Citations

Abstract Background: Endocrine therapies remain central to estrogen receptor alpha-positive (ERα+) breast cancer treatment, yet their predominantly cytostatic effects highlight the need for combinatorial approaches that promote tumor cell death. We therefore tested BH3 mimetics as single agents and in combination with (Z)-endoxifen (ENDX) across endocrine sensitive and endocrine resistant ERα+ breast cancer models. Methods: Six BH3 mimetics were tested in MCF7 and T47D cells and matched ENDX-resistant and fulvestrant-resistant derivatives. ENDX combinations were assessed using 2D proliferation and synergy assays, 3D RASTRUM® bioprinted cultures, migration assays, and in vivo MCF7 xenograft studies. Results: BH3 mimetics showed broad anti-proliferative activity as single agents in endocrine sensitive and resistant models. ENDX combinations were predominantly additive in 2D assays, with limited synergy confined to specific dose windows. In 3D culture, selected combinations of an MCL1 inhibitor (MCL1i) or navitoclax with ENDX produced greater suppression than single agents and, in several conditions, reduced growth below baseline, consistent with loss of viability. Combination treatment also enhanced suppression of migration under select concentrations. In MCF7 xenograft studies, ENDX robustly suppressed tumor growth; however, adding MCL1i or navitoclax did not provide additional antitumor benefit at the doses, schedules, and timeframes tested. Conclusion: BH3 mimetics show activity in ERα+ breast cancer models in vitro , including endocrine resistant models. Although ENDX combination benefit did not translate in vivo under the conditions tested, these data support further study of optimized BH3 mimetic combinations, including evaluation of dose, ratio, schedule, tumor target engagement, and other clinically advanced agents.

Abstract Breast and ovarian cancers are major causes of cancer mortality in women with frequent relapse after curative intent treatment, underscoring the need for novel therapeutics. To this end, we identified CTPS1 as an essential gene in multiple breast and ovarian cancer models, including those resistant to chemotherapy and PARP inhibitors. CTPS1 and CTPS2 are responsible for de novo synthesis of cytidine triphosphate (CTP) which is required for DNA/RNA polymerization, phospholipid synthesis and protein glycosylation. We found CTPS1 to be the predominant isoform expressed in cancer cells, with significantly higher levels in advanced and resistant disease models compared to normal tissue, benign lesions and treatment naïve cancers. Using CTPS1 siRNAs and CTPS1-dTAG (degradation tag) cell lines, we discovered that depletion of CTPS1 induces S-phase cell cycle arrest followed by apoptosis. Leveraging a first-in-class, highly selective, and orally bioavailable CTPS1 inhibitor (STP938) developed by Step Pharma, we identified nanomolar IC50 values across a panel of cell lines, both in 2D and 3D culture systems, and potent anti-neoplastic activity ex vivo and in vivo using patient-derived xenograft (PDX) models. STP938 also synergized with standard-of-care chemotherapy agents and PARP inhibitors, including in therapy-resistant models. Given the essentiality of CTP for biosynthetic processes, we performed bulk and single cell RNA-seq, total and phospho-proteomics, metabolomics, and lipidomic strategies to examine changes induced by acute and chronic STP938 exposure. This revealed that cancer cells are able to overcome pharmacologic CTPS1 inhibition by rewiring of pyrimidine and purine synthesis pathways, upregulation of the pentose phosphate pathway, and extensive remodeling of lipid synthesis pathways including shifts in PC/PE content, increased lysophospholipid production, and altered mitochondrial lipid composition (decreased cardiolipin and increased PG). To identify genetic drivers of STP938 resistance and synthetic lethal vulnerabilities, we performed a genome-wide CRISPR knockout screen together with a PRISM drug sensitivity screen. Genes regulating dNTP biosynthesis, S-phase progression, and RNA processing were identified as primary escape mechanisms, while DHODH inhibitors, purine and nucleoside analogs, and mitosis modifiers were implicated as synthetic lethal opportunities. Notably, we discovered that 25% of ovarian tumors lack CTPS2 protein expression, suggesting near complete reliance on CTPS1 for survival. Thus, a Phase 1a/b clinical trial of STP938 (NCT06297525) has been initiated, including an expansion cohort for patients with CTPS2-null ovarian tumors. These laboratory discoveries and ongoing clinical efforts aim to enable broader uptake of this novel therapeutic approach to improve long-term outcomes in patients with CTPS1-dependent disease. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Epp, Esther Rodman, Noa Odell, Hannah Smith, Nicole Pearson, Xiaonan Hou, Matthew Goetz, Scott Kaufmann, S. John Weroha, Phillip Beer, John Hawse. CTPS1 as a metabolic vulnerability and novel therapeutic target in breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4560.

