Philadelphia Research Articles

A Framework for Using Regional Scenarios in Racial Equity Planning

Problem, research strategy, and findings Planning under uncertainty and planning for racial equity both require imagination. In confronting the uncertain future, regional planners often opt for exploratory scenario planning: telling multiple future stories to prepare for any outcome. For equity planning, imagination supports vision beyond the deeply unequal present condition, wherein policy tinkering is insufficient for necessary change. And yet, scenario planning and equity planning are estranged. Although regional planners wish to engage both, they lack guidance on how. I look to close this gap by providing a framework (the Framework) for using scenarios in equity planning. The Framework builds on the five types of racial equity, a six-stage hybrid scenario process, and the three outcomes of public-sector scenarios. Using the Framework, I assessed the inclusion of equity in the Delaware Valley Regional Planning Commission’s Dispatches From Alternative Futures scenario plan. This plan successfully raises racial equity as a concern for the future of the Philadelphia (PA) region. However, the stakeholder group was not sufficiently diverse for deliberative equity and the scenario planners did not use tools that could assess the distributional outcomes. Neither epistemic nor restorative equity were a significant part of the scenario plan, leaving open the possibility for co-designed scenarios for a racial equity future. Takeaway for practice Metropolitan planning organizations (MPOs) can use the Framework to ensure that exploratory scenarios advance equity goals. Although the Framework can supply a starting point for MPOs, the most important voices are those from Black, Indigenous, and people of color (BIPOC) and other marginalized communities. Scenario planners should consider how they can reach out to community members not just as stakeholders but as co-creators of a more epistemically equitable scenario planning process, one that addresses the uncertainties and aspirations that already percolate within these communities.

Abstract PO-007: A phase 1 study of romidepsin, azacitidine, dexamethasone, and lenalidomide (RAdR) for relapsed/refractory T-cell malignancies

Abstract Romidepsin, azacitidine and lenalidomide are all active in T-cell malignancies (TCM) demonstrating single agent objective response rates (ORR) of 20 to 30% (Coiffier 2012; Morschhauser 2013; Dupuis 2022). Combinations of these targeted agents improves the rates of complete response (CR) but have significant toxicity due to indefinite schedules. We hypothesized that use of a multi-targeted therapy regimen such as RAdR given in fixed duration cycles would be safe and effective. We tested escalating doses of lenalidomide (5-20mg) with fixed doses of romidepsin, azacitidine and dexamethasone in a phase 1 study. Patients (pts) first received a 7-day window of lenalidomide for cytokine correlative research followed by six cycles of romidepsin (12mg/m2; days 1 and 10), azacitidine (300mg; days 1-10), dexamethasone (40mg; days 1 and 10), and lenalidomide (5-20mg; days 1-10) in 21-day cycles for up to 6 cycles. The primary endpoint was the maximum tolerated dose (MTD) of lenalidomide and the primary objective was the overall safety of the regimen. A 3+3 design was used with an additional 9 pts treated at the MTD. 26 pts were enrolled with a median age of 61 years (range, 28-80) including 31% over 65. Eight (31%) pts had PTCL, NOS, 6 (23%) had mycosis fungoides (MF), 5 (19%) had angioimmunoblastic T-cell lymphoma, and 4 (15%) ATLL. Median prior lines of therapy were 2 (range, 1-8), 31% had received >3 prior lines and 2 patients had received prior allogeneic transplantation. The median number of cycles was 4 (range, <1-6). The most frequent reasons for stopping therapy were progressive disease (38%), completion of planned therapy (27%) and adverse events (23%). The most frequent grade 1-2 non-hematologic adverse events were vomiting (52%), diarrhea (48%) and dysgeusia (48%), and AE grade 3+ included infection (16%) and hypokalemia (16%). Grade 3+ neutropenia occurred in 24% of cycles and both grade 3+ anemia and thrombocytopenia in 12% of cycles. Two DLTs were observed, grade 4 thrombocytopenia at dose level 1 and grade 3 abdominal pain at dose level 2. The MTD of lenalidomide was determined to be 20mg. In evaluable pts (N=21) the ORR was 66% and CR rate was 14%. After a median follow-up of 23 months. Median progression free survival (PFS) and overall survival (OS) were 4 months and not reached, respectively. 2-year OS was 53%. Two pts (PTCL, NOS and MF) completed the planned 6 cycles of therapy and remain in remission without consolidation at 30 months. Five pts were successfully bridged to SCT, all of whom remain alive and without progression. In pts treated at the MTD, the ORR was 89% and the CR rate was 22%. The multi-targeted therapy regimen RAdR is safe and can achieve CR after fixed duration in patients with relapsed refractory TCM. Toxicity is predominantly gastrointestinal and hematologic. Most patients do not achieve durable CR with RAdR alone, but it may be effective bridging therapy to consolidation. This study was supported by the Intramural Research Program of the NIH and was registered at Clinicaltrials.gov [NCT04447027]. Citation Format: Max Gordon, Milos Miljkovic, Samuel Ng, Rahul Lakhotia, Christopher Melani, Kevin Conlon, James Phelan, Bonita Bryant, Stefania Pittaluga, Elaine Jaffe, Louis Staudt, Wyndham Wilson, Mark Roschewski. A phase 1 study of romidepsin, azacitidine, dexamethasone, and lenalidomide (RAdR) for relapsed/refractory T-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-007.

