Approximately 80% of breast cancers express estrogen receptor alpha (ERα), yet ERα-positive disease remains associated with high recurrence and late relapse. (Z)-Endoxifen (ENDX), the most abundant active metabolite of tamoxifen, is a potent, orally bioavailable, selective estrogen receptor modulator currently in development for breast health indications. During its synthesis, structurally related compounds are generated as byproducts termed here as new chemical entities (NCEs): AT416E, AT416Z, AT402E, AT402Z, and AT300, which have not been previously studied. We evaluated these NCEs alongside ENDX using multiple ERα-positive breast cancer cell lines, including models harboring clinically relevant ESR1 mutations. Activity was assessed using 2D and 3D proliferation, apoptosis, cell cycle profiling, migration, invasion, ERα transcriptional activity, RNA sequencing and synergy with abemaciclib. Several NCEs exhibited potent anti-estrogenic and anti-cancer effects. In various settings, these compounds matched or exceeded ENDX’s activity, highlighting their therapeutic promise. RNA sequencing analysis revealed both shared and compound-specific transcriptional programs. Some NCEs also combined favorably with abemaciclib, producing additive to synergistic effects comparable to or greater than ENDX. Together, our findings support further investigation of select NCEs for ERα-positive breast cancer, particularly for tumors with ESR1 activating mutations and as potential second- or third-line options for recurrent disease.

Succinate dehydrogenase (SDH)-deficient paraganglioma and pheochromocytoma (PPGL) are rare neuroendocrine neoplasms for which no effective targeted therapies currently exist. To uncover potential therapeutic targets, we performed an unbiased CRISPR-Cas9 genetic screen in immortalized mouse chromaffin cells (imCCs) with and without Sdhb loss. Our screen identified genes that differentially affect cell proliferation in Sdhb-deficient versus normal imCCs. Subunits of the transcriptional Mediator complex emerged as potential tumor suppressors, as their loss selectively promoted growth of Sdhb-deficient cells. The neddylation pathway, required for ubiquitin-mediated selective protein degradation, plays a critical role in Sdhb-deficient imCC growth and survival: loss of the neddylation regulator Ube2m led to increased proliferation, while loss of Ube2f suppressed growth of Sdhb-deficient imCCs. Neddylation inhibitors MLN4924 (Pevonedistat) and HA-9104 reduced UBE2F activity and selectively inhibited growth of Sdhb-deficient imCCs. This unexpected result highlights the neddylation pathway as a promising druggable vulnerability to be studied in SDH-deficient PPGL.

Objectives/Goals: Ovarian cancer is the most lethal gynecologic cancer with emergence of recurrent and resistant disease being frequent. We sought to identify novel ways to effectively kill chemotherapy-resistant forms of ovarian cancer and to prevent recurrence following curative intent with surgical and adjuvant regimens. Methods/Study Population: Our group identified CTPS1 as a novel dependency in triple negative breast cancer (TNBC) and ovarian cancer. We found CTPS1 was highly expressed in breast and ovarian tumor tissues and therapy-resistant models. We demonstrated siRNA knockdown of CTPS1 reduced proliferation in sensitive and resistant cell lines. In collaboration with Step Pharma, who developed the first CTPS1 inhibitor (STP938), we performed genome-wide CRISPR KO screens on STP938-sensitive and -resistant cells. Top synthetic lethal hits identified included TSC1, TSC2, and PPP2R2A. Synergy assays were conducted with STP938 in combination with mTOR inhibitors (Everolimus, Sirolimus, and Temsirolimus) and the PPP2R2A inhibitor LB-100 to evaluate the therapeutic benefit of combination therapies. Results/Anticipated Results: We identified CTPS1 as a novel vulnerability in many ovarian cancer cell line models, including models of chemotherapy and/or PARP inhibitor resistance. The first-in-class CTPS1 inhibitor (STP938) was shown to be a highly effective anticancer agent in sensitive and resistant forms of ovarian cancer, both in vitro and in vivo. Genome-wide CRISPR KO screens identified central components of the mTOR pathway (TSC1 and TSC2), as well as PPP2R2A, as synthetic lethal targets in the setting of STP938 exposure. We anticipate that combinatorial treatment of mTOR inhibitors and PP2R2A inhibitors with STP938 will further enhance efficacy and reduce the risk of recurrence when used in the adjuvant treatment of ovarian cancer patients. Discussion/Significance of Impact: This study will validate synthetic lethal interactions from our CRISPR screen, reveal genes that could be mutated in ovarian cancer patients that may be driving resistance to STP938, and uncover combination therapies that can be used to increase efficacy in ovarian cancer patients treated with STP938.