Abstract Despite therapeutic advances, ovarian cancer (OC) recurrence remains common and outcomes for advanced disease are poor, highlighting the need for novel therapeutic targets. To this end, we identified CTPS1 as a highly expressed and essential gene for the survival of many OC cell lines, including models resistant to chemotherapy and PARP inhibitors. CTPS1 is one of two enzymes, alongside CTPS2, responsible for the de novo synthesis of cytidine triphosphate (CTP), a critical nucleotide required for DNA and phospholipid biosynthesis. We discovered that CTPS1 is the predominant isoform expressed in OC cells, with significantly higher levels of expression in advanced and resistant disease compared to normal tissue, benign lesions and treatment naïve cancers. Intriguingly, 25% of all OC samples were found to completely lack CTPS2 protein expression suggesting their near complete reliance on CTPS1 for survival. Using CTPS1 siRNAs and genetically engineered OC cells with a degradation tag (dTAG) fused to the C-terminus of endogenous CTPS1 alleles, we showed that CTPS1 knockdown or proteasomal degradation induces an S-phase cell cycle arrest followed by apoptosis. Leveraging a first-in-class, highly selective, and orally bioavailable CTPS1 inhibitor (STP938) developed by Step Pharma, we identified nanomolar-range IC50 values across a panel of over 20 OC cell lines and validated its anti-neoplastic activity in ex vivo patient-derived xenograft (PDX) models. STP938 synergized with standard-of-care chemotherapy and PARP inhibitors, even in resistant models, further supporting its clinical potential. To elucidate STP938’s mechanism of action, we performed multi-omic profiling (bulk RNAseq, phospho- and total proteomics) on sensitive and isogenic resistant OC lines, as well as single-cell RNAseq of an OC PDX tumor following in vivo treatment with vehicle or STP938, revealed dysregulation of DNA replication, cell cycle, and metabolic pathways. As expected, STP938 rapidly depleted CTP levels, which remain suppressed for 72 hours, suggesting insufficient salvage pathway compensation which ultimately compromised cancer cell viability. As CTP is also a direct precursor for activated intermediates in phospholipid biosynthesis. Mass-spectrometry based lipidomics and metabolomics were employed to interrogate the impact of STP938 in these processes where numerous changes were found. To uncover mechanisms of resistance to STP938 and reveal therapeutic vulnerabilities, we conducted genome-wide CRISPR knockout screens in multiple treatment naïve and STP938 resistant models and analysis of results are ongoing. In collaboration with Step Pharma, a Phase 1a/b clinical trial of STP938 is currently enrolling patients with advanced solid tumors, including an expansion cohort for CTPS2 null OC patients (NCT06297525). These laboratory discoveries and ongoing clinical efforts aim to enable broader uptake of this novel therapeutic approach to improve long-term outcomes in patients with CTPS1-dependent disease. Citation Format: Xiyin Wang, Michael J. Emch, Lauren A. Voll, Rebecca Epp, Esther P. Rodman, Noa Odell, Nicole A. Pearson, Xiaonan Hou, Matthew P. Goetz, Scott H. Kaufmann, S. John Weroha, Philip Beer, John R. Hawse. Exploiting CTPS1 dependency for the treatment of ovarian cancerPR005: Exploiting CTPS1 dependency for the treatment of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr PR005.

