Hawse, John R.

Abstract Breast cancer (BC) is the most common cancer diagnosed in American women. The majority of breast cancers express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). However, 15-20% lack expression of all three and are called triple-negative breast cancer (TNBC). TNBC accounts for a disproportionately large amount of morbidity and mortality due to its aggressive nature and the paucity of effective adjuvant maintenance therapy, driving an urgent need for novel therapeutics. Z-endoxifen (ENDX) is a potent tamoxifen metabolite that dually targets ERα and protein kinase C beta I (PKCβI). We recently described next-generation molecules, the first-in-class endoxifen-PROTAC (ENDX-PROTAC) compounds which incorporate ENDX as the warhead and cereblon (CRBN) as the E3 ubiquitin ligase recruitment moiety (Jayaraman 2024, SABCS). We used the PRISM screen of over 900 cancer cell lines to determine that ENDX-PROTACs have broad antitumor activity in both TNBC and ER+ breast cancer in addition to, leukemia, pancreatic, lymphoma, lung, melanoma, and ovarian cancer cells, among others. ENDX-PROTACs primarily target a key translation termination factor, G to S phase transition 1 (GSPT1). Here, we demonstrate for the first time that these compounds also cause marked DNA damage. The lead compounds of this series, EPrC-007 and EPrC-009 demonstrated potent anti-proliferative effects in MDA-MB-231, BT549, and MDA-MB-436 TNBC cell lines. EPrC-007 induced substantial DNA double strand breaks in MDA-MB-231 cells as assessed by γ-H2AX Western blotting. Interrogation of DNA repair pathways revealed activation of ATM/CHK2 and downregulation of ATR/CHK1, suggesting inhibition of DNA repair as a potential mechanism. Additionally, EPrC-007 also induced γ-H2AX foci in MDA-MB-231 and BT549 cells, but not in the MCF10A normal breast tissue cell line, suggesting selective anti-tumor activity. Since we suspect that EPrC-007 blocks DNA repair machinery, we next sought to determine if these molecules would synergize with DNA damaging agents or other DNA repair inhibitors. Promisingly, EPrC-007 demonstrated remarkable synergy with olaparib (poly-(ADP-ribose) polymerase (PARP) inhibitor), hydroxyurea (ribonucleotide reductase inhibitor), and cisplatin (direct DNA damaging agent), but not camptothecin (topoisomerase I inhibitor). Importantly, only olaparib co-treatment demonstrated synergy in the PARP- and PROTAC-resistant HCC1143 TNBC cell line. Furthermore, the combination of EPrC-007 with olaparib induced markedly more γ-H2AX foci than either agent alone, confirming synergy at the DNA damage level. Taken together, our studies demonstrate the remarkable potential of ENDX-PROTACs in the single-agent setting as well as in combination with PARP inhibitors. Citation Format: Aamod S. Dekhne, Swaathi Jayaraman, Lucy Rai Thulung, Becky Bruinsma, Baustin M. Welch, Samuel Wyatt, Esther P. Rodman, Sayantani Bhattacharya, Thomas R. Caulfield, John R. Hawse, Matthew J. Schellenberg, Matthew P. Goetz. Novel endoxifen-based proteolysis targeting chimera (PROTAC) small molecules synergize with olaparib and exhibit potent antitumor activity in triple negative breast cancer through induction of DNA damage [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 1666.