Abstract Introduction: Epoxy fatty acid (EpFA) mechanisms of hormone therapy resistance (HTR) in ER+HER2- breast cancer (BC) are poorly understood. EETs are EpFAs that contribute to BC progression and HTR by promoting oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to HTR. In this study, two fulvestrant resistant (FR) subclones of the letrozole resistant (LR) MCF-7AC1 line were isolated (termed LR/FR) and were incidentally resistant to the CDKi palbociclib (PR) (termed MCF-7AC1 LR/FR/PR or multiply resistant - MCF-7AC1 MR). These HTR cell lines exhibited CYP3A4 upregulation (2.3- to 4.3-fold) and increased cellular (±)14,15-EET levels, which were suppressed by biguanide treatment (P<0.05). Structure activity studies led to the design of hexyl cuban-1-yl biguanide (HCB) and fluorinated HCB derivatives C5F2-HCB and C6F3-HCB, which inhibited CYP3A4-mediated EET biosynthesis and growth of the HTR lines. HCB fluorination improved pharmacokinetics (PK) and toxicity. Methods: HCB and fluorinated HCBs were tested for impact on EET biosynthesis, OXPHOS, PK, and growth inhibition and reversal of hypoxia in the MCF-7AC1 MR tumor model. Results: Fulvestrant selection for MCF-7AC1 MR cell lines resulted in undetectable cyclin D1 and estrogen receptor (ERα) expression, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated (P<0.001). MCF-7 MR cells demonstrated 1.8, 1.4, and 1.2- fold higher levels of (±)8,9, (±)11,12, and (±)14,15-EET compared to parental MCF-7 cells (P<0.05). The most potent fluorinated HCB, C6F3-HCB, suppressed cellular levels of (±)8,9-EET, (±)11,12-EET, and (±)14,15-EET by 53-60% (P<0.029). The MCF-7AC1 MR cells were more potently inhibited by HCBs and fluorinated derivatives compared to the MCF-7 cell line. Comparing an MCF-7 MR line vs. MCF-7, HCB exhibited IC50 values of 4.1 vs. 7.0 uM, C5F2-HCB 10 vs. 25 uM, and C6F3-HCB 5.4 vs. 12 uM (P values all <0.05). C5F2-HCB exhibited the best PK and treatment of the MCF-7AC1 MR xenograft model showed reversal of hypoxia (P<0.05) and transient inhibition by C5F2-HCB (P=0.025 at 20 days). P/F also transiently inhibited this model (P=0.006 at 20 days). C5F2-HCB and P/F showed synergy in vitro (CI=0.46) leading to testing of C5F2-HCB/P/F in vivo enabled by acceptable toxicity of the triplet in mice. This combination showed synergy in vivo vs. standard of care palbociclib/fulvestrant by nonparametric statistical tests (Mann Whitney; P=0.0389; Kolmogorov-Smirnov; P=0.0473), providing a pathway for possible clinical development of the combination. Conclusions: MR BC cell lines exhibit upregulation of CYP3A4 and biosynthesis of EETs, indicating a role for EETs in HTR. The C5F2-HCB/P/F exhibits activity against the highly resistant MCF-7 MR xenograft with acceptable toxicity indicating an avenue for clinical development. Citation Format: Zhijun Guo, Jianxun Lei, Christian T. Wells, Allison M. Makovec, Andrew C. Yates, Swaathi Jayaraman, John R. Hawse, Joshua A. McCarra, Qing Cao, Michael J. Pryzbilla, Brenda L. Koniar, Beverly J. Norris, Craig M. Florey, Robert J. Schumacher, Michael A. Farrar, Kaylee L. Schwertfeger, Elizabeth A. Ambrose, Henry Wong, Gunda I. Georg, Antonino D'Assoro, Matthew P. Goetz, David A. Potter. Fluorinated cuban-1-yl biguanides overcome hormone therapy resistance to standard of care fulvestrant, and palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB196.