Abstract PO-023: CD70 deregulation in follicular lymphoma at diagnosis is associated with relapse and opens new avenues for dual CD19-CD70 CAR-T therapy

Abstract Follicular lymphoma (FL) is the most common indolent B-cell lymphoma. Despite most patients achieve a complete response with first-line treatment, an important fraction of them relapse, which may worsen the prognosis and increase the risk of a histological transformation to aggressive lymphoma. Therefore, there is a need to better stratify the patients at the diagnosis and develop tailored therapies for high-risk patients. Here we have assessed the immune profile (Nanostring® nCounter) in FFPE-derived biopsies in a series of FL patients at diagnosis, homogenously treated with immunochemotherapy regimen (mostly R-CHOP), and followed-up at Hospital Clínic de Barcelona for more than 12 years. By comparing patients who experienced relapse (n=20) with those who did not relapse at any time (NR, n=12), we have found 31 differentially expressed genes, 25 of them up-regulated in the relapse group. Among them, we have further focused on the role of CD70 in FL pathogenesis. mRNA CD70 was higher in those patients who eventually relapsed and correlated with inferior PFS. Moreover, CD70high FL cases bear a limited number of recurrent somatic mutations. We next sought to determine the specific subpopulations expressing CD70 by multiplex immunofluorescence (Vectra Polaris). Interestingly, we have demonstrated it is mainly expressed in tumor cells correlating with inferior PFS. Moreover, a fraction of T cells from the tumor microenvironment also expresses CD70. Intriguingly, higher levels of CD70 were detected in both CD4+ and CD8+ T cells in the relapse group, including T regulatory and T follicular helper cells. CD70 interacts with CD27, its only known ligand, and can be mutually activated. While CD27 RNA levels did not change between groups, CD27 protein was increased in the B cell population in patients who eventually relapse and in TFH cells, while it was down-regulated in CD8+ and CD4+ T helper non-follicular cells. Furthermore, we found an association between the percentage of CD70+ cells within B cell population and CD70+ in CD4+ and CD8+ cells (R=0.51, p < 0.05). To investigate the role of CD70 in FL pathogenesis we generated two CD70 KO cell lines and FL patient derived cells (n=4) using CRISPR/Cas9 technique. By co-culturing T cells from healthy donors with CD70+ or CD70- FL cell lines, we observed CD70+ tumor cells promote CD70 expression in T cells. In primary samples, we have demonstrated that CD70-KO B cells exhibit reduced response to proliferative stimuli. Moreover, RNAseq analysis followed by GSEA pathway assignment indicated that CD70 expression was related to increased angiogenesis, glycolysis and hypoxia. Finally, to advance towards personalized therapies for this high-risk patient and taking advantage of our institution CAR-T program, we have generated a dual CD19-CD70 CAR-T combining an approved academic product, ARI-0001, and a truncated CD27 protein. Finally, we have demonstrated that this novel CAR-T is effective in both cell lines and ex vivo FL spheroids (PDLS, Dobaño-López, Blood Cancer J 2024), while in vivo studies are ongoing. Citation Format: Ferran Araujo-Ayala, Mireia Bachiller, Raluca Alexandru, Pablo Mozas, Salut Colell, María Villalba-Esparza, Judith Mateos-Jaimez, Ferran Nadeu, Andrea Rivero, Maria Ros, Cèlia Dobaño-López, Juan Garcia Valero, Neus Serrat, Marta Gimenez-Alejandre, Ariadna Giro, Daniel Hodson, Armando López-Guillermo, Elias Campo, Dolors Colomer, Alba Maiques-Diaz, José Ignacio Martín-Subero, Sílvia Beà, Laura Magnano, Sonia Guedan, Carlos E. de Andrea, Patricia Pérez-Galan. CD70 deregulation in follicular lymphoma at diagnosis is associated with relapse and opens new avenues for dual CD19-CD70 CAR-T therapy [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-023.

Abstract PO-015: The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation, immune evasion and small nucleolar RNA processing to sustain the survival of DLBCL cells

Abstract Introduction.The ETS1 and FLI1 transcription factors are both located in the 11q24.3 region, which is commonly amplified in 25% of diffuse large B cell lymphomas (DLBCL) (Bonetti et al, 2013) and largely co-regulate a series of genes involved in B-cell signaling, differentiation and cell cycle (Priebe et al, 2020; Sartori et al, 2021). While FLI1 is expressed at a higher level in DLBCL of the germinal center B-cell (GCB) type than in the activated B-cell-like (ABC) DLBCL, ETS1 is more expressed in the latter subgroup. We and others have reported preclinical anti-tumor activity in lymphomas and other tumors with small molecules blocking the binding of ETS factors and RNA helicases (Erkizan et al, 2009; Spriano et al, 2019). Methods.To explore interaction partners, protein precipitation was conducted utilizing streptavidin beads to capture strep-tagged ETS1 in the ABC DLBCL cell line, HBL-1, followed by Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). Shotgun proteomics analysis identified biologically relevant ETS1 protein interactors, with a spectral count above four, that were validated by normal and reverse co-immunoprecipitation experiments. Transcriptome analysis was done via RNASeq and smallRNASeq after DDX21 siRNAs silencing in four DLBCL cell lines, two derived from ABC (HBL1 and U2932) and two from GCB (OCI-Ly1 and VAL). Direct targets were validated by DDX21 chromatin immunoprecipitation (ChIP). Results. The ETS1 interactors included proteins associated with RNA processing. Among these, we focused on the novel interactor DDX21, an RNA helicase also regulated by pETS1 (Chung et al, 2023) and FLI1 (Sartori et al, 2021). DDX21 was found differentially expressed between GCB and ABC DLBCL, with higher expression in the latter (P<0.001). When we silenced DDX21 with siRNAs, toxicity was seen in ABC cell lines. To better characterize the role of DDX21 as a transcriptional factor in DLBCL we obtained RNASeq transcriptome after DDX21 silencing. Transcriptional profiling and functional annotation of transcripts regulated by DDX21 revealed genes coding proteins involved in cell cycle (FDR <0.001), ribosomes (FDR <0.001) and immune evasion (FDR <0.001). Based on the role of DDX21, smallRNAseq was also performed and miRNAs of some members of the let7 family (hsa-let-7d, hsa-let-7e and hsa-let-7f) were found downregulated by DDX21, together with miR-532. Among the upregulated miRNAs miR-222 and miR-155 were the most interesting. Different snoRNAs were also found deregulated, SNORA37 was further validated as a DDX21 upregulated direct target by RNA and ChIP qPCR. Conclusions. In ABC DLBCL, ETS1 interacts with proteins involved in spliceosome and in ribosome biogenesis, including DDX21. More expressed in ABC than GCB DLBCL, DDX21 appears essential for the survival of ABC lymphoma cells by regulating cell cycle, RNA processing and immune evasion. Several miRNAs and snoRNAs were found deregulated by DDX21. The interaction between ETS1 and DDX21 offers an additional therapeutic opportunity against DLBCL cells. Citation Format: Giulio Sartori, Valdemar Priebe, Luciano Cascione, Elaine Y.L. Chung, Mélanie Favre-Juilland, Sara Napoli, Alberto Arribas, Stefano Giuffrida, Andrea Rinaldi, Margot Thome Miazza, Francesco Bertoni. The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation, immune evasion and small nucleolar RNA processing to sustain the survival of DLBCL cells [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-015.

