGMT+/– background and observed MMR loss-mediated resistance to TMZ and abrogation of its synergy with ATRi. Unlike TMZ, KL-50 retained a profound synergy with ATRi in an MGMT-dependent manner irrespective of MMR status. We are currently investigating our novel KL-50 and ATRi combination in MGMT/MMR isogenic cell line-based in vivo AML models and in AML patient-derived xenografts to establish it as a promising novel biomarker-targeted therapy for AML patients. Citation Format: Prateek Bhardwaj, Ranjini Sundaram, Amro Baassiri, Martin Matthews, Jennifer VanOudenhove, Susan Gueble, Stephanie Halene, Ranjit Bindra. Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML) [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 7123.

Abstract 7119: Clinic-ready Polθ inhibitor SYX1097 suppresses solid tumors in vivo

Abstract A large fraction of tumors with BRCA1 or BRCA2 mutations only have weak responses to PARP inhibitor (PARPi) treatment, and often develop PARPi resistance. To address these challenges, we have developed SYX1097, a selective and potent oral Polθ inhibitor as a single agent or in combination with PARPi for treating tumors with BRCA mutations. Both Polθ and PARP1 play key roles in microhomology-mediated end joining for DNA double-strand repair and in single-strand DNA gap filling during DNA replication. Using in vitro cellular assays and in vivo animal models, we show that SYX1097 alone or combined with PARPi without obvious side effects. SYX1097 dramatically inhibited proliferation in BRCA -/- isogenic cells with IC50 <20 nM, while only had very weak effect on their parental cells (>10000nM). SYX1097 also enhanced tumor cell sensitivity to Olaparib 5 to 15 times. SYX1097 in vivo efficacious started at 50mg/kg mono-therapy, and generate >50% tumor regression at 5mg/kg when combined with Olaparib. In a 20-day repeated dosing study, SYX1097 200mg/kg BID was fully tolerated by mice. When considering additive hematological toxicity, comparing with “ATRi + PARPi”, “SYX1097 + PARPi” manifested deep and durable tumor regression without significant body weight loss or reticulocytes decrease. Moreover, SYX1097 is found synthetic lethal with defect of genes not involved in homologous recombination repair and able to suppress tumors with specific gene defect in vivo, which indicated SYX1097 can be used as single agent guided by specific biomarker.SYX1097 has a clean safety profile tested with Safety-Screen 44TM Panel, and is well tolerated in mice, rats and dogs. It possesses remarkable pharmacokinetic properties in various species, with no hematological toxicity observed during the preclinical toxicity studies. SYX1097 is advancing towards IND filing in 2024.Collectively, SYX1097 is a promising small molecule that may be utilized as a single agent or in combination with PARPi for cancer treatment not only limited by HR deficiency. Citation Format: Yilan Zhang, Kai Jiang, Xiaokang Xu, Liu Wu, Chongxun Ge, Yue Xie, Hu He, Song Liu, Xuzhen Tang, Song Shi. Clinic-ready Polθ inhibitor SYX1097 suppresses solid tumors in vivo [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 7119.

Abstract 7074: Perturbed RNA N6-methyladenosine methylome in SETD2 mutant clear cell renal carcinoma

Abstract Clear cell renal carcinoma (ccRCC), which arises from the epithelial cells of the proximal tubule represents 70% of all renal cell carcinoma. Mutations that drive ccRCC in conjunction with near ubiquitous and early VHL loss include, PBRM1 (33 - 45%), BAP1 (15%) and SETD2 (13%). Loss of SETD2 leads to diminished occupancy of H3K36me3 in active gene bodies, which is progressively deregulated in primary RCC and metastasis, linking loss of H3K36me to tumor progression and metastasis. N6-Methyladenosine (m6A) is an important post-transcriptional regulator of RNA transcript stability and half-life. Preliminary findings have shown H3K36me3 (deposited by SETD2), targeting the core m6A methyltransferase machinery (METTL3, METTL14 and WTAP) to the mRNA by virtue of its interaction to METTL14 subunit. The goal of this study is to investigate if H3K36me3 depletion as observed in SETD2 mutant ccRCC, leads to a deregulated m6A methylome. We further investigate how deregulated methylome in key pathways leads to cancer initiation and progression in ccRCC. To accomplish our goals, we generated isogenic ccRCC cell lines with H3K36me3 depletion by knocking out SETD2. RNA-immunoprecipitation and sequencing for m6A (MeRIP) showed global deregulation of m6A methylome in SETD2KO isogenic ccRCC cell lines. We also show a number of pro-tumorigenic genes and pathways including cell invasion, inflammation and stemness being differentially methylated and expressed in SETD2KO ccRCC cell lines. Furthermore, we show SETD2KO cells being more sensitive to METTL3 inhibition suggesting the relevance of m6A hypermethylated transcripts in promoting pro-tumorigenic and growth promoting processes in SETD2KO ccRCC cell lines. Citation Format: Shafiq Shaikh, Ryan Wagner, Ryan Hlady, Keith D. Robertson. Perturbed RNA N6-methyladenosine methylome in SETD2 mutant clear cell renal carcinoma [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 7074.

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P<0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p<0.001) and hyperproliferative Ki67+ GCB cells (p<0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value <0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p<0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p<0.01), and vs. these cell lines cultured in vitro (all p<0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p<0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p<0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p<0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [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 7586.