Epithelial ovarian cancer continues to be a highly fatal female reproductive malignancy despite treatment with aggressive chemotherapy, anti-angiogenic agents, PARP inhibitors, and surgical debulking strategies. JAK/STAT signaling is reported to be oncogenic in ovarian cancer and constitutively active in a proportion of chemotherapy-resistant ovarian cancer cell lines and human tumors. However, the individual contributions of specific JAK/STAT pathway components, in particular STAT1 and STAT3, in mediating oncogenic activity are not well understood. To delineate the precise molecular functions of STAT1 and STAT3 in the two most common ovarian cancer subtypes, high-grade serous and endometrioid, OVSAHO and MDAH-2774 cell lines were treated with IFNγ or IL6, to activate canonical STAT1 and STAT3 signaling respectively, with and without STAT1 and STAT3 knockdown. RNA-sequencing and ChIP-sequencing were utilized to globally define STAT1 and STAT3 specific transcriptomes and cistromes. STAT1 and STAT3 specific gene signatures were derived and applied to publicly available datasets to evaluate their predictive/prognostic value. Through these global and unbiased assessments, unique and overlapping STAT1 and STAT3 transcriptional profiles, and their associated pathways, were identified. From these data, STAT1 and STAT3 gene signatures were developed that are representative of transcriptionally active STAT1 and STAT3. STAT1 specific gene signatures were found to be more lowly expressed in ovarian tumors compared to normal ovarian tissue while STAT3 specific signatures were higher in malignant tissue. Elevated levels of STAT1 and STAT3 signaling were associated with non-responsiveness to initial chemotherapy treatment. Finally, STAT1 and STAT3 signatures were associated with worse progression free survival for ovarian cancer patients, while only STAT1 signatures were associated with worse overall survival. Importantly, these results were specific to the gene signatures indicative of STAT activity and differed from assessment of STAT1 and STAT3 gene expression alone. These studies further inform the precise mechanisms of STAT1 and STAT3 action in ovarian cancer and have defined gene signatures that have both prognostic and predictive value. Additionally, these results can be leveraged for the identification and development of pharmacologic approaches that elicit specific effects on the JAK/STAT signaling pathway and/or selectively alter the functions of individual STAT transcription factors.

Airway remodeling in asthma is characterized by increased extracellular matrix (ECM) production and deposition by airway smooth muscle (ASM) cells. Existing studies have shown contrasting effects of 17β-estradiol (E2) in regulating ASM cellular remodeling via differential activation of estrogen receptors (ERs: α and β). Even though downstream metabolites of E2 (2-hydroxyestradiol: 2-HE and 16-hydroxyestradiol: 16αHE2) are gaining recognition for their biological roles in various cellular systems, their role in ASM remodeling remains largely unexplored. Here, we explore the effects of 2-HE and 16αHE2, a highly potent metabolites, on ECM remodeling in ASM. ECM mRNA's/proteins expression and deposition were determined by Western blotting, qRT-PCR, and In-Cell Western analysis. Interaction of metabolites with ERs was performed using a docking study and their impact on regulation of an estrogen response element (ERE) was monitored via a luciferase reporter assay. Further, the ER-specific effect of metabolites was validated using shRNA-mediated ERα and ERβ knockdown ASM cells. 16αHE2 exposure showed no notable changes in transforming growth factor-β (TGF-β)-induced ECM proteins expression and deposition, whereas 2-HE exposure blunted the TGF-β effects. Molecular docking unveiled the binding of 16αHE2 with ERα, while 2-HE more strongly bound to ERβ, which was also confirmed by ERE-luciferase assay. In ERβ knockdown ASM cells, 2-HE inhibited the TGF-β-induced phosphorylation of SMAD2/3, AKT, and ERK1/2. However, 16αHE2 failed to elicit any of these effects. Furthermore, 2-HE significantly decreased the TGF-β-induced transcriptional activities of AP-1 and NF-κB. Overall, our findings suggest 2-HE blunts TGF-β-induced ECM through ERβ; therefore, it may serve as a novel therapeutic target for airway remodeling and asthma.