Abstract PO-019: Ubiquitin E3 ligase OSTM1 regulates the cAMP/PKA/CREB pathway and suppresses B-cell malignancies

Abstract B-cell malignancies (BCM) often arise from genomic alterations introduced during different stages of B-cell development. Here, we performed a whole genome targeting CRISPR/Cas9 screen in the immortalized yet non-transformed IL-3-dependent Ba/F3 murine pro-B cell line to identify genes whose loss confers IL-3 independence, reasoning that these genes may function as tumor suppressor genes (TSGs) in BCM. One of the candidate genes is osteoporosis-associated transmembrane protein 1 (OSTM1), an E3 ubiquitin ligase whose loss-of-function causes autosomal recessive osteoporosis. Informatics analysis of patient data showed that OSTM1 is frequently deleted across BCM, which co-occurs with the deletion of a well-characterized TSG Cdkn2a. Genetic silencing of OSTM1 in Ba/F3 cells conferred IL-3-independent survival and proliferation, as well as splenomegaly, lymphadenopathy, and abnormal peripheral blood cell count, indicative of transformation in vivo. Using mass spectrometry screens, we identified phosphodiesterase 3B (PDE3B) as an interacting partner of OSTM1. OSTM1 ubiquitylates PDE3B and promotes its proteasomal degradation. The OSTM1-PDE3B interaction and PDE3B degradation are negatively regulated by OSTM1 N-glycosylation. As PDE3B catalyzes the conversion of cAMP to AMP, it negatively regulates the cAMP-dependent PKA/CREB/CREBBP tumor suppressive pathway. Indeed, overexpression of PDE3B accelerated cell proliferation while PDE3B silencing in OSTM1 KO cells reversed the transformation phenotype in cell lines and in mouse allograft tumors. Importantly, a strong correlation between OSTM1 disruption, increased PDE3B stability, and decreased PKA/CREB/CEBBP signaling was observed in cell lines and in BCM patient datasets. Finally, CD19-Cre mediated B-cell specific ablation of OSTM1 cooperated with CDKN2A deletion to drive spontaneous immature B-cell lymphomagenesis in mice. Collectively, these results indicate that OSTM1 is a novel TSG which targets PDE3B for proteasomal degradation. Loss of OSTM1 enhances PDE3B stability and abrogates the tumor-suppressive role of cAMP/CREB/CREBBP pathway which promotes BCM. Citation Format: Muhammad Usama Tariq, Julia Shen, Jaeyong Jung, Namratha Sheshadri, Junrong Yan, Rongrong Li, Kevin Lu, Tong Liu, Hong Li, Yue Lynn Wang, Ping Xie, Wei-Xing Zong. Ubiquitin E3 ligase OSTM1 regulates the cAMP/PKA/CREB pathway and suppresses B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-019.

Abstract PO-031: Systematic discovery of chemotherapy response biomarkers in Peripheral T Cell Lymphomas

Abstract Background. Identifying which patients will benefit from individual therapies remains a general challenge in oncology. Predictive biomarkers of drug sensitivity, when known, can increase the rate of success of trials, and allow stratification of patients for targeted combination therapies, as recent shown by the GUIDANCE-01 trial in Large B-Cell Lymphoma. Peripheral T-Cell Lymphomas (PTCL) could particularly benefit from this approach, being heterogeneous diseases against which many therapies are active in unpredictable subsets of patients. A central challenge of biomarker discovery is finding true determinants of drug response, which may be multiple genes, among thousands of molecular features that may accidentally correlate. Here, we aim to mitigate this challenge by combining functional genetic screens and gene expression profiling in diverse PTCL cultures, to enable the systematic discovery of drug sensitivity biomarkers. Methods. To discover genes whose expression directly modifies drug sensitivity, we conducted genome-wide CRISPR screens with PTCL cells under treatment with five therapies with clinical activity in PTCL: Romidepsin, Pralatrexate, Gemcitabine, Oxaliplatin, and Bortezomib. To discover genes whose basal expression significantly correlates with specific drug sensitivities, we measured RNAseq and drug response functions for a diverse panel of 27 PTCL cultures. Genes in the intersection of both experiments are causally related to and correlate with drug responses in PTCL, and have the statistical benefit of detection by independent methods. Results. Functional genetic screens on therapy provided mechanistic insights for each drug of interest, and identified genes whose perturbation affects drug sensitivity. Whereas hundreds of genes nominally correlate with drug sensitivity in cell line panels, for each drug a shortlist of only 5 to 20 genes are both CRISPR hits and correlate with response across PTCL cultures. For each drug tested, multi-gene sensitivity scores constructed from hits stratify PTCL cultures into statistically distinct sensitive and non-sensitive subgroups (Mann Whitney P-Values <0.01). These genes’ relationships to drug mechanisms were often supported by prior studies, including functions in DNA repair, cell cycle regulation, and solute carriers required for cellular entry of specific therapies, such as SLC19A1 for Pralatrexate and SLC43A2 for Oxaliplatin. Conclusions. Combining functional genetic screens and correlative drug sensitivity measurements narrowed the search for predictive biomarkers of drug sensitivity, and may be a general means of refining biomarker hypotheses for validation in clinical datasets. Although chemotherapy sensitivity is commonly polygenic, multi-gene scores based on few mechanistically-related genes were able to significantly distinguish drug sensitive from resistant PTCLs. These approaches provide novel hypotheses for drug-gene relationships that may serve as the basis for molecular subtyping of individual lymphomas for precision regimens. Citation Format: Adam C Palmer, Jacob C Pantazis. Systematic discovery of chemotherapy response biomarkers in Peripheral T Cell Lymphomas [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-031.

