Smith, Hannah

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 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.