Triple negative breast cancer (TNBC) and ovarian cancer share many molecular features and are primarily treated with surgical resection and aggressive chemotherapy regimens. Unfortunately, survival rates for patients with advanced metastatic disease are poor, highlighting the need for innovative therapeutic approaches. Methods:Using the DepMap database, we first sought to identify genes that were highly expressed and more essential for proliferation/viability in TNBC cells relative to other breast cancer subtypes. Candidate genes were validated using gene-specific siRNAs in a panel of TNBC and estrogen receptor positive breast cancer cells. CTPS1 expression, and its functional significance, was further evaluated in ovarian cancer models, including chemotherapy- and PARP inhibitor-resistant cell lines. Pharmacologic inhibition was assessed using STP938, a first-in-class selective CTPS1 inhibitor, in TNBC and ovarian cancer cells as well as in ex vivo and in vivo patient-derived xenografts (PDX). Results:Six genes (CTPS1, HUS1, PRKRA, RAD1, RAD9A, and RHOA) were identified as potential TNBC selective dependencies. Among these, CTPS1 was prioritized for further study given that it was highly expressed, further upregulated in chemotherapy- and PARP inhibitor-resistant cell lines, and resulted in the greatest anti-neoplastic effects when depleted. Knockdown of CTPS1 confirmed its selective essentiality and resulted in rapid and durable S-phase cell cycle arrest. Pharmacologic inhibition of CTPS1 with STP938 led to robust anti-neoplastic effects at nM concentrations across both chemotherapy-sensitive and -resistant TNBC and ovarian cancer cell lines. Significant anti-neoplastic activity was observed in 6 independent ex vivo ovarian cancer PDX models. Further, STP938 significantly inhibited progression of an ovarian cancer PDX model in vivo. Conclusion:These findings identify CTPS1 as a critical dependency in TNBC and ovarian cancer. Selective pharmacologic inhibition of CTPS1 using STP938 is a potent inhibitor of tumor cell proliferation/viability and has anti-cancer activity in patient derived ex vivo and in vivo tumor models. These findings suggest that therapeutic targeting of CTPS1 represents an alternative approach for the management of patients with advanced and aggressive forms of these diseases.

Protein kinase C (PKC) isozymes are ubiquitous kinases that direct diverse cellular pathways and are important drug targets for the treatment of cancer and neurological diseases. PKCs are auto-regulating enzymes governed by phospholipid and Ca2+ signals via a mechanism that has remained enigmatic due to a paucity of structural information. Herein we present a series of structures of the full-length human PKCβI and PKCβII isozymes. These structures reveal the molecular basis by which PKCs maintain an auto-inhibited state, convert to a defined and ordered active conformation via a "lipid-lever" mechanism of allosteric activation, and how isoform-specific differences alter their allosteric regulatory mechanisms. We show that endoxifen, a recently identified PKCβI inhibitor, can alter the allosteric regulatory mechanism of PKCβI, providing a proof of concept for allosteric regulators of PKCs. Collectively, our data describe a foundational molecular model of second messenger-mediated allosteric regulation of PKCs that underpins PKC function, misregulation, and mechanisms of inhibition.

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.