Abstract 316: A novel, high-throughput full-length scRNA-seq workflow for improved biomarker discovery

Abstract Objective: Single-cell RNA-seq (scRNA-seq) analysis has been widely applied in oncology research for biomarker discovery. Although droplet-based methods are commonly used for such studies owing to their high throughput, they still miss important insights due to their lack of full-length transcript coverage. While full-length methods are available, to date, they have not been able to meet the throughput demands of many researchers. Moreover, both droplet and full-length scRNA-seq methods do not currently provide adequate readouts for non-coding genes, thereby limiting investigation of gene regulatory networks to protein coding genes. To close these gaps, we have developed a new high-throughput full-length scRNA-seq workflow that comprehensively profiles both protein-coding and non-coding genes in up to 60,000 cells within two days. Methods: Our new high-throughput workflow uses two rounds of combinatorial indexing, starting with a 96-well plate format for the first barcoding step followed by an automated second barcoding step in a 5,184-nanowell chip using an automated nanodispensing system. Initial testing demonstrated that our method could handle up to 60,000 cells without generating significant levels of doublets due to barcode collisions. To further illustrate the capacity of the new scRNA-seq approach, we profiled a total of approximately 11,000 isogenic A549 cells that either express WT TP53 or are TP53 null. In addition, both isogenic cell lines were treated with epigenetic therapy or mock treatment. Libraries were generated and sequenced using an Illumina® NextSeq®2000 sequencer. The sequencing data was then analyzed to define differential gene expression for both protein-coding and non-coding transcripts as a function of TP53 genotype and treatment condition, using Cogent™ NGS software. Results: Preliminary analysis showed that, on average, approximately 11,000 genes and 40,000 transcripts were detected per single cell at a read depth of 100,000 reads per cell. UMAP-based clustering confidently separated the cells according to their genotypes and treatment conditions using either protein-coding genes or non-coding genes. Furthermore, differential expression analysis identified both protein-coding and non-coding transcripts with significant expression differences, underscoring biological significance. Conclusion: Our new high-throughput full-length scRNA workflow enables preparation of high-quality full-length RNA-seq libraries for up to 60,000 cells with only two rounds of barcoding and shows high sensitivity and specificity in gene/transcript detection and quantification. The technology significantly improves the ability to identify new biomarkers by enabling comprehensive profiling of both protein-coding and non-coding full length transcripts. Citation Format: Peng Xu, Joseph Liu, Yana Ryan, Kazuo Tori, Xuan Li, Hima Anbunathan, Mike Covington, Tomoya Uchiyama, Mohammad Fallahi, Xuan Qu, Xiaoyun Xing, Ting Wang, Bryan Bell, Shuwen Chen, Yue Yun, Andrew Farmer. A novel, high-throughput full-length scRNA-seq workflow for improved biomarker discovery [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 316.

Abstract 4709: The RASMULTI(ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12X_mutant cancers

Abstract Oncogenic RAS binds to multiple effector proteins to activate downstream signaling pathways, including the MAPK and PI3K axes. While RAS-mediated MAPK activation has a well-validated role in driving cancer progression, the contribution of PI3K activation has been more difficult to assess. Understanding the degree to which different KRAS-mutant genotypes stimulate PI3K activity may inform biomarker and combination strategies for RAS inhibitors currently undergoing clinical evaluation.RMC-7977 is a RASMULTI(ON) inhibitor that targets GTP-bound RAS(ON) and blocks effector protein binding. RMC-7977 exhibits similar potency for wild-type and oncogenic mutant RAS and is a powerful preclinical tool, representative of the investigational RASMULTI(ON) inhibitor RMC-6236, for comparing how RAS-mutant genotypes differ with respect to signal activation. MAPK signaling proceeds through a protein phosphorylation cascade that is readily detectable using standard cell biology methods, but PI3K is a lipid kinase whose activity is not amenable to protein characterization techniques. An assay of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) was developed to directly measure the activity of PI3K in the context of RAS inhibition.In isogenic LIM1215 cell lines expressing different KRASG12X mutants (KRASG12D, KRASG12V, KRASG12C and KRASG12R), RAS inhibition by RMC-7977 completely suppressed PI3K activity in cells with wild-type KRAS and KRASG12D, had a moderate inhibitory effect in KRASG12C and KRASG12V, and had no effect in KRASG12R. To confirm that this inhibition is dependent on a direct interaction between RAS(ON) and PI3K, CRISPR engineering was used to introduce mutations in PIK3CA to perturb the RAS-binding domain. In cells with disrupted RAS-PI3K interactions, the basal levels of PIP3 are significantly reduced and no longer change in response to treatment with RMC-7977.Based on the isogenic cell line results, KRASG12D and KRASG12R-mutant pancreatic cancer cell lines were profiled for their response to RAS inhibition. RMC-7977 decreased the levels of MAPK signaling in both KRASG12D and KRASG12R genotypes, but PI3K activity was only inhibited in KRASG12D lines. MEK inhibitors only inhibited MAPK signaling and had no effect on PI3K activity in either genotype. The simultaneous inhibition of MAPK and PI3K signaling by RMC-7977 led to deeper suppression of cyclin D levels and a more pronounced G1 cell cycle arrest as compared with MAPK inhibitors.We demonstrate that the coupling between RAS activation and PI3K signaling varies between oncogenic mutants, with KRASG12D showing the greatest degree of PI3K stimulation. In KRASG12D-mutant pancreatic cancer cell lines, a RASMULTI(ON) inhibitor suppresses both PI3K and MAPK signaling. These preclinical results may be used to inform potential, rational, mechanism-based combination treatment strategies for RAS(ON) inhibitors. Citation Format: Priyanka S. Bapat, Kyle Seamon, Andrea Gould, David Wildes. The RASMULTI(ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12X_mutant 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 4709.