Abstarct Introduction: (Z)-endoxifen (ENDX) is a potent orally bioavailable selective estrogen receptor modulator (SERM) and the most active metabolite of tamoxifen. Given its effective anti-estrogen activity and favorable safety profile, ENDX is being developed as a therapeutic solution in breast health. During its synthesis, two chemical biproducts (compound AT416 and AT402) with Z- and E- isomeric forms are produced. This study aimed to assess the anti-estrogenic activity of these four ENDX-related compounds with ENDX in ERα+ breast cancer. Methods: Compounds AT416E/Z are formed by acetylation of the final API with ethyl acetate during Step 3 of the manufacturing process of ENDX. Compounds AT402 E/Z are formed by coupling of impurity AT300 with propiophenone as a side reaction during Step 2 of the ENDX manufacturing process. The anti-cancer activities of the four ENDX-related compounds were tested across multiple ERα+ breast cancer cell lines (MCF7, T47D, UCD12), including those with ESR1 mutations (Y537S and D538G) known to confer endocrine resistance. Studies included cell proliferation, migration and invasion assays, cell cycle progression assessments, induction of apoptosis, activity of an estrogen response element (ERE) luciferase reporter construct and RT-qPCR analyses of ERα target gene expression. Results: In estrogen-containing media, ENDX exhibited strong anti-proliferative activity in MCF7 cells, regardless of ESR1 mutations. In MCF7 cells cultured in estrogen-deficient media, the four compounds demonstrated more pronounced anti-proliferative effects than ENDX. In T47D cells with the ESR1 Y537S mutation, AT416E showed superior anti-proliferative activity compared to ENDX, while in T47D cells with the D538G mutation, these two compounds exhibited comparable effects. In UCD12 cells, ENDX displayed the strongest anti-proliferative effects. Regarding the inhibition of migration and invasion, AT416E consistently outperformed ENDX in both T47D and MCF7 cells. ENDX was the most effective at inducing G1 phase cell cycle arrest followed. Both AT402E and AT402Z elicited pro-apoptotic effects comparable to ENDX in MCF7 cells, while in T47D cells, ENDX was the most effective at inducing apoptosis, followed by AT402E and AT402Z. In T47D and MCF7 cells, luciferase-based transactivation assays showed that ENDX was the most potent anti-estrogen followed by AT402Z. Finally, based on RT-qPCR analyses, ENDX was a more effective inhibitor of estrogen-induced ERα target gene expression, compared to the biproducts. Summary: These studies have revealed that four novel compounds derived from the manufacturing process of ENDX confer notable anti-tumor activity, with some performing comparably to or better than ENDX in several cancer-relevant cellular readouts. Future studies will validate these findings in additional in vitro/in vivo breast cancer models with the goal of identifying the basis for their varied anti-estrogenic and anti-cancer effects. Such discoveries should allow for the rational design of novel molecules with more potent therapeutic efficacy. Citation Format: Natalie Farris, Lena Batoon, Rajeev S. Muthyala, H. Lawrence Remmel, Sandra S. Hammer, John R. Hawse, Steven C. Quay. Anti-cancer activity of (Z)-endoxifen-related compounds in ERα+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A007.

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 Introduction: Eicosanoid mediated mechanisms of hormone therapy resistance in ER+HER2- breast cancer are poorly understood. Cytochrome P450 arachidonic acid (AA) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to breast cancer progression by promoting mitochondrial oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to hormonal therapy resistance. In this study, the well-known letrozole resistant (LR) MCF-7 AC1 cell line overexpressing aromatase was selected for fulvestrant resistance (FR). A clonal cell line resistant to letrozole and fulvestrant (LR/FR) also exhibited resistance to palbociclib (PR) (MCF-7 AC1 LR/FR/PR). Xenograft tumors of this cell line were resistant to letrozole, fulvestrant, and palbociclib. Structure activity studies and modeling led to the development of CYP3A4 AA epoxygenase inhibiting biguanides hexyl cuban-1-yl biguanide (HCB) and fluorinated hexyl derivatives C5F2-HCB and