Ovarian cancer is the deadliest gynecological malignancy, owing to its late-stage diagnosis and high rates of recurrence and resistance following standard-of-care treatment, highlighting the need for novel treatment approaches. Through an unbiased drug screen, we identified the kinase inhibitor, lestaurtinib, as a potent antineoplastic agent for chemotherapy- and PARP-inhibitor (PARPi)-sensitive and -resistant ovarian cancer cells and patient derived xenografts (PDXs). RNA-sequencing revealed that lestaurtinib potently suppressed JAK/STAT signaling and lestaurtinib efficacy was shown to be directly related to JAK/STAT pathway activity in cell lines and PDX models. Most ovarian cancer cells exhibited constitutive JAK/STAT pathway activation and genetic loss of STAT1 and STAT3 resulted in growth inhibition. Lestaurtinib also displayed synergy when combined with cisplatin and olaparib, including in a model of PARPi resistance. In contrast, the most well-known JAK/STAT inhibitor, ruxolitinib, lacked antineoplastic activity against all ovarian cancer cell lines and PDX models tested. This divergent behavior was reflected in the ability of lestaurtinib to block both Y701/705 and S727 phosphorylation of STAT1 and STAT3, whereas ruxolitinib failed to block S727. Consistent with these findings, lestaurtinib additionally inhibited the serine/threonine kinases, JNK and ERK, leading to more complete suppression of STAT phosphorylation. Concordantly, combinatorial treatment with ruxolitinib and a JNK or ERK inhibitor resulted in synergistic antineoplastic effects at dose levels where the single agents were ineffective. Taken together, these findings indicate that lestaurtinib, and other treatments that converge on JAK/STAT signaling, are worthy of further pre-clinical and clinical exploration for the treatment of highly aggressive and advanced forms of ovarian cancer.

Estrogen receptor alpha-positive (ER+) breast cancer remains a major clinical challenge due to the development of de novo and acquired resistance to endocrine therapy. (Z)-endoxifen (hereafter endoxifen), the most abundant active tamoxifen metabolite, has emerged as a promising drug candidate due to its superior anti-estrogenic activity and favorable side effect profile. This study aimed to elucidate the gene signature(s) associated with endoxifen resistance by employing gene expression analysis, expression signature generation, pathway enrichment analysis, and correlation analysis, using PandaOmics, a commercially available target-discovery platform. Changes in gene expression and pathways in resistant cells were compared to those seen in sensitive cells upon endoxifen treatment. Resistant cells were characterized by stronger inhibition of the estrogen response, partial retention of endoxifen's antiproliferative effects, acquired activation of proinflammatory pathways and epithelial-mesenchymal transition (EMT), activation of the mTOR pathway (contrasting with its inhibition in sensitive cells), and elevated levels of PKCβ. These resistance-specific changes may potentially drive an endoxifen resistance phenotype and, therefore, proteins involved in these pathways may be proposed as potential therapeutic targets for overcoming endoxifen resistance in breast cancer.