Abstract 2373: Functional and specific T-cell engagers against a peptide-MHC tumor target

Abstract Accessing peptides from intracellular tumor antigens displayed on MHC class I (pMHC) can expand the T-cell engager (TCE) target pool for solid tumor applications. A challenge limiting TCE development for pMHC targets is the identification of rare, high-affinity, and high-specificity pMHC-binders. In this work, we describe TCEs in a 1 × 1 format with potent tumor-cell killing activity and high specificity for MAGE-A4, a pMHC tumor antigen expressed in multiple cancer types. We identified functional MAGE-A4 x CD3 TCEs with high specificity and affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA-A:02*01). We strategically selected MAGE-A4-binders with high binding specificity for pMAGE-A4230-239, but not MHC-I or many closely related MHC-restricted peptides. We then paired them with diverse and developable CD3-binders with a range of binding affinities, subunit specificities, and binding kinetic profiles. High-throughput in vitro functional characterization of hundreds of these TCEs enabled identification of molecules with optimal T-cell dependent cellular cytotoxicity in MAGE-A4-expressing tumor cell lines, with comparable tumor-killing activity and cytokine release to a clinical-stage TCE in a 2 × 1 format. Previous studies have highlighted unique challenges associated with pMHC targets, including their potential for cross-reactive binding to pMHCs on healthy tissues, and the absence of pharmacologically relevant species for preclinical toxicity testing. To address these challenges, we have designed and implemented an in vitro and in silico workflow to identify antibody binding to potential off-target peptides. Using this approach, we selected TCEs with several pMHC binding orientations and high specificity and affinity. These TCEs killed tumor cell lines endogenously expressing MAGE-A4 pMHCs and not isogenic cells with MAGE-A4 knocked out. Results demonstrate that our high-resolution, multiparametric antibody discovery capabilities can identify pMHC-binding antibodies with desired functional and specificity attributes. This approach unlocks the discovery and development of optimal TCEs against complex pMHC antigens. Citation Format: Davide Tortora, Peter Bergqvist, Allison Goodman, Ryan Blackler, Nathalie Blamey, Stefania Carrara, Lauren Chong, Gabrielle Conaghan, Cindy-Lee Crichlow, Valentine de Puyraimond, Harveer Dhupar, Patrick Farber, Jessica Fernandes Scortecci, Kate Gibson, Rodrigo Goya, Ahn Lee, Franco Li, Tova Pinsky, Craig Robb, Patrick Rowe, Antonios Samiotakis, Eduardo Solano Salgado, Ping Xiang, Irene Yu, Kelly Bullock, Tara Fernandez, Stephanie K. Masterman, Kush Dalal, Tim Jacobs, Bryan C. Barnhart. Functional and specific T-cell engagers against a peptide-MHC tumor target [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 2373.

Abstract 7590: Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair

Abstract Colorectal Cancer (CRC) is the third most common cause of cancer mortality in the US. A major problem in CRC management is relapse and progression to metastatic disease leading to poor overall survival. The tumor suppressor p53 is mutated in most CRCs and often drives metastasis and therapy resistance. Current therapeutic options targeting p53 mutant cancers show poor efficacy and frequently exhibit high toxicity. To address this clinical problem, we developed a novel therapeutic strategy for selective targeting of p53 mutant cancers. The strategy combines a thymidine analogue (e.g., trifluorthymidine/TFT, a component of TAS102) and poly (ADP-ribose) polymerase inhibitor (PARPi). We observed that TAS102 does not block DNA replication, but rather prompts post-replicative base-excision DNA repair (BER), and PARPi increases double-strand DNA breaks (DSBs) in p53 mutant cancer cells. Normal p53 wild type (p53wt) cells are arrested in the G1 phase and repair DNA. Thus, the TAS102-PARPi combination selectively targets p53-mutant cancer cells, resulting in the accumulation of DSBs and cell death. This novel strategy was examined in p53wt and p53-mutant tumor cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. The drug combination was significantly more effective in inhibiting p53 mutant tumor growth and in prolonging survival compared to monotherapies. This strategy is currently being tested in a first-in-human dose-escalation Phase I study (NCT04511039) in patients with advanced CRC. Further investigation revealed that the TAS102-PARPi combination induced DNA damage response (DDR) and the G2 checkpoint, leading to G2 arrest in p53 mutant cancer cells. We observed that TAS102-PARPi induced phosphorylation of CDC2 (also known as CDK1) but did not increase phosphorylation of histone H3 (a marker of mitosis), therefore indicating activation of the G2 checkpoint. Furthermore, the roles of ATM and ATR kinases in the DDR and G2 responses were clarified using highly specific kinase inhibitors. The analysis of the DDR/G2 signaling response in isogenic p53wt and mutant CRC cell lines showed that inhibition of WEE1 kinase can selectively abrogate the G2 checkpoint in p53-mutant cells, resulting in marked DNA damage and cell death. Importantly, the blockade of WEE1 strongly synergized with the TAS102-PARPi combination, further enhancing its potency and efficacy against p53-mutant cancer cells. In summary, our research provides a foundation for a novel therapeutic strategy for p53 mutant cancers that may provide a paradigm shift in the treatment of these deadly cancers, increasing efficacy while minimizing toxic adverse effects. Citation Format: Mohammed M. Alruwaili, Natsumi Naranjo, Priyanka Rajan, Kyeong Beom Jo, Dae-Kyum Kim, Thomas Melendy, Robert Straubinger, Christos Fountzilas, Andrei Bakin. Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair [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 7590.