C6F3-HCB. Fluorination on the hexyl moiety led to broader inhibition of EET regioisomer biosynthesis. These agents were tested for inhibition of proliferation, signaling, and OXPHOS/glycolysis balance in the MCF-7 AC1 LR/FR/PR cell line. Results: In the MCF-7 AC1 LR/FR/PR clonal cell line cyclin D1 and estrogen receptor (ER) expression were undetectable, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated 11, 40, 13, 10, and 12- fold (n=3, p<0.001) compared to the MCF-7 cell line. LC-MS analysis demonstrated 1.8, 1.4, and 1.2- fold higher total cellular levels of (±)8,9, (±)11,12, and (±)14,15-EET in the MCF-7 AC1 LR/FR/PR cell line compared to the MCF-7 cell line (n=3, p<0.05). C6F3-HCB suppressed cellular levels of EETs: (±)8,9-EET by 55% (n=3, p<0.031), (±)11,12-EET by 53% (n=3, p<0.029), and (±)14,15-EET by 60% (n=3, P<0.0001). The MCF-7 AC1 LR/FR/PR cell line was more potently inhibited by hexyl-cuban-1-yl biguanides compared to the MCF-7 cell line, exhibiting lower IC50 values (MCF-7 AC1 LR/FR/PR IC50 vs. MCF-7 IC50; HCB 4.1 vs. 7.0 uM; for C5F2-HCB 10 vs. 25 uM; C6F3-HCB: 5.4 vs. 12 uM; P values all <0.05). Measurement of spare respiratory capacity revealed that the MCF-7 AC1 LR/FR/PR cell line had 4.2% spare respiratory capacity compared to 34% for MCF-7, indicating a lower OXPHOS reserve for the resistant cells. Additionally, the MCF-7 AC1 LR/FR/PR cell line displayed a 2.4-fold higher extracellular acidification rate (ECAR) than the MCF-7 cell line, indicating a higher glycolysis rate (Warburg effect) in the resistant cells. Conclusion: Selection for fulvestrant resistance of MCF-7 AC1 cells resulted in upregulation of CYP3A4 and biosynthesis of hormone therapy resistance associated EETs while correlating with greater sensitivity to fluorinated hexyl-cuban-1-yl biguanides that inhibit EET biosynthesis. Citation Format: Zhijun Guo, Jianxun Lei, Allison Makovec, Swaathi Jayaraman, John R. Hawse, Carol Lange, Elizabeth Ambrose, Gunda I. Georg, Tony D'Assoro, Goetz P. Matthew, David A. Potter. Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB334.

Abstract Background: Ovarian function suppression (OFS) combined with tamoxifen (TAM) or an aromatase inhibitor (AI) is standard for premenopausal (PM) ER+, HER2- breast cancer (BC). However, > 40% PM patients (pts) are intolerant of OFS. For these pts, TAM is the only FDA approved option. In the neoadjuvant endocrine setting, pts with endocrine sensitive disease (ESD) (Ki-67 ≤ 10% at 4 weeks) have 5yr dDFS > 97%; however, only 45% of TAM pts vs >75% with AI+OFS achieve 4 wk ESD (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent anti-estrogen superior to TAM and AI in xenograft models and with antitumor activity in endocrine-resistant postmenopausal pts. ENDX additionally targets protein kinase C beta 1 (PKCβ1) at concentrations >500 ng/mL, resulting in AKT inhibition and apoptosis (Jayaraman npj Breast Cancer 2023). We hypothesize that ENDX dual targeting of ERα and PKCβ1 will obviate the need for OFS and be non-inferior to AI plus OFS for PM pts with ER+/HER2- BC. Methods: EVANGELINE (NCT05607004) is an ongoing phase 2 multicenter neoadjuvant study with pharmacokinetic (PK) run-in assessing ENDX in PM women with ER+/HER2- BC. The primary objective for the PK run-in is to identify a dose (40 or 80 mg/day) resulting in ENDX steady state concentrations (Css) of 500-1000 ng/mL (to target both ERα and PKCβ1) without significant toxicity. Following PK run-in, the randomized phase II goal is to assess whether the ESD rate with ENDX is non-inferior to exemestane plus goserelin. Women with ESD at wk4 continue treatment for 24 weeks followed by surgery. Here we report the results from the 40 mg/day PK run-in. Results: Seven PM women (6 White, 1 Asian) aged 28-51 (median 46) received ENDX 40 mg/day. Pt characteristics on study were: ER > 90% (all pts), median Ki-67 = 13 (range 4-33%), cTstage (cT2: 6 pts, cT3: 1 pt), and tumor grade (G1: 1 pt, G2: 6 pts). The median ENDX 28 day Css (ng/mL) was 263.6 (range 180.3-376.6). One pt discontinued due to wk4 Ki-67 remaining > 10%. The remaining 6 had ESD, with either wk4 Ki-67 remaining ≤ 10% (3 pts) or decreasing to ≤ 10% (3 pts) and after 24 wks underwent surgery with surgical Ki-67 ≤ 3% (range 0-3%). MRI central review (wk12 and wk24) demonstrated target lesion