Endocrine therapy (ET) with aromatase inhibitors (AI) plus ovarian function suppression (OFS) is standard for premenopausal women with ER+/HER2- breast cancer. However, many premenopausal women are intolerant of AI + OFS, leaving tamoxifen (with or without OFS) as their only option. Extensive studies have demonstrated that premenopausal women treated with neoadjuvant ET whose tumors exhibit Ki-67 ≤ 10% following 4 weeks of therapy (defined as endocrine sensitive disease (ESD)) achieve 5-year distant disease-free survival > 96%. Problematically, only 41% of patients reach this ESD threshold with tamoxifen compared to 78% with AI+OFS (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent selective estrogen receptor modulator (SERM) that inhibits ERα at plasma concentrations of 3-5 ng/ml and dually targets PKCβ1, a suspected oncogene in breast tumors, at concentrations ≥ 500 ng/ml. We therefore developed the EVANGELINE study (NCT05607004) is an open label, randomized neoadjuvant phase II study that includes a pharmacokinetic (PK) Run-in, aiming to evaluate whether ENDX +/- goserelin is non-inferior to exemestane plus goserelin with respect to ESD rate. Following the completed PK run-in portion of the study, where 40mg/day or 80mg/day ENDX were assessed, the 40 mg/day dose was selected for further evaluation in the second part of the trial. The second part is comprised of a randomized treatment cohort, for patients with a Ki-67% >10% at preregistration randomized to either 40mg (Z)-endoxifen + goserelin or exemestane + goserelin neoadjuvant treatment and a Single Arm Treatment Cohort for patients who have a Ki-67 ≤ 10% at pre-registration assigned to 40mg/day (Z)-endoxifen monotherapy neoadjuvant treatment. To investigate ENDX further in premenopausal women, a multicenter study in the United States, was designed(EVANGELINE; NCT05607004). This phase 2, open-label, randomized, neoadjuvant trial includes a PK run-in phase designed to assess whether the 4-week ESD rate with (Z)-endoxifen + goserelin is non-inferior to that of exemestane + goserelin in premenopausal women with Stage IIA or IIB ER+/HER2- breast cancer with diagnostic Ki67 > 10%. Additionally, a single arm Phase 2, open-label neoadjuvant Cohort was designed to assess the 4-week ESD rate with ENDX monotherapy in premenopausal women with Stage IIA or IIB ER+/HER2- BC with diagnostic Ki67 ≤10%. Subjects will undergo neoadjuvant treatment for six months before receiving surgical treatment in the involved breast. This study will additionally assess safety, tolerability, response rates, surgical outcomes, and monitor disease progression. Comprehensive biomarker evaluations will be conducted using paired tumor biopsies and blood samples collected at baseline and week four, as well as 6 month surgical specimens. Enrollment in the PK run-in phase (n-22) was completed in Fall 2024 with the second portion of the trial slated to begin early in 2025. Matthew P. Goetz, Vera J. Suman, Claudia Lopez, Hayley Erickson, Lonnissa Nguyen, Sandra S. Hammer, Lida Mina, Pooja Advani, Roberto Leon-Ferre, Karthik Giridhar, Felipe Batalini, Daniel Flora, Jason M. Jones, Nusayba A. Bagegni, Katie N. Hunt, Mara Piltin, Amy Degnim, James N. Ingle, Judy C. Boughey, Sarah A. Buhrow, Joel M. Reid, Matthew Schellenberg, John R. Hawse, Steven C. Quay. A randomized phase 2 non-inferiority trial of (Z)-endoxifen + goserelin and exemestane + goserelin as neoadjuvant treatment for premenopausal women with ER+/HER2- breast cancer (EVANGELINE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT237.

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. 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.