Abstract 3234: A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with the lowest five-year survival rate of all cancers. Around 10% of pancreatic cancer diagnoses are thought to be familial pancreatic cancer (FPC). BRCA2 pathogenic variants have been identified to be closely associated with FPC. Personalized medicine approaches that are tailored to FPC patients based on their mutation profiles could improve patient outcomes and minimize adverse effects. This study aims to identify novel potential drug candidates for BRCA2-deficient PDAC and dissect its underlying molecular mechanisms. To discover new vulnerabilities for BRCA2-deficient pancreatic cancer, we performed high-throughput drug screening (HTS) using isogenic murine Brca2 knockout (KO) and control PDAC cells. The HTS assay revealed that JQ1, a small molecule inhibitor of the bromodomain and extra-terminal domain protein (BET) family, selectively induced cytotoxicity in Brca2 KO cells. JQ1 significantly decreased the cell viability of Brca2 KO cells in vitro and suppressed the growth of Brca2 KO tumors in vivo compared to the controls. The transcriptomic analyses showed that JQ1 treatment resulted in upregulation of the gene sets associated with macroautophagy. Multi-orthogonal autophagy assays, including live cell imaging of fluorescence-based autophagy reporter, also supported that Brca2 KO cells had a constitutively higher basal level of autophagic activities compared to the controls, and JQ1 further induced autophagic flux in Brca2 KO cells. Moreover, blocking the autophagy process by pharmacological inhibition or knocking down essential autophagy genes rescued JQ1-induced cell death in Brca2 KO cells, indicating that the increased autophagy is responsible for JQ1-mediated cell death in Brca2 KO cells. In conclusion, we found that BRCA2 deficiency elevated autophagic flux, and extensive autophagy was further activated by BET inhibition, culminating in autophagy-dependent cell death in Brca2-deficient pancreatic cancer cells. Our findings suggest that BET inhibition could be a promising therapeutic strategy for treating BRCA2-deficient pancreatic cancer. Citation Format: Suyakarn Archasappawat, EunJung Lee, Keely Ji, Jocelyn Pena, Virneliz Fernandez Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic 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 3234.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian 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 6441.

Abstract 4366: Loss of EP300 triggers IL-1α signaling and subsequent activation of the IL-6/JAK/STAT3 axis to drive oncogenesis in bladder cancer

Abstract Bladder Cancer (BLCA) is the sixth most common cancer in the United States with 81,180 new cases and 17,100 deaths in 2022. Next Generation Sequencing of BLCA has revealed an enrichment of alterations in genes classified as chromatin modifiers compared to other solid tumors. Among these is EP300, the gene encoding the p300 histone acetyltransferase, which is mutated in ~12% of BLCA with the majority putative loss of function frameshift and nonsense mutations. To investigate the biological role of EP300 loss-of-function in BLCA pathogenesis, we generated isogenic EP300 null RT112 bladder cancer cells using a 2-step CRISPR knockout (KO) and knockin strategy. EP300 KO cells demonstrated increased proliferation, anchorage independent cell growth, tumor formation in mice, and enhanced invasive potential. To investigate the mechanism(s) whereby EP300 loss-of-function enhances oncogenicity, we performed an unbiased Tandem Tag Mass Spectrometry (TMT MS) screen to identify pathways dysregulated in EP300 KO versus parental cells. EP300 loss-of-function was found to induce IL-6/JAK/STAT3 signaling via upregulated expression of IL-1α, a canonical pro-inflammatory cytokine. Knock down of IL1A gene expression was sufficient to downregulate STAT3 activation in EP300 KO cells, whereas ectopic stimulation using IL-1α increased STAT3 activation in parental RT112 cells, suggesting that IL-1α was the predominant upstream activator of JAK/STAT3 signaling in our model. Pharmacological inhibition of downstream targets of IL-1α signaling such as IRAK1/4 were also able to inhibit STAT3 signaling in EP300 KO cells in a dose and time dependent manner. Further investigations indicated that activated IL-1α signaling not only drove transcriptional upregulation of IL-6 but also the production of soluble IL-6R, activating IL-6 trans signaling. Finally, to elucidate how IL-1α upregulation is triggered by EP300 loss-of-function, we performed immunoprecipitation and immunofluorescence assays, which revealed that IL-1α binds directly to p300. Additionally, ectopic p300 expression in EP300 KO clones dramatically reduced IL-1α protein expression, indicating that p300 acts as a negative regulator of IL-1α in a bladder cancer context. In summary, EP300 loss-of-function relieves p300’s transcriptional and/or physical-tethering inhibition on IL-1α signaling, subsequently activating the IL-6/JAK/STAT3 pathway to drive oncogenesis in BLCA. Citation Format: James A. Rodrigues, Jiaqian Luo, Amanda R. Sabel, Ziyu Chen, Xinran Tang, Fengshen Kuo, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene Pietzak, Gopa Iyer, David B. Solit, Sizhi P. Gao. Loss of EP300 triggers IL-1α signaling and subsequent activation of the IL-6/JAK/STAT3 axis to drive oncogenesis in bladder 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 4366.