Background: Targeted therapies, such as endocrine agents, have significantly improved outcomes for patients with estrogen receptor alpha-positive (ERα+) breast cancer. Unfortunately, for patients with triple-negative breast cancer (TNBC), which lack expression of ERα and HER2, there remains a dearth of targeted adjuvant agents. We discovered that estrogen receptor beta (ERβ) is expressed in approximately 20% of TNBC cases, and its activation has been shown to inhibit proliferation, invasion, and migration in preclinical models. However, it remains unclear whether ERβ-targeted therapies maintain efficacy following the development of chemoresistance. Methods: To address this question, we generated ERβ+ TNBC cell line models with acquired resistance to paclitaxel or doxorubicin. We then assessed their response to ERβ-targeted therapies and analyzed transcriptomic changes associated with chemoresistance and ERβ ligand treatment. Results: Chemotherapy-resistant ERβ+ TNBC cells retained sensitivity to ERβ-targeted therapies and, in some cases, exhibited enhanced responsiveness. ERβ expression did not compromise chemotherapy efficacy in treatment-naïve cells. Chemotherapy-resistant cells had a vastly altered transcriptome and surprisingly, a heavily reduced ERβ transcriptome, compared to sensitive cells despite the maintenance of ERβ-driven anti-neoplastic activity. Conclusions: These findings suggest that ERβ remains a relevant drug target in chemotherapy-refractory disease and has aided in the refinement of a minimal ERβ transcriptomic signature associated with response to ERβ-targeting agents, further informing the primary mechanisms through which ERβ elicits its tumor suppressive effects.

Abstract Estrogen receptor alpha (ERα)-positive breast cancer accounts for up to 80% of cases. Despite advances in CDK4/6 inhibitors (CDK4/6i) and endocrine therapies, recurrence and late relapse remain significant challenges. Recurrence often arises from disseminated cancer cells that persist despite 5-10 years of systemic adjuvant treatment. While CDK4/6i and endocrine therapies induce prolonged growth arrest resembling senescence, they do not significantly kill cancer cells. Senescent/pseudo-senescent cancer cells are implicated in aggressive variant formation, recurrence, and metastasis. We explored eliminating breast cancer cells via senolysis while they are in therapy-induced senescent state to prevent their potential to escape, repopulate, or cause long-term adverse effects. Live-cell imaging of MCF7 and T47D cells treated with abemaciclib and endocrine therapies (endoxifen or estrogen deprivation to mimic aromatase inhibition) at clinically relevant concentrations for eight days demonstrated prolonged growth cessation and G1 cell cycle arrest even two weeks post-drug withdrawal. These cells displayed a senescence-like gene expression profile, with increased CDKN2B (p15), CDKN1A (p21), BCL2, reduced KI67, and increased TGFβ expression, a known Senescence-Associated Secretory Phenotype (SASP) factor linked to deep senescence. These results confirm effective senescence induction by various CDK4/6i and endocrine therapy combinations. Further analysis using established senescence markers (e.g., nuclear size, telomere-associated foci, satellite distension, and loss of nuclear HMGB1 and LaminB1) revealed unique senescence phenotypes across therapy combinations. Cell viability assays and live imaging of Caspase 3/7 activation demonstrated differential vulnerabilities of senescent/senescent-like ERα+ cancer cells (MCF7, T47D, UCD4, UCD12) to 13 senolytics (1 μM), including natural flavonoids and inhibitors targeting pro-survival factors, tyrosine kinases, and the Notch pathway. For example, MCF7 and UCD4 cells responded to MCL1i and RSL3, T47D to BCLXLi and RSL3, and UCD12 to BCLXLi and Navitoclax. Notably, senolytic cocktails (e.g., RSL3, GNF7, Venetoclax, Dasatinib) at reduced concentrations (0.25 μM each) effectively eliminated senescent/senescent-like ERα+ cancer cells across all cancer cell lines, achieving 100% elimination in T47D cells. Collectively, these findings demonstrate that despite variability in therapy-induced senescence, senolytic combinations effectively target senescent ERα+ cancer cells. This provides preliminary evidence for the potential integration of this strategy into standard breast cancer therapy with a goal to improve recurrence-free survival and increase the cure rates for ERα+ breast cancer patients. In vivo studies are underway to evaluate this strategy in preclinical models. Citation Format: Lena Batoon, Angelo J. Guilatco, Anna Detry, Xiyin Wang, Megan M. Weivoda, John R. Hawse. CDK4/6 inhibitors and endocrine therapies induce senescence with drug- and cell line-specific vulnerabilities in ERα+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 519.