Abstract PO-021: Modeling marginal zone lymphomagenesis

Abstract Introduction: Marginal zone lymphomas (MZL) are the second most prevalent subtype of indolent Non-Hodgkin Lymphomas (iNHL), yet lack a unique cytogenetic identifying abnormality. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) pathway. PI3K inhibitors have demonstrated impressive clinical activity in several indolent lymphomas including MZL, underscoring the pivotal role of the PI3K-AKT-mTOR (PAM) pathway in MZL pathogenesis. Furthermore, iNHL overexpress the antiapoptotic protein Bcl-2. Methods: We developed a genetically engineered mouse model with heterozygous global knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1), resulting in heightened PAM pathway activation across all tissues. We closely monitored these mice for tumor formation via regular physical assessments over several months. Upon tumor detection, mice were euthanized, and tumors were histologically examined. For a more precise representation of B-cell biology akin to the human disease, we employed the Cre-LoxP system to create the CD19-Cre-PTENfl/fl/LKB1fl/fl and CD19-Cre-PTENfl/fl/Eµ-Bcl2. Results: Thirty mice of global KO PTEN+/- LKB1+/- succumbed to or were euthanized due to disease progression, defined as either lymph node enlargement and/or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Median survival time was 6 months (95% CI: 6, 8). Immunohistochemical analysis of the 51 lymph nodes revealed iNHL in 61.5% (N=32), with all of the MZL subtype. The penetrance rate was 37%. Then, we looked at the absolute B cells, and the major B subtypes in age-matched mice (N=3 per group): WT, CD19-Cre-PTENfl/fl/LKB1fl/fl, CD19-Cre-PTENfl/fl/Eµ-Bcl2. We observed the following preliminary results in the absolute B cells (23,489,812; 1,550,430; 140,476,875), Marginal zone cells (1,087,883; 452,470; 42,658,701), follicular cells (19,078,598; 203,286; 57,212,627), B1 cells (962,192; 939,386; 1,803,060), and plasma cells (211,725; 202,790; 10,531,585) respectively. Then, Kruskal-Wallis tests were used to test the main effect of these three models (WT, CD19-Cre-PTENfl/fl/LKB1fl/fl and CD19-Cre-PTENfl/fl/Bcl2) on absolute cell counts for each cell type: B cells (p=0.027), follicular (p=0.061), Marginal zone (p=0.027), B1 (p=0.43), and plasma cell (p=0.066). The test results indicate that there were statistically discernible differences in absolute cell counts among the three models for B cells and MZ. Conclusion: Marginal zone lymphoma remains an incurable lymphoma with a dearth of reliable preclinical models. Our data provides, for the first time, evidence supporting the involvement of the PAM pathway in MZL pathogenesis, laying the groundwork for further research elucidating the disease biology and rational therapeutic strategies for this incurable malignancy. Citation Format: Emily Harris, Ariel Sindel, Jian He, Roy Sabo, Alden Chesney, Guanhua Lai, Adolpho Mauro, Rebecca Martins, Jennifer Koblinski, Joelene Windle, Victor Yazbeck. Modeling marginal zone lymphomagenesis [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-021.

Abstract PO-034: Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs

Abstract Cancer cells need metabolic adaptations to survive in hostile environment such as low nutrient and drug treatment conditions. DLBCLs (Diffuse large B cell lymphoma) originate from a metabolic competitive/challenging environment of germinal center (GC) when B cells undergo positive selections against antigens. However, it is largely unknown whether and how metabolic flexibility (the ability to use different nutrients) and plasticity (the ability to process the same nutrient differently) contribute to DLBCLs. Our previous study identified that SIRT3, the only mitochondrial protein deacetylase, is critical to survival and proliferation of DLBCLs by promoting glutaminolysis. Our mitochondrial localized SIRT3 inhibitor, YC8-02, has pan-toxicity to all DLBCL cells tested regardless of different subtypes with somatic mutations. Here, we observed glucose as an alternative nutrient source to compensate in the TCA cycle in SIRT3 deficient cells. DLBCLs exhibited resistance to SIRT3 inhibition in high glucose condition, while SIRT3 inhibited DLBCLs highly depend on glucose metabolism. The combination of YC8-02 with glucose metabolism inhibitors induces synergistic killing to DLBCLs. As glucose is the main carbon source of cells, it is unknown how DLBCLs manage the flexibility of using glucose for different purposes. Using in vivo metabolic imaging (MALDI), we observed that, in contrast to normal GC B cells, lymphomas specifically use the glucose for de novo nucleotide synthesis. SIRT3 inhibition decreased nucleotide levels in DLBCL cells in vitro and in vivo. The metabolic flux tracing showed that DLBCL cells requires SIRT3 activity to activate a glucose/serine/one-carbon metabolism pathway for de novo nucleotide biosynthesis, and Inhibition of SIRT3 decreased the glucose and serine being used for nucleotides in DLBCLs. Serine as another potent source for nucleotides can rescue DLBCLs from SIRT3 inhibition. Furthermore, we validated that overexpression of enzymes, like MTHFD2, in one carbon metabolism pathway can rescue the proliferation in SIRT3 deficient cells. High nucleotides levels in medium also help DLBCLs survival in YC8-02 treatments. To overcome the metabolic flexibility on environmental nucleotides, we used inhibitors of nucleoside transporters (ENT1/2), dipyridamole and draflazine, which can synergize with YC8-02 to kill DLBCLs in vitro and eradicate systemic lymphomas in vivo. In summary, our results illustrate that DLBCLs with active SIRT3 exhibit metabolic plasticity by repurposing glucose to nucleotide biosynthesis, while lymphoma cells can also reply on metabolic flexibility to manage nutrient usage and gain extracellular nucleotides for survival under SIRT3 inhibition. Our combination treatment SIRT3 and ENT inhibitors can be a promising therapy for DLBCLs by restraining their metabolic plasticity and flexibility. Citation Format: Meng Li, Noel R Park, Chris Chin, Coraline Mlynarczyk, Hening Lin, Shawn Davidson, Ari M Melnick. Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-034.