decreases in all pts with 1 CR, 1 PR and 4 SD (RECIST). Treatment related toxicities included grade 3 headache (n=1), grade 2 amenorrhea (n=1), and grade 2 hot flashes (n=1). The median (range) baseline estrone (n=5) was 54 pg/mL (19-114) with median (range) fold increase from baseline of 9.0 (1.3-23.2) at wk4 and 4.7 (0.4 - 25.9) at wk24. The median baseline estradiol level (n=5) was 29 pg/mL (19-209) with median fold increases from baseline of 17.9 (0.4-57.0) at wk4 and 8.1 (0.04 - 56.6) at wk24. Additional surgical and blood biomarker data will be presented at the meeting. Conclusions: ENDX (40 mg/day) exhibits promising antitumor activity for PM ER+/HER2- BC but with ENDX Css below target. Enrollment is ongoing to the 80 mg/day dose level. Citation Format: Matthew P. Goetz, Vera J. Suman, Heather Fraser, Lida Mina, Pooja Advani, Roberto Leon-Ferre, Karthik Giridhar, Felipe Batalini, Katie N. Hunt, Swaathi Jayaraman, James Jakub, Patricia Cronin, Mara Piltin, Amy Degnim, James N. Ingle, Judy C. Boughey, Sarah Buhrow, Joel Reid, Matthew Schellenberg, John Hawse, Steven Quay. Neoadjuvant (Z)-endoxifen in premenopausal ER+, HER2- breast cancer: Evaluation of the first pharmacokinetic cohort of the EVANGELINE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT205.

Abstract The protein kinase C (PKC) family of enzymes are indispensable serine-threonine kinases involved in signal pathways implicated in many different cellular processes. Dysregulation of PKC isoforms may contribute to the development and progression of breast cancer. Z-Endoxifen (Z-ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM) exhibited potent antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancers. Mechanistically, we discovered that ENDX inhibits PKCβI and downstream AKT signaling. Based on insights from our recently determined crystal structure of PKCβI, we investigated the effects of ENDX on PKCβI in estrogen receptor-positive (ER+) breast cancer. In vitro FRET-based biochemical assays revealed that ENDX inhibits the kinase activity of conventional (PKCβI) as well as a novel (PKC;) PKC isoform with a similar IC50. However, the PKCβI catalytic domain was found to be less sensitive to ENDX compared to full-length PKCβI. We identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory model using X-ray crystallography. Since the spatial assembly of PKC is crucial for its kinase activity, we assessed the effects of ENDX on the intracellular localization of PKCβI using live cell confocal imaging. Sub-cellular tracking of YFP-tagged PKCβI in ER+ MCF7 cells paradoxically revealed that ENDX promoted the translocation of PKCβI to the plasma membrane in both a dose and time-dependent manner. Furthermore, co-treatment with inhibitors of PHLPP phosphatases, which regulate PKC, alleviated this translocation. Based on our prior data demonstrating greater antitumor activity of the Z vs E isomer of ENDX, we discovered that Z-ENDX inhibits PKCβI kinase activity more effectively than its E-isomer using in vitro kinase assays. Further, E-ENDX failed to promote PKCβI translocation to the membrane.Taken together, these results suggest that allosteric effects of Z-ENDX trigger PKCβI recruitment to the plasma membrane where it is normally active yet suppresses its kinase activity. These findings indicate that Z-ENDX induces a non-productive structure of the enzyme and "breaks" the well-known mechanism of PKC activation upon binding to the cell membrane. Moreover, Z-ENDX likely triggers PHLPP-mediated PKCβI dephosphorylation, and ultimately PKCβI degradation, suggesting that Z-ENDX represents a new mechanistic basis for targeting and downregulating PKCβI, and potentially other PKC family members, in cancer cells. This newly discovered function of Z-ENDX likely contributes to its superior anti-cancer effects when compared to TAM and other endocrine therapies. Citation Format: Sayantani Sarkar Bhattacharya, Huy V. Huynh, Jasmin K. Farmakes, Taylor L. Witter, Elizabeth S. Bruinsma, Swaathi Jayaraman, Anh T. Cong, John R. Hawse, Matthew P. Goetz, Matthew J. Schellenberg. Protein kinase C beta 1 (PKCβ1) is allosterically inhibited and paradoxically translocated to the membrane by z-endoxifen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB051.

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.