Breast and ovarian cancer represent the most common and lethal cancers in women. Despite chemotherapy and targeted agents, recurrence is frequent and outcomes for patients with advanced disease are dismal, underscoring the need for novel therapeutic strategies. Using an unbiased approach with DepMap CRISPR screens across more than 1000 cancer cell lines, we identified six genes (CTPS1, RHOA, PRKRA, RAD9A, HUS1, and RAD1) that were essential for triple negative breast cancer (TNBC) cells, but not ERα+ breast cancer cells, that were also associated with poor outcomes. Validation of these effects by siRNA knockdown studies revealed that suppression of CTPS1 elicited the most potent inhibitory effects, characterized by S phase cell cycle arrest and apoptosis. Similar effects were also found in ovarian cancer cells which share many molecular features. CTPS1, along with CTPS2, is responsible for enzymatic conversion of UTP to CTP, an essential nucleoside triphosphate required for multiple cellular processes including DNA/RNA synthesis, phospholipid synthesis and glycosylation. CTPS1 was found to be highly expressed in tumor tissues, for both breast and ovarian cancer. RT-PCR assessment of CTPS1 expression in a panel of over 40 breast and ovarian cancer lines confirmed variable, but elevated expression relative to normal models. Notably, chemotherapy-resistant and PARP inhibitor (PARPi)-resistant models exhibited the highest levels of CTPS1 expression among all cells analyzed. In partnership with Step Pharma, which has developed a first-in-class highly selective CTPS1 inhibitor (STP938), we have demonstrated that STP938 elicits potent anti-proliferative effects at nM concentrations across many cell line and patient derived models, both in 2D and 3D culture systems. Furthermore, STP938 exhibited strong anti-cancer activity in chemotherapy- and PARPi-resistant ovarian cancer models, both as a monotherapy and in combination with standard-of-care treatments. RNA-seq analysis in cell models following STP938 treatment or CTPS1 knockdown/knockout showed significant dysregulation of DNA replication and cell cycle related genes, suggesting potential synergy with drugs targeting replication stress or DNA damage response pathways. Single-cell sequencing further revealed specific cell-cell interactions/cross talk/pathways that are disrupted by STP938 in vivo using ovarian cancer patient-derived xenograft. The first-in-human clinical trial for STP938 began in 2022 for relapsed/refractory B- and T-cell lymphomas (NCT05463263). In August 2024, a second phase I trial opened for advanced solid tumors, including an expansion cohort for CTPS2-null ovarian cancer (NCT06297525) at Mayo Clinic. These laboratory discoveries, and ongoing clinical studies, are expected to pave the way for a broader uptake of this novel therapeutic approach aimed at improving outcomes in patients with highly aggressive forms of these diseases. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Epp, Esther Rodman, Noa Odell, Nicole Pearson, Xiaonan Hou, Matthew Goetz, Scott Kaufmann, John Weroha, Philip Beer, John Hawse. Exploiting CTPS1 dependency for the treatment of breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6388.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, primarily due to its complex tumor microenvironment (TME), which drives both disease progression and therapy resistance. Understanding the molecular mechanisms governing TME dynamics is essential for developing new treatment strategies for this devastating disease. In this study, we uncover an oncogenic role for Galectin-1 (Gal1), a glycan-binding protein abundantly expressed by activated pancreatic stellate cells (PSCs), a key component of the PDAC TME that orchestrates tumor progression. Our findings reveal that Gal1 expression is elevated in the nucleus of human PSCs in both tissue samples and cultured cell lines. Using chromatin immunoprecipitation followed by sequencing analysis (ChIP-seq), we identify Gal1 occupancy at the promoters of several cancer-associated genes, including KRAS , a pivotal oncogene involved in PDAC pathogenesis. We demonstrate that Gal1 binds to the KRAS promoter, sustaining KRAS expression in PSCs, which, in turn, maintains PSC activation and promotes the secretion of protumorigenic cytokines. Mechanistically, Gal1 is required to preserve histone H3 lysine 4 monomethylation levels and to recruit the histone methyltransferase MLL1 to target promoters. Collectively, our findings define a nuclear function of Gal1 in modulating the transcriptional landscape of cancer-associated genes in PSCs within the PDAC TME, mediated through an epigenetic mechanism. These insights enhance our understanding of PDAC pathology and open potential avenues for therapeutic interventions targeting intracellular Gal1.