Abstract 108: Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model

Abstract Mutations in STK11 that lead to loss of its protein LKB1 occur in 15-20% of non-squamous non-small cell lung cancer (NSCLC) and are frequently co-mutated with oncogenic KRAS mutations. KRAS and STK11 co-mutant NSCLC is associated with poor prognosis, low PD-L1 expression, low T-cell infiltration, and poor response to immune-checkpoint inhibitors (ICI). Loss of LKB1 leads to altered transcriptional programs with the activation of CREB-dependent gene transcription and changes in the tumor microenvironment including recruitment of suppressive myeloid cells. We used the syngeneic Kras mutant murine CMT167 tumor cell line that is wild-type for Tp53 and Stk11 and sensitive to ICI and generated isogeneic Stk11 knockout cell lines (CMT167-Stk11-KO) by CRISPR/Cas-9 deletion. Loss of Stk11 recapitulated LKB1-loss transcriptional signatures and rendered CMT167 tumors resistant to ICI. Single-cell RNA sequencing (10x) of implanted tumors demonstrated significantly less CD8 T-cells and increased neutrophils (PMN) in Stk11-KO versus Stk11-WT tumors. Analysis of differentially expressed genes in the tumor compartment identified increased expression of complement pathway genes including the central mediator C3 in CMT-167-Stk11-KO tumors. Examination of a well characterized clinical cohort of human KRAS-mutant NSCLC patient samples with and without STK11 mutation confirmed the increased expression of complement C3 in STK11-mutant tumors. Analysis of a human NSCLC cell line panel demonstrated that C3 expression was modulated by LKB1. Knockout of C3 in CMT167-Stk11-KO tumor resulted in dramatic inhibition of tumor growth and re-sensitized CMT167-Stk11-KO tumors to ICI treatment in WT mice. Using C3-/- knockout mice, this effect was determined to be reliant on tumor-derived C3 and dependent on adaptive immunity as no difference in growth of CMT167-Stk11-KO tumors occurred in nude mice or in WT mice after CD8 T-cell depletion. We then compared the transcriptome of Stk11-KO tumor cells with and without C3 deletion, and observed that C3 upregulates the expression of Cxcl1, Cxcl2 and Cxcl3 which mediate PMN recruitment and activation. Since these chemokines ligate Cxcr2, we asked whether Cxcr2 mediated tumor growth and ICI resistance in Stk11-KO tumors. While treatment with single-agent Cxcr2 inhibitor and anti-PD-1 therapy had no effect on tumor growth, the combination resulted in significant suppression of tumor growth in vivo. These results support a role for tumor-derived C3 suppressing CD8 T-cell immunity, potentially indirectly through recruitment of PMN or inducing PMN suppressor function. Our results also provide rationale for targeting tumor-derived complement and inhibiting Cxcr2 to enhance ICI efficacy as novel therapeutic approaches in patients with STK11-mutant NSCLC. Citation Format: Sora Suzuki, Catrina Ting, Bojidar Kandar, Te-An Chen, Anm Nazmul Khan, Thejaswini Giridharan, Brahm Segal, Edwin H. Yau. Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model [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 108.

Abstract 4536: MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma

Abstract 5’-methylthioadenosine phosphorylase (MTAP), is deleted in 15% of all cancers (estimated 100,000 patients per year in the United States), including 12-15% of cholangiocarcinoma (CCA) cases. MTAP deficiency accumulates 5’-methylthioadenosine (MTA) that subsequently binds protein arginine methyl transferase 5 (PRMT5) and inhibits its activity. This renders selective targeting of MTAP null tumors with agents (MRTX1719, AMG193, TNG462, TNG908) targeting MTA bound PRMT5 (PRMT5:MTA), while sparing surrounding normal MTAP wild-type (WT) tissue. Early signs of clinical activity was seen with MRTX1719, including objective responses in patients with MTAP loss tumors, including biliary tract cancer from the phase I/II study (NCT05245500). Despite these advances, prolonged targeted therapy poses the risk of development of acquired resistance demanding the need for better therapeutic strategies. To address this pressing need, we co-treated CCA cell lines with MRTX1719 and PARP inhibitor olaparib. We hypothesize that since PRMT5 is a critical regulator of splicing and DNA damage pathways, inhibition of PRMT5 disrupts these downstream processes creating an additional vulnerability to DNA damage inducing agents such as PARP inhibitors. Our preliminary data combining MRTX1719 with olaparib in three MTAP loss CCA cell lines (RBE, YSCCC, TFK1) show synergy (combination index; CI<1), and significant reduction in cell viability as well as colony formation ability when compared to monotherapy in vitro. To further gain mechanistic insights into the biology of MTAP loss, we stably expressed MTAP in MTAP null CCA cell line, RBE and demonstrated that MTAP loss drives tumorigenesis in vivo highlighting its tumor suppressor function in CCA. Mass spectrometry-based profiling of histone modifications from the isogenic RBE cell lines showed heightened methylation and reduced acetylation marks in the absence of MTAP. We also noted substantial differences in the distribution of splicing events, with increased intron retention, 3’ alternative splicing and decreased exon skipping as a result of MTAP loss. Similar differences in proportion of splicing events were also observed in MTAP loss CCA patient derived xenografts as compared to WT models. Collectively, these data implicate MTAP to be a crucial player in modulation of the chromatin and splicing events that may drive therapeutic response to PRMT5 inhibition. Citation Format: Pooja A. Shah, Ajay Kumar Saw, William Padron, Ming Zhao, Argun Akcakanat, Kurt Evans, Funda Meric-Bernstam, Milind Javle, Jordi Rodon, Kunal Rai. MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma [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 4536.