Abstract PR07: SETD1B mutations confer apoptosis resistance and BCL2 independence in B-cell lymphoma

Abstract The translocation t(14;18) activates BCL2 and is considered the initiating genetic lesion in over ninety percent of follicular lymphomas (FL). Surprisingly, FL patients respond poorly to clinically approved inhibitor, venetoclax. The mechanisms underlying BCL2 independence in FL are poorly understood, and it has been speculated that epigenetic rewiring of cell death programs may play a role. We performed separate genome-wide CRISPR/Cas9 screens to explore the mechanisms of resistance to Venetoclax and an experimental MCL-1 inhibitor (S63845) and unveiled the histone lysine methyltransferase SETD1B (KMT2G) as an unexpected mechanism of resistance to both inhibitors in lymphoma. We show that mutations and deletions affecting SETD1B occur in 7% of FLs and 16% of diffuse large B cell lymphomas (DLBCL). In vivo, SETD1B acts as a tumor suppressor gene and cooperates with the related histone-lysine N-methyltransferase 2D (KMT2D) gene leading to a more aggressive disease presentation. This oncogenic cooperation is reflected in the genomes of human lymphoma, where SETD1B mutations typically co-occur with KMT2D mutations. Mechanistically, SETD1B is required to express pro-apoptotic BCL2 family proteins like BIM and BIK. Inhibitors of the KDM5 histone H3K4 de-methylase restore the expression of BIM and BIK, venetoclax sensitivity and lead to synergistic drug effects in SETD1B deficient lymphoma xenografts. Our results highlight a role for SETD1B in the epigenetic regulation of cell death proteins in B cell lymphomas, connecting the loss of SETD1B to lymphoma development and reduced sensitivity to venetoclax. Citation Format: Ana Portelinha, Shenqiu Wang, Sara Parsa, Man Jiang, Sagarajit Mohanty, Soumya Sharma, Neeraj Arya, Omid Tavana, Jiayu Wen, Jude Fitzgibbon, Ahmet Dogan, Ana Younes, Ari M. Melnick, Hans-Guido Wendel. SETD1B mutations confer apoptosis resistance and BCL2 independence in B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR07.

Abstract PR013: Detecting pairwise and higher-order antagonistic epistatic effects among somatic cancer genotypes to discover synthetic lethality

Abstract The mutual exclusivity of somatic variants in cancer suggests that current somatic variants can exert antagonistic epistatic effects on the selection of new mutations, contributing to a complex landscape of evolutionary trajectories. However, quantifying pairwise and higher-order epistatic effects—which are essential to estimation of the trajectory of likely cancer genotypes—has been a challenge. We have developed a continuous-time Markov chain model that enables the estimation of mutation origination and fixation, dependent on somatic cancer genotype. We coupled the continuous-time Markov chain model with an approach that deconvolutes the underlying mutation rates and selection intensities across trajectories of oncogenesis. By elucidating the routes of variant evolution and the strengths of selective forces at play, our approach lays the groundwork for quantifying antagonistic epistatic effects of specific somatic genotypes. We applied our approach to report the variant mutation rates, selection intensities, and consequent substitution rates for lung adenocarcinoma, comparing smoker and nonsmoker tumor cohorts. We describe the distinct influences of smoking on underlying mutation rates and on physiology affecting the selective regime of lung tissue. Inference of the most likely routes of site-specific variant evolution, as well as estimation of the selection strength operating on each step along the route represents a key component for prioritization, development, and implementation of personalized cancer therapies that target synthetic lethality. Citation Format: Jorge A. Alfaro-Murillo, Krishna Dasari, Jeffrey P. Townsend. Detecting pairwise and higher-order antagonistic epistatic effects among somatic cancer genotypes to discover synthetic lethality [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR013.