Background: Aromatase inhibition (AI) with ovarian function suppression (OFS) is standard for premenopausal women (PrW) with ER+/HER2- primary breast cancer (BC). However, > 40% are intolerant of OFS leaving tamoxifen (TAM) as their only option. In the neoadjuvant endocrine setting, PrW with endocrine sensitive disease (ESD) (week 4 Ki-67 ≤ 10%) have 5-year disease free survival > 97%; however, only 35% achieve ESD with TAM vs 76% with AI+OFS (Gluz SABCS 2023). Z-endoxifen (ENDX) is a selective estrogen receptor modulator targeting ERα at 3-5 ng/ml and PKCβ1 ≥ 500 ng/ml, the latter resulting in AKT inhibition and apoptosis (Jayaraman NPJ Breast Cancer). EVANGELINE (NCT05607004) is an open label, randomized neoadjuvant phase II study with a PK Run-in to evaluate whether ENDX is non-inferior to exemestane plus goserelin in terms of ESD rate. The PK Run-in was designed to identify an ENDX dose that targets both ERα and PKCβ1, defined as day 28-day ENDX steady state concentration (Css) > 500 ng/ml in ≥ 5/6 patients (pts), and to assess antitumor activity and toxicity. In the PK Run-in, pts received either ENDX 40 mg/day (as monotherapy) or 80 mg/day (+/- goserelin). Pts with ESD remained on ENDX for 6 cycles (24 weeks) followed by surgery. We reported (Goetz AACR 2024) that among 7 pts (age 28-51; median 46) enrolled at the 40 mg/day dose, the median ENDX 28-day Css was 264 ng/mL (range:180-377), and ESD rate was 86% (6/7 pts), with either week 4 Ki-67 remaining ≤ 10% (n=3) or decreasing to ≤ 10% (n=3) with 1 pt remaining >10%. Grade 2 or higher AEs included grade 2 amenorrhea (n=2), grade 2 hot flashes (n=1) and grade 2 hypertension (n=1). The 6 pts with ESD completed 24 weeks of ENDX followed by surgery. Here we present the surgical findings of these 6 pts including Cell Cycle Arrest (CCA), Residual Cancer Burden (RCB), as well as the preliminary 4 week ESD rate for the 80 mg/day +/- goserelin cohorts. Methods: All pts who started protocol treatment are included in the summarized results. Results: The 6 pts treated with 24 weeks of ENDX 40 mg/day underwent surgery with no complications. cT stage was T2 in 5 pts and T3 in 1 pt. Central review of pre- and week 24 breast MRIs using RECIST criteria found tumor diameter reductions of 15 to 100% resulting in 1 CR, 1 PR and 4 SD. Median pre-Ki67 was 11 (range: 4-33%). Surgical CCA (Ki67 ≤ 2.7%) rate was 67% (4/6). The remaining 2 pts had Ki-67 of 3% (n=1) or in the patient with a near pCR, too few cells to quantitate (n=1). RCB was 1 (n=1), II (n=3), and III (n=2). PEPI scores will be reported at the meeting. At the 80 mg/day dose, ten pts were randomized to either ENDX alone (n=5, age 44-53; median 46) or ENDX + goserelin (n=5, age 37-52; median 44) with similar ER expression (all > 80%), cT, cN category and tumor grade in each arm. Ki-67 testing was completed pre- and week 4 for 9 pts where pre Ki-67 was ≤ 10% in 3 pts, > 10% in 4 pts, and not available in 2 pts (inadequate tumor cellularity). Following drug treatment, week 4 Ki-67 was maintained or fell to ≤ 10% in 6 pts, remained > 10% (1 pt), was indeterminate in 2 pts (inadequate tumor cellularity). PK data (80 mg/day) was available from the first 9 pts, and demonstrated a median 28 day ENDX Css of 344 ng/ml (range 161-959) with 3/9 pts > 500 ng/ml. At the 80 mg/day dose, there were no grade 3-4 toxicities. Grade 2 toxicities included hot flashes (2 pts), dyspepsia (1 pt), amenorrhea (1 pt), oligomenorrhea (1 pt), and nausea (1 pt). No VTE events were reported. Conclusions: Neoadjuvant ENDX, when administered to PrW, demonstrated substantial antitumor activity when administered as monotherapy (40 or 80 mg/day) or with OFS (80 mg/day). Increasing the ENDX dose from 40 to 80 mg improved the likelihood of achieving an Endx Css > 500 ng/ml without significantly increasing toxicity. We will present the final results from the 80 mg/day cohort at the meeting. Citation Format: Matthew Goetz, Vera J. Suman, Joel M. Reid, Lida A. Mina, Pooja Advani, Arezoo Mirad, Roberto Leon Ferre, Karthik Giridhar, Felipe Batalini, Sarah Buhrow, Stephanie Safgren, Swaathi Jayaraman, Patricia Cronin, Mara Piltin, Amy C. Degnim, Sarah Premji, James N. Ingle, Tufia Haddad, Amye J. Tevaarwerk, Jason Jones, Daniel Flora, Harjinder Singh, Nusayba Bagegni, Katie N. Hunt, Judy C. Boughey, Matthew Schellenberg, John Hawse, Steven C. Quay. Neoadjuvant Z-endoxifen for Premenopausal Estrogen Receptor (ER)+, Human Epidermal Growth Factor Receptor (HER2)- Breast Cancer (BC): Evaluation of the Pharmacokinetic (PK) Run-in for the EVANGELINE Study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-11-04.