Abstract 7058: NSD2 overexpression drives multiple myeloma dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome

Abstract Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the oncogenic histone methyltransferase NSD2. Genome-wide CRISPR screens in isogenic MM cells with high and low NSD2 expression identified adenylate kinase 2 (AK2), an enzyme critical for transport of ATP from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased NADP(H) critical for conversion of ribonucleotides to deoxyribonucleosides by the enzyme ribonucleotide reductase (RNR). Consequently, metabolomic studies revealed depletion of deoxynucleotide triphosphates (dNTPs) in AK2-deficient t(4;14) MM cells, which resulted in replication stress, DNA damage and apoptosis. Exogenous supplementation with deoxynucleosides rescued AK2 depleted MM cells confirming the role of AK2 in dNTP homeostasis. In addition, NSD2 overexpression drives a large genome-wide increase in chromatin methylation, which depletes the methyl donor S-adenosylmethionine (SAM). Accordingly, we found that high NSD2 expression in MM cells disrupts creatine biosynthesis, a process dependent on SAM. The creatine/phosphocreatine system represents an alternative means of transporting high energy phosphate from the mitochondria. Hence, creatine supplementation restored NADP(H) levels and rescued AK2-deficient t(4;14) MM cells indicating that the hypersensitivity of these cells to AK2 depletion is driven by creatine deficiency due to NSD2 overexpression. Furthermore, AK2 inhibition impaired ATP-dependent protein folding increasing sensitivity of t(4;14) MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM. Citation Format: Amin Sobh, Elena Encinas, Alisha Patel, Greeshma Surapaneni, Charlotte Kaestner, Janai Poullard, Monica Clerio, Karthik Vasan, Tzipporah Freeman, Dongwen Lv, Daphne Dupere-Richer, Alberto Riva, Benjamin Barwick, Daohong Zhou, Lawrence Boise, Constantine Mitsiades, Baek Kim, Richard Bennett, Navdeep Chandel, Jonathan Licht. NSD2 overexpression drives multiple myeloma dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome [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 7058.

Abstract 2704: cGAS-STING drives myeloid inflammation in chromosomally unstable esophageal cancer

Abstract Esophageal adenocarcinoma (EAC) is an aggressive disease characterized by chromosomal instability (CIN), whereby cells continuously acquire chromosomal abnormalities that promote therapeutic resistance, immune evasion and metastasis. CIN in cancers also fuels the misplacement of genomic DNA in the form of structural abnormalities of the nucleus, such as micronuclei (MN), that lead to chronic activation of the cytosolic DNA sensing cGAS-STING pathway. However, how chronic cGAS-STING pathway activation is tolerated and shapes the tumor landscape of CINhigh EAC cancers is poorly understood. Using publicly available RNA-seq and genome sequencing data of EAC tumours and esophageal cancer cell lines as well as a panel of EAC cell lines, we demonstrate that the core cGAS-STING pathway machinery is rarely inactivated in EAC despite high levels of baseline CIN and aneuploidy. Through transcriptomic profiling of an isogenic EAC precursor cell line model of variable CIN and cGASKO/WT EAC cell lines we show that both chronic and transient CIN-driven cGAS-STING activation converge on the expression of CXCR1/2 ligands and other pro-inflammatory cytokines and chemokines rather than typical anti-tumor type I interferon signalling in EAC cells. Indeed, a novel transcriptional signature of cGAS+ micronuclear burden-correlated genes correlates with orthogonal measures of CIN in EAC tumours and esophagogastric cancer cell lines and is associated with an enrichment in myeloid-derived cells as well as poor prognosis. Correspondingly, using multiplexed immunofluorescence (IF) we find that a high preponderance of cGAS+MN and STING expression in human EAC samples is associated with decreased tumor purity, increased myeloid cell and macrophage infiltration, and increased peripheral blood neutrophil counts, indicative of tumor-promoting myeloid inflammation. Ongoing multiplexed IF and single-nuclei RNAseq analysis of cGAS+MNhigh and cGAS+MNlow EAC tumors will further help characterise the components that comprise the CIN EAC tumor microenvironment (TME) and the CIN- and cGAS-STING-dependent cell-cell interactions that govern it. Taken together, our findings provide an explanation for the observed maintenance of cGAS and STING in EAC and identify disruption of cGAS-STING-dependent myeloid cell inflammation and recruitment as a potential therapeutic target for CINhigh EAC. Citation Format: Bruno Beernaert, Erkin Erdal, Rose Clark, Tong Liu, Ester M. Hammond, Eileen E. Parkes. cGAS-STING drives myeloid inflammation in chromosomally unstable esophageal 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 2704.