Abstract IA009: Single strand DNA GAP accumulation as a functional biomarker for DNA Repair Inhibitors

Abstract Deficiency of homologous recombination (HR)-mediated DNA repair occurs through genetic or epigenetic inactivation of the BRCA1 and BRCA2 (BRCA1/2) genes. HR-deficiency also provides unique opportunities for targeted therapy, through the mechanism of synthetic lethality, as exemplified by the hypersensitivity of BRCA1/2-mutated tumors to PARP inhibitors (PARPi). To date, several PARP inhibitors have been approved for clinical use. The promising clinical response of patients with germline BRCA1/2-mutations prompted the use of PARPi for patients with somatic BRCA1/2 mutations as well. In addition, these results extended the use of PARPi for various types of ovarian, breast, pancreatic, and prostate tumors with HR defects. PARPi resistance is emerging as the major obstacle to clinical effectiveness in patients with HR-deficient tumors. PARPi resistance results from several independent mechanisms, leading to the restoration of Homologous Recombination and/or Replication Fork stabilization. The absence of alternative options for patients with tumors with innate or acquired resistance underlines the urgency to develop additional therapeutics. More recent studies have identified new synthetic lethal opportunities for BRCA1/2 deficient tumors. Knockout of the genes for POLQ (DNA polymerase theta) or for USP1 (Ubiquitin Specific Protease 1) results in killing of these tumors, and inhibitors of these enzymes are emerging as promising agents for HR-deficient tumors. POLQ and USP1 expression is particularly high in subtypes of breast and ovarian tumors with defects in HR. As a result, POLQi or USP1i in HR-deficient tumors induces cell death. POLQi or USP1i can synergize with PARPi in killing HR-deficient tumors or PARPi-resistant tumors. BRCA1/2 deficient tumors have an increase in single strand DNA gaps (ssGAPs) near replication forks. Treatment with PARPi results in an increase in the size and number for these ssGAPs, ultimately leading to DSBs and cell death. Interestingly, PARPi resistance results, at least in part, from the ability of resistant cells to close the ssGAPs through enhanced POLQ and USP1 activity, thereby providing a rationale for the combination of PARP1, POLQi, and USP1 in HR-deficient cancers. USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Moreover, this synergy was observed in a set of patient-derived ovarian cancer organoids (PDOs), thus confirming the sensitivity of BRCA1-deficient cells to inhibition by these agents. The accumulation of ssDNA gaps after treatment with a USP1i, PARP, or POLQi correlated with the sensitivity to these drugs in all models tested. Ovarian cancer PDOs provide a powerful tool for detecting drug synergy and for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to DNA Repair inhibitors as monotherapy or in combination in ongoing clinical trials. Citation Format: Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for DNA Repair Inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA009.

Abstract IA003: Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase

Abstract The WRN helicase serves as a key target in the treatment of cancers characterized by microsatellite instability (MSI) because it plays a crucial role in resolving harmful non-canonical DNA structures that arise in cells with defective mismatch repair systems. Despite the critical functions of human DNA and RNA helicases, no drugs targeting these enzymes have been approved, largely due to the difficulties in identifying potent and selective inhibitors. In this study, we present the chemoproteomics-based identification of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This inhibitor specifically interacts with a cysteine residue (C727) within the helicase domain, which undergoes inter-domain movement during the DNA unwinding process. VVD-133214 reacts with the WRN protein in a nucleotide cooperative manner, promoting stable, compact structures that impede the enzyme's dynamic flexibility essential for its helicase activity. Inhibition of WRN by VVD-133214 leads to extensive double-stranded DNA breaks, nuclear enlargement, and ultimately cell death, specifically in MSI-high cells but not in microsatellite stable cells. The inhibitor demonstrated good tolerance in mice and significant tumor reduction in various MSI-high colorectal cancer cell lines and patient-derived xenograft models. Our findings highlight an allosteric strategy to inhibit WRN function that avoids interference from the endogenous ATP cofactor in cancer cells and positions VVD-133214 as a promising therapeutic candidate for patients with MSI-high cancers. Citation Format: Matthew P. Patricelli, Kristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A. Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Shota Kikuchi, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodilles, Seth M .Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N Williams, Martha K. Pastuszka, John J. Sigler, Piergiorgio Pettazzoni, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne, Jean-Marc Plancher, Isabel Cuartas, Joan Seoane, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Todd M. Kinsella. Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA003.

Abstract B015: Combinatorial genetic screens to map synthetic lethal interactions and identify new cancer drug targets in KRAS mutant cancers

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR014) of the Conference Program/Proceedings. Citation Format: Rand Arafeh, Laura Chang, Lydia Sawyer, Helen Wang, James McFarland, Joshua Dempster, Peter DeWeirdt, John Doench, William C Hahn. Combinatorial genetic screens to map synthetic lethal interactions and identify new cancer drug targets in KRAS mutant cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B015.

Abstract A012: Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders

Abstract Monosomy 7 is among the most frequent cytogenetic abnormalities in hematological disorders and is associated with short survival and drug resistance. Despite its high prevalence and detrimental impact, the therapeutic vulnerabilities underlying monosomy 7-associated blood disorders remain largely elusive, impeding progress toward improved patient care. The homeostatic cellular requirement for a normal dosage of essential genes creates an opportunity to target vulnerabilities that arise due to reduced levels of proteins encoded by a haploinsufficient essential gene. Briefly, the loss of one copy of a dosage-sensitive essential gene (gene X), in combination with the inhibition of itself or a related gene (gene Y), or an associated pathway results in lethal consequences for cells. The remarkable and selective clinical efficacy of lenalidomide for the treatment of del(5q) MDS has demonstrated how allelic haploinsufficiency underlies the sensitivity to this drug by synthetic lethality. Differential expression analysis of gene and protein expression in primary AML samples with monosomy 7 revealed significant downregulation of multiple proteasome pathway members at the protein level, but not at the RNA level. Primary AML samples with -7/del(7q) exhibited increased sensitivity (low IC50) to the proteasome inhibitor bortezomib, as evidenced by two independent ex vivo drug screening cohorts (the Beat AML and the FIMM study). Chromosome 7 harbors four proteasome subunits, PSMA2, PSMC2, PSMG3, and SEM1. We performed gene expression, protein expression, copy number analysis, and individual gene knockout experiments. The results have revealed PSMA2 to be a haploinsufficient essential gene on chromosome 7. PSMA2 knockout confers leukemia a growth disadvantage for multiple AML cell clines in both TP53 wild-type and knockout backgrounds. We generated PSMA2 isogenic hemizygous deletion and diploid single-cell clones. PSMA2 hemizygous deletion cells exhibited approximately half the protein expression compared to diploid controls, confirming that PSMA2 is a haploinsufficient gene. PSMA2hemizygous deletion single-cell clones showed significantly enhanced sensitivity to all three evaluated proteasome inhibitors (bortezomib, ixazomib, and carfilzomib), aligning with the sensitivity observed in primary -7/del(7q) leukemia samples. PSMA2 hemizygous deletion cell clones displayed increased p38 and decreased pERK levels upon treatment with proteasome inhibitors, potentially contributing to their increased sensitivity to proteasome inhibitors. Proteomics analysis and in vivo validation is ongoing. As such, we have identified haploinsufficient essential gene PSMA2 mediated proteasome pathway vulnerability in monosomy 7 associated leukemia and further showed that proteasome inhibitors as promising therapeutic approaches for treating hematological disorders associated with monosomy 7. Citation Format: Haijiao Zhang, Basil Allen, Daniel Bottomly, Peter Ryabinin, Schannon K. Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A012.