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide. Triple negative breast cancer (TNBC) is an aggressive subtype that is defined by the lack of expression of estrogen and progesterone receptors in the absence of HER2 amplification or overexpression. TNBC represents 15-20% of all breast cancers and occurs more frequently in young premenopausal women. TNBC often ends with a poor clinical outcome due to high histological grade, rapid recurrence in many patients combined with few treatment options. These realities highlight the urgent need to identify novel therapeutics vulnerabilities that can be leveraged to improve TNBC patient outcomes. Methods: We utilized the publicly available DepMap and Kaplan Meier datasets to identify essential genes that were unique to TNBC cells and whose expression levels were associated with worse patient outcomes. siRNA based approaches were used to confirm the relevance of top hits in mediating cancer cell phenotypes including viability, cell-cycle profiles and induction of apoptosis among others. A novel drug currently being developed was identified for the top hit in these studies and was subsequently employed in multiple in vitro approaches to characterize the mechanisms of action in TNBC as well as initial efficacy studies using clinically relevant ex vivo patient derived models. Results: Using the DepMap database, we interrogated genome-wide sgRNA knockout screens conducted in over 40 breast cancer cell lines to identify specific genes whose expression was required for cell viability in TNBC cells, but not other breast cancer cell lines. This endeavor identified 6 genes (CTPS1, HUS1, PRKRA, RAD1, RAD9A and RHOA) that were preferentially essential in TNBC cells. Further profiling of these genes revealed that CTPS1 expression is higher in cancer relative to normal tissue and that its expression is further upregulated in chemotherapy- and PARPi-resistant models. Knockdown of CTPS1 in TNBC cells was shown to decrease cell proliferation and viability with no effects observed in estrogen receptor+ cell lines. Using a first-in-class, highly selective and orally bioavailable CTPS1 inhibitor (STP938) developed by Step-Pharma, we identified IC50s in the low nM range for many TNBC cell lines, including cell models that are resistant to existing standard-of-care therapies. Inhibition of CTPS1 was shown to arrest cells in S phase. Conclusions: Our studies have identified CTPS1 as an essential gene in TNBC. CTPS1 encodes an enzyme responsible for the catalytic conversion of UTP (uridine triphosphate) to CTP (cytidine triphosphate), an essential step in the biosynthesis of nucleic acids. These findings are timely in light of the recent and ongoing development of the first-in-class CTPS1 inhibitor, STP938, for which first-in-human studies are underway for refractory B- and T-cell lymphomas. Based on our initial studies using this drug, we provide pre-clinical evidence of its efficacy in multiple models of primary and advanced TNBC. Efforts are currently underway to move this work toward a clinical trial for TNBC patients. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Russell, Esther Rodman, Philip Beer, John Hawse. Identification of novel genetic/therapeutic vulnerabilities in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-08.

