Wang, Xiyin

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 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.

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.

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.