Abstract PR014: Combinatorial genetic screens to map synthetic lethal interactions and identify new cancer drug targets in KRAS mutant cancers

Abstract The success of precision oncology depends on our ability to translate accumulating genomic data into actionable treatment options in a personalized manner. The first step requires the identification of a specific genomic signature, then matching this signature with the most effective therapy. Unfortunately, more often than not, we do not have a specific cancer drug that is effective for the mutation found in a specific tumor. We desperately need to identify new targets so that we can develop new drugs to deliver on the promise of precision cancer medicine. In addition, we need to develop a rational way to combine drugs that does not depend on trial and error. Combinatorial genetic screening CRISPR-Cas9 now allows us to identify synthetic lethal interactions in which simultaneous perturbation of two genes leads to cell death. However, multiplex gene editing to reveal these synergies between genes is a major challenge. For model systems like yeast, high-throughput methods and technologies have made it possible to create genetic networks with around 23 million double mutants (6000 genes x 6000 genes) and has resulted in the most detailed genetic interaction network to date consisting of ∼900,000 genetic interactions. This landscape of genetic interaction network of yeast has taken the scientific research community 15 years. In human cells, there are ∼20,000 genes and 400 million combinations, making this very complex network impossible to screen and test all these 400M combinations. To systematically interrogate such genetic interactions, we designed a dual CRISPR-Cas9 perturbation library targeting the top 1000 genes upregulated in KRAS mutant cancers (lung, pancreas and colon cancers). We performed these combinatorial double knockout screens on 100K (100x1000) unique gene pairs and identified 27 pairs whose co-disruption results in a loss of cellular fitness. We next validated those top synthetic lethal pairs of genes by performing secondary screens using CRISPR-Cas12a on more KRAS mutant and KRAS WT cell lines. Overall, our findings will provide insight into potential combinational targets in KRAS mutant tumors and will highlight the synthetic lethality effects that occurs among the novel targets, and other proliferation, metastasis, immune and metabolism modules. Citation Format: Rand Arafeh, Laura Chang, Lydia Sawyer, Helen Wang, James McFarland, Joshua Dempster, Peter DeWeirdt, John Doench, William C. Hahn. Combinatorial genetic screens to map synthetic lethal interactions and identify new cancer drug targets in KRAS mutant cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR014.

Abstract A010: Proteasome inhibitors induce a BAX and BAK independent, non-canonical apoptosis

Abstract In the intrinsic pathway of apoptosis, death stimuli (including drugs, cellular stress, viral infections, etc.) trigger the oligomerization of BAX and BAK resulting in pore formation in the outer mitochondrial membrane to release cytochrome c from the intermembrane space. The released cytochrome c then activates APAF-1 and CASPASE9 to form a 1.4 MDa complex known as apoptosome, which recruits and activates downstream caspases that dismantle the cell. However, we have found that proteasome inhibition leads to a BAX and BAK independent apoptotic cell death. We show using immunofluorescence, fluorogenic assays, and flow cytometry that the proteasome inhibitors, bortezomib and MG-132, trigger a caspase dependent cell death that requires the apoptosome components APAF-1 and CASPASE9, but not cytochrome c. Furthermore, size exclusion chromatography revealed that unlike classical death stimuli, which induce the mega-dalton canonical apoptosome fomation, proteasome inhibitors induce a minor shift in APAF-1, forming a smaller, non-canonical apoptosome. Current efforts are focused on the identification of the APAF-1 activator. Our results indicate the existence of a non-canonical caspase-dependent cell death pathway, which could be a novel therapeutic target in malignancies by bypassing the BCL-2 family of apoptotic regulators. Citation Format: Tresor O Mukiza, Ricardo Enrique-Rodriguez, Amit Budhraja, Lauren Brakefield, Tudor Moldoveanu, Joseph T Opferman. Proteasome inhibitors induce a BAX and BAK independent, non-canonical apoptosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A010.