Abstract Breast and ovarian cancer represent the most common cancer types in women worldwide and are among the top fatal cancers. Despite the use of cytotoxic chemotherapy and targeted agents, recurrence is frequent and survival rates for patients with advanced metastatic disease are dismal. These realities highlight the need to identify novel biomarkers and therapeutic vulnerabilities in such tumors and to develop alternative treatment strategies. We took an unbiased approach to tackle this problem in which we leveraged publically available (DepMap) and in-house generated genome-wide CRISPR screens conducted in over 1000 cancer cell lines. We initially focused on the identification of essential genes that were unique to triple-negative breast cancer (TNBC) cells and whose expression levels were associated with worse patient outcomes. This interrogation revealed six genes (CTPS1, RHOA, PRKRA, RAD9A, HUS1, and RAD1) meeting these initial criteria. The essentiality of these genes was validated using gene-specific siRNAs in a panel of TNBC cells and knockdown of CTPS1 was shown to elicit the most inhibitory effect. CTPS1 depletion resulted in a rapid S-phase cell cycle arrest followed by apoptosis, not only in TNBC cells but also in highly aggressive ovarian cancer cells which share many molecular features with TNBC. CTPS1 converts uridine triphosphate to cytidine triphosphate, forming an essential nucleic acid required for multiple cellular processes. CTPS1 mRNA expression was substantially elevated in tumor vs. normal tissue, for both breast and ovarian cancer. Using over 40 breast and ovarian cancer cell lines, RT-PCR analyses confirmed increased expression of CTPS1 in TNBC and most ovarian cancer cell lines compared to other disease sub-types and “normal” cell line/tissue controls. Interestingly, chemoresistant and PARP inhibitor-resistant models exhibited the highest levels of CTPS1 among all cells analyzed. We subsequently identified and acquired STP938, a first-in-class highly selective CTPS1 inhibitor being developed by Step-Pharma. STP938 was found to elicit potent anti-cancer effects at nM concentrations across many cell line and patient-derived models, in both 2D and 3D culture systems. RNAseq analysis in aggressive cell line models following STP938 treatment or CTPS1 knockdown revealed significant regulation of genes related to DNA replication and the cell cycle pathways suggesting that combinatorial/sequential treatments of STP938 with replication stress/DNA damage response related drugs may be synergistic. STP938 is currently being developed for the treatment of lymphoma, and our findings support the repurposing of STP938 for breast and ovarian cancer, an avenue that we are currently developing. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Russell, Esther Rodman, Nicole Pearson, Xiaonan Hou, Scott Kaufmann, Saravut Weroha, Philip Beer, John Hawse. CTPS1: An unexplored vulnerability in breast and 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 1878.

Abstract Ovarian cancer is the deadliest gynecological cancer with a low 5-year overall survival rate that affects over 300,000 women globally each year. Given lack of routine screening procedures, ovarian cancer patients are frequently diagnosed with late-stage disease that rapidly progress despite initial responses to front line therapy. PARPi and VEGFi targeted therapies have improved progression free survival in small subsets of patients but have largely failed to improve overall survival. Since the robust uptake of PARPis in the front line setting, resistance to this class of drugs is a growing problem. The literature is filled with many novel treatment approaches, but the vast majority fail to gain traction in pre-clinical models and early phase trials. A contributing factor to these disappointments relates to the prevalent use of 2D monoculture model systems that do not recapitulate the tumor microenvironment. To address this issue, we utilized the Rastrum 3D cell printer to grow ovarian cancer cells and PDX models in bio-functional matrices which provide optimal stiffness and extracellular matrix components specific to the ovarian tumor microenvironment. We found that ovarian cancer cells grown under these conditions exhibit dramatic up-regulation of the JAK/STAT signaling pathway, including autocrine/paracrine signaling factors, which correlated with increased sensitivity to JAK/STAT inhibitors. This affect was lost when STAT1 and STAT3 knockout cell lines were grown in this 3D environment. Ovarian tumors are known to be rich in cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). We incorporated primary CAFs into this 3D culture system and found they could promote ovarian cancer cell growth only in cancer cells with intact JAK/STAT signaling further indicating the essential role of this pathway in mediating tumor progression. Co-culture with M2 macrophages was also found to induce ovarian cancer cell growth, particularly in resistant models. Conversely, co-culture with M1 macrophages inhibited treatment naïve ovarian cancer cell growth but not resistant cell line growth. Single-cell RNAseq of the complex 3D cultures elucidated profound differences in the cross-talk between these cell types that is dependent on cancer cell sensitivity to standard-of-care agents. Finally, the addition of JAK/STAT inhibitors was found to substantially alter this cross-talk between cell types and inhibit cancer cell viability. These data further implicate the importance of JAK/STAT signaling, specifically in clinically relevant 3D models, in supporting ovarian cancer progression and response to chemotherapy and PARPi treatment. Ongoing studies are aimed at confirming these findings in pre-clinical mouse models and to elucidate the most effective JAK/STAT inhibitors in vivo with the goal of informing the design of future clinical trials. Citation Format: Esther Rodman, Xiyin Wang, Hannah Smith, Xiaonan Hou, Scott Kaufmann, John Weroha, John Hawse. 3D models of chemotherapy- and PARPi-resistant ovarian cancer indicate essential roles for JAK/STAT signaling in mediating drug resistance [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 1224.