Abstract 4684: A comprehensive platform for identification of KRAS-specific synthetic lethal targets using patient-derived cells

Abstract Colorectal cancer (CRC) remains a global health concern, constituting a large portion of cancer incidence and mortality. Despite advances in comprehending the genetic and epigenetic landscape of CRC and progress achieved in developing KRAS-specific inhibitors poised for clinical evaluation, a significant unmet medical need persists. This underscores the necessity for innovative therapeutic interventions in this field, as effective therapeutic strategies targeting specific molecular vulnerabilities are currently lacking. Our study aims to address this gap by focusing on the identification of synthetic lethal (SL) inhibitors specific to the KRAS pathway. We have established a platform utilizing human intestinal stem cell (hISC)-derived CRC model cells, patient-derived xenografts (PDXs), and clinical samples for genomic and functional analyses. Employing CRISPR/Cas9 technology, we introduced common CRC mutations into hISCs, generating isogenic CRC model cells with diverse mutational backgrounds including KRAS G12D. Additionally, we developed PDXs from CRC patients and collected biopsy samples from primary and metastatic tumors. Through RNA-seq and whole-exome sequencing (WES), we identified molecular signatures and pathways associated with different mutational variants. Our innovative platform enables high-throughput screening, incorporating both small molecule screening and CRISPR/Cas9 dropout screening with a genome-wide library on CRC model cells. Notably, our focus on KRAS-specific SL inhibitors distinguishes our approach, aiming for targeted therapies tailored to specific CRC subtypes. Validation of driver mutations, such as APC, KRAS, and TP53, in patient-derived material showcased the relevance of our model. High-quality screening results, meeting industry-standard QC criteria, allowed us to identify essential and tumor suppressor genes critical for CRC. Importantly, the use of normal hISCs enabled the identification of genes crucial only for transformed cells. The platform serves as a powerful tool for target discovery and validation in CRC, emphasizing the significance of primary material-derived cells. Beyond CRC, our findings have broader implications for other cancer types and personalized medicine approaches. By focusing on the identification of KRAS-specific SL inhibitors, our research contributes to the development of novel therapies for CRC, addressing the urgent need for targeted interventions in this heterogeneous malignancy. Citation Format: Marcin Dulęba, Eliza Zimoląg, Joanna Szuszkiewicz, Marcin Serocki, Joanna Szklarczyk, Olga Dracz, Zuzanna Kurzejamska, Diego Coelho, Michał Combik, Oleksii Bryzghalov, Joanna Krawczyk, Agata Stachowicz, Michał Mikula, Krzysztof Brzózka, Rafał Dziadziuszko, Tomasz Rzymski, Andrzej Mazan. A comprehensive platform for identification of KRAS-specific synthetic lethal targets using patient-derived cells [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 4684.

Abstract 1563: Anoikis associated metabolic modulation by UCA1 in CRC

Abstract Colorectal Cancer (CRC) is the second most lethal cancer in estimated deaths for 2023 according to the American Cancer Society. This is partially due to the low survival rate once CRC is diagnosed at a distant location. This warrants early diagnostic markers and an understanding of the mechanisms underlying the metastatic process, anoikis resistance being one of them. Our laboratory analysis of the TCGA database comprising 519 human CRC tissues shows lncRNA UCA1 overexpressed in tumors compared to normal, and higher expression of lncRNA UCA1 is responsible for the progression, metastasis, and poor survival of CRC patients. In our anchorage-independent growth model (anoikis resistant), using isogenic (oncogenic and metastatic) cell lines, we observed a very high expression of lncRNA UCA1 along with GluT1 with increased cell survival. Cancer cells, however, appear to rapidly increase their glucose uptake and directly ferment it into lactate, even in the presence of oxygen and functional mitochondria. UCA1 has been found to play an essential role in the development of metastasis and dysregulation of glycolysis in various cancers. To verify if lncRNA UCA1 modulates anoikis resistance and glucose metabolism, we generated SW480+UCA1 overexpression (OE) and SW620+shUCA1 knockdown (KD) stable cell lines using lentiviral transduction. The OE GFP-expressing cell lines were further sorted, and grown in puromycin. The KD cell lines were selected with puromycin for the generation of stable cell lines. The stable cell lines were characterized through different phenotypic assays. UCA1 OE cell lines have a higher glucose uptake and the UCA1 KD cell lines have a lower glucose uptake. The lactate production is high in the KD and low in OE cell lines. The OE and KD cell lines along with the control cell lines were subjected to an anoikis model, and glucose and lactate analysis were performed accordingly. RT-PCR and western blot analysis were performed for the target genes responsible for cell survival, stemness, and glucose metabolism. We are analyzing the Extracellular Acidification Rate (ECAR), Oxidative Consumption Rate (OCR), ATP production rate the rate and potential max rate of glycolysis, and the mitochondrial stress of the recombinant cell lines using the “Agilent Seahorse XF”. The role of lncRNA UCA1 in anoikis resistance leading to metastasis and modulating glucose metabolism will provide new insights into the pathophysiology of CRC metastasis. This information will be beneficial for developing more unique strategies for the treatment of this metastatic disease. Citation Format: Sophia Leslie, Kyle Doxtater, Samantha Lopez, Bilal Hafeez, Tamer Oraby, Timothy Loy, Manish K. Tripathi. Anoikis associated metabolic modulation by UCA1 in CRC [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 1563.