Abstract B002: POLB knockout is synthetic lethal with PARP inhibition leading to complete and durable responses in BRCA-mutant tumor xenografts

Abstract Despite the clinical benefit of PARP1/2 inhibitors (PARPi), which are FDA-approved for the treatment of certain BRCA-mutant cancers, many patients achieve incomplete disease control and develop progressive disease. Motivated by this clinical need, we utilized our CRISPR target discovery screening platform to identify novel targets that synergize with PARP inhibitor treatment. By conducting parallel screens in both BRCA-mutant and wildtype cells, we identified DNA polymerase beta (POLB) as a novel target that - when combined with PARPi - selectively kills BRCA-mutant lines while sparing normal cells. POLB knockout and cDNA rescue experiments using both BRCA1 and BRCA2-mutant isogenic cell lines further demonstrated that the catalytic activity of POLB is required for synthetic lethality with PARPi. Most strikingly, POLB knockout combined with sub-therapeutic doses of PARPi, led to profound tumor regression and prevented in vivo tumor regrowth, even after cessation of drug treatment. Mechanistically, POLB knockout is associated with increased single and double strand DNA breaks, accumulation of poly-ADP-ribose polymers, cell cycle arrest and apoptosis. Together, these results suggest that POLB inhibitors in combination with PARPi have the potential to drive deep and durable responses providing a novel therapeutic option for cancer patients with BRCA1/2-mutations. Citation Format: Madhavi Bandi. POLB knockout is synthetic lethal with PARP inhibition leading to complete and durable responses in BRCA-mutant tumor xenografts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B002.

Abstract IA022: Mechanisms of splicing dysregulation and dependency in cancer

Abstract Dysregulated RNA splicing is a hallmark feature of cancer. Much like mutational signatures in tumors, RNA mis-splicing patterns are widely heterogeneous and segregate tumors into clades, suggesting there are distinct mechanisms underlying these splicing aberrations. However, the mechanisms driving such distinct RNA splicing dysregulation remain largely unknown. Herein, we discovered that copy-number variation of essential splicing factors is a pervasive cause of splicing dysregulation, and in some tumor contexts, confers deep dependencies on RNA-binding proteins (RBPs) that converge on these mechanisms. Using a systematic chemical biology approach, we delineate how splicing RBPs participate in distinct types of RNA quality control in cells, and when disrupted, produce surprisingly unique patterns of RNA mis-splicing. These RBP-specific mis-splicing patters are common in distinct cancer types. Notably, unbiased genome-wide analysis revealed that RBP-specific mis-splicing patterns found in tumors were significantly associated with copy number loss of a network of functionally linked splicing factors. These splicing factors often reside on tumor suppressor loci that are frequently deleted in breast and other cancers, suggesting that copy number loss of essential splicing RBPs is a collateral event with selected loss of tumor suppressors. Importantly, RBP-specific mis-splicing signatures also segregate tumor models that are dependent on the concordant RBP, suggesting that these tumors evolve defects in RBP function that predispose them to further perturbation. Together, our work suggests that deletions of tumor suppressor loci may drive collateral dysregulation of RNA splicing through partial loss of splicing RBPs and provokes the hypothesis that these mis-splicing signatures may predict actionable dependencies in the cancers that harbor them. Citation Format: Kristen Karlin. Mechanisms of splicing dysregulation and dependency in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA022.

Abstract B020: Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells

Abstract Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Citation Format: Gabriele Picco, Yanhua Rao, Angham Al Saedi, Yang Lee, Shriram Bhosle, Carmen Herranz-Ors, Samantha Walker, Kieron May, Matthew Coelho, Jon Houseley, Benjamin Schwartz, Mathew J. Garnett. Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B020.

Abstract A021: Hunting for synthetic lethal partners in DNA damage response-altered endometrial cancer

Abstract DNA damage response (DDR) genes are frequently altered in cancer, which may lead to protein loss-of-function (LoF) and result in particular DDR defects that can be therapeutically targeted via the concept of synthetic lethality (SL). There is a high prevalence of DDR alterations in endometrial cancer, a gynecologic malignancy with increased incidence and mortality in recent decades, and an unmet clinical need for a wider range of treatment options to reduce adverse effects and combat therapy resistance. In this study, a large-scale, agnostic bioinformatic screening approach was employed to discover novel potential SL targets specific to DDR LoF biomarkers in endometrial cancer for subsequent experimental validation in vitro. SL analysis was performed with the in-house Target / Biomarker / Lineage (TBL) bioinformatic pipeline (Division of Cancer Therapeutics, The Institute of Cancer Research) using datasets obtained from the Cancer Dependency Map (DepMap). The TBL pipeline statistically analyzed cancer cell line target gene dependency probabilities, inferred from Project Achilles and Project Score genome-wide CRISPR-Cas9 dropout screens, and integrated this with mutation and copy number variation genomic datasets. Potential interactions were filtered for target-biomarker mutual exclusivity using patient data from The Cancer Genome Atlas (TCGA). The ability of the TBL pipeline to identify known SL interactions was demonstrated through in silico identification of ARID1B/ARID1A in ovarian cancer and SMARCA2/SMARCA4 in lung cancer as significant hits. In addition, the identification of WRN/ARID1A as a significant hit in uterine cancer, given that ARID1A loss has been significantly associated with microsatellite instability (MSI) in endometrial cancer, is consistent with previous findings of WRN helicase as a SL partner with MSI in endometrial cancer. Analysis and downstream refinement of endometrial cancer significant hits led to the identification of seven final candidate target-biomarker gene pairs. These putative SL partners comprise novel potential endometrial cancer targets, with various functions ranging from nucleotide biosynthesis to epigenetic remodeling, and include novel potential and established LoF biomarkers relevant to endometrial cancer biology, namely DNA mismatch repair. In vitro validation of the final candidates by genetic or pharmacologic approaches to assess target gene dependency in endometrial cancer cell lines with altered or wildtype biomarker status will be discussed. These experiments will provide key experimental data to validate our bioinformatic approach to SL target-biomarker discovery, and may identify novel SL partners involving DDR LoF alterations in endometrial cancer that could lead to the development of new molecular targeted therapies and improved treatment options for this patient subpopulation. Citation Format: Julie Zhou, Konstantinos Mitsopoulos, Olivia Rossanese. Hunting for synthetic lethal partners in DNA damage response-altered endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A021.