Abstract Ovarian cancer is the most fatal of all female reproductive cancers due to its late detection, aggressive nature, and propensity to develop resistance to current standards of care. Despite initial responses to first line platinum- and paclitaxel-based chemotherapies, >70% of ovarian cancers recur with increasingly resistant disease. Targeted therapies, including PARP-inhibitors (PARPi) and VEGF-inhibitors, are being included in the front-line setting; and while they have improved progression-free survival in a subset of patients, they have thus far failed to improve overall survival in patients without specific genetic signatures. With the broad uptake of PARPi, PARPi-resistance is becoming common. Once resistance develops, nearly all patients die of their disease and there is an obvious need to elucidate the basis for chemotherapy- and PARPi-resistance and devise novel approaches to overcome resistance. Using a small molecule drug screen, we identified lestaurtinib, a known tyrosine kinase inhibitor as a potent inhibitor of treatment naive and highly advanced models of ovarian cancer. Lestaurtinib was originally developed to target FLT3, a protein not expressed in any ovarian cancer models we’ve tested. To identify lestaurtinib targets, we preformed phospho-proteomics in parallel with RNAseq and discovered the JAK/STAT signally pathway as a top hit. We confirmed that essential components of JAK/STAT signaling are constitutively activated in chemotherapy- and PARPi-resistant cells, suggesting that induction of this pathway may drive resistance. Importantly, we found that lestaurtinib-sensitive patient-derived-xenografts (PDXs) exhibited higher expression of multiple JAK/STAT signaling pathway members compared to PDXs that did not respond to lestaurtinib. Genetic knockdown of STAT1 and STAT3 via siRNA, or knockout via CRISPR/Cas9, resulted in significant growth inhibition of sensitive and resistant models, confirming their importance in maintaining cell viability and progression. Combining lestuartinib with standard-of-care cisplatin or olaparib, a PARPi, was shown to be synergistic in multiple models, indicating that pharmacological inhibition of JAK/STAT signaling has the potential to alleviate drug resistance. Finally, we assessed a panel of JAK/STAT inhibitors that interfere at various levels of the pathway and found profound differences in their ability to block STAT-phosphorylation and inhibit cell proliferation. Thus, it is critical to further define the mechanisms by which these drugs function to selectively suppress JAK/STAT signaling in order to elucidate specific nodes of the pathway that drive ovarian cancer progression. Ongoing studies are aimed at identifying the unique roles of STAT1 and STAT3, the specific contributions of their tyrosine and serine phosphorylation, and the relevance of up-/down-stream mediators of the JAK/STAT pathway on ovarian cancer cell growth with the goal of informing future clinical trials. Citation Format: Esther Rodman, Michael Emch, Archit Bajaj, Xiaojia Tang, Xiaonan Hou, Scott Kaufmann, Krishna Kalari, John Weroha, John Hawse. Exploiting JAK/STAT signaling to inhibit highly advanced and resistant forms of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B002.

Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to effective cancer management. Many potential therapies have been proposed and tested—including appetite stimulants, targeted cytokine blockers, and nutritional supplementation—yet highly effective therapies are lacking. Innovative approaches to treating cancer cachexia are needed. Members of the Kruppel-like factor (KLF) family play wide-ranging and important roles in the development, maintenance, and metabolism of skeletal muscle. Within the KLF family, we identified KLF10 upregulation in a multitude of wasting contexts—including in pancreatic, lung, and colon cancer mouse models as well as in human patients. We subsequently interrogated loss-of-function of KLF10 as a potential strategy to mitigate cancer associated muscle wasting. In vivo studies leveraging orthotopic implantation of pancreas cancer cells into wild-type and KLF10 KO mice revealed significant preservation of lean mass and robust suppression of pro-atrophy muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other factors implicated in atrophy, calcium signaling, and autophagy. Bioinformatics analyses identified Transforming growth factor beta (TGF-β), a known inducer of KLF10 and cachexia promoting factor, as a key upstream regulator of KLF10. We provide direct in vivo evidence that KLF10 KO mice are resistant to the atrophic effects of TGF-β. ChIP-based binding studies demonstrated direct binding to Trim63 , a known wasting-associated atrogene. Taken together, we report a critical role for the TGF-β/KLF10 axis in the etiology of pancreatic cancer–associated muscle wasting and highlight the utility of targeting KLF10 as a strategy to prevent muscle wasting and limit cancer-associated cachexia.