Abstract 443: Unsaturated fatty acids promote cell cycle progression and proliferation of platinum resistant ovarian cancer cells

Abstract Introduction: Ovarian cancer (OC) depends on fat as fuel for metastasis and growth. We have previously demonstrated that cisplatin resistant (Pt-R) OC cells uptake higher amounts of fatty acids (FAs) compared to sensitive (Pt-S) cells- a process which facilitates cancer cell survival under cisplatin-induced oxidative stress. Here we aimed to determine which class of FAs promotes the Pt-R phenotype and to identify the mechanism through which FAs support cancer survival. Materials and methods: We used two isogenic OC cell lines pairs, sensitive and resistant to cisplatin: OVCAR5 Pt-S (IC50 = 2.881μM) vs Pt-R (IC50 = 6.831 μM) and PEO1 (IC50 = 4.901 μM) vs PEO4 (IC50 = 8.285 μM). Cells were cultured under low serum conditions, supplemented with 50 µM oleic (OA) or 50 µM palmitic acid (PA), and used for viability assays, RNA-Seq, and cell cycle analysis. Quantitative RT-PCR measured expression of key FAs transporters in Pt-S and Pt-R cells. Results: A viability assay showed that Pt-R cells were less viable under serum depletion compared to Pt-S OC cells. OA (unsaturated), but not PA (saturated), promoted proliferation of both OC cell lines, and rescued Pt-R vs Pt-S cells; 268.4% vs 91.6% for OVCAR-5 and 38.1% for PEO4 vs 12.1% for PEO1, suggesting that Pt-R cells depend more on unsaturated FAs compared to Pt-S cells. Quantification of key FAs transporters demonstrated that Pt-R cell expressed higher levels of FABP4, FABP5 and CD36 compared to Pt-S cells. Treatment with an FABP inhibitor reduced the Pt IC50 in Pt-R cells (from 11.893 μM to 5.0415 μM in OVCAR5 and from 8.285 μM to 6.968 μM in PEO4). RNA-Seq analysis analysed transcriptomic changes induced by 24 hour treatment with OA in OVCAR5 cells. There were 923 differentially expressed protein-coding genes, with 300 upregulated and 623 downregulated transcripts (FDR<0.05). Pathway analysis revealed cell cycle related pathways like: cell cycle mitotic, cell cycle checkpoints, G2/M checkpoints among the top 10 most significantly upregulated pathways and E2F1 was identified as the main effector. Cell cycle analysis confirmed that OA induced increased percentages of cells in S- and G2/M phases - from 6.95% to 11.6% (p<0.05), and 13.6 to 17.6% (P<0.05), respectively. Treatment with an E2F1 inhibitor significantly reduced the OA-induced cell proliferation. Conclusion: Pt-R OC cells are dependent on unsaturated FAs compared to Pt-S cells and upregulate key transporters to allow increased uptake of FAs. OA supports proliferation of Pt-R cells and cell cycle progression from G1 to S- and G2/M phases. Ongoing experiments focus on key mechanisms activated by unsaturated FAs in Pt-R cells. Citation Format: Ana Maria Isac, Guangyuan Zhao, Horacio Cardenas, Daniela Matei. Unsaturated fatty acids promote cell cycle progression and proliferation of platinum resistant ovarian cancer cells [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 443.

Abstract 1214: Investigating therapeutic strategies to promote immune rejection of KRASG12C inhibitor-resistant subpopulations in lung cancer

Abstract Oncogenic KRAS signaling not only promotes progression and survival of cancer cells, but it can also play a major role in suppressing anti-tumor immunity through several mechanisms, including secretion of cytokines, reduction of antigen presentation and suppression of tumor-intrinsic interferon responses. Consequently, treatment with KRASG12C inhibitors alleviates oncogenic KRAS driven immune suppression, resulting in a profound remodeling of the tumor microenvironment (TME). In immunogenic mouse models, these changes can lead to the generation of some complete responses which are dependent on the engagement of the immune system. In this project we aim to investigate whether the induction of immune responses triggered by KRASG12C inhibition can effectively target inhibitor-resistant subclones. To achieve this, we mimic the development of drug resistance by combining reporter-traced isogenic KRASG12C and KRASG12D mutant lung cancer cells, where the KRASG12D mutant cells are inherently resistant to KRASG12C inhibitors. Growth of resistant cells was monitored via in vivo luciferase imaging and end point flow cytometry. KRASG12C inhibition as monotherapy, either with the active-state KRASG12C inhibitor RMC-4998 or the inactive-state inhibitor adagrasib, led to a proliferative advantage of the drug-resistant subpopulation, suggesting that any immune response to KRASG12C inhibition in the bulk tumor population was insufficient to target the resistant cell subclones. To investigate if immune mediated elimination of resistant cells could be achieved using combinations, we assessed two strategies commonly evaluated in clinical trials with KRASG12C inhibitors: anti-PD1 blockade and SHP2 inhibition. Of note, KRASG12D mutant cells do not respond to these therapies when grown subcutaneously. Treatment of RMC-4998 in combination with either anti-PD1 or the SHP2 inhibitor RMC-4550 was able to induce complete eradication of both KRASG12C and KRASG12D mutant lung cancer cells, despite the resistant cells being insensitive to these therapies when treated in the absence of sensitive cells. Furthermore, both combinations induced immune memory towards the resistant cells. These long-term effects were dependent on the immune system as they were not observed in immune deficient mice. Immune profiling of treated tumors revealed that both combinations can result in a more profound remodeling of the TME and potentiate the immune responses. Overall, our preclinical results demonstrate that the anti-tumor responses generated by the combination of KRASG12C inhibitors with SHP2 inhibition and/or immune checkpoint blockade can result in the bystander immune mediated killing of subclones of KRASG12C inhibitor resistant cells, providing a paradigm for the development of therapeutic combinations that may be more able to counter the development of inhibitor resistance. Citation Format: Mona Tomaschko, Christopher Moore, Claire E. Pillsbury, Kangbo Ng, Sophie de Carne, Sareena Rana, Andrea de Castro, Panayiotis Anastasiou, Miriam Molina-Arcas, Julian Downward. Investigating therapeutic strategies to promote immune rejection of KRASG12C inhibitor-resistant subpopulations 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 1214.