Jayaraman, Swaathi

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.

Abstract Introduction: Heme-binding biguanides inhibit cytochrome P450 monooxygenase-mediated biosynthesis of the epoxy fatty acid (EpFA) (±)14,15-EET, which promotes progression of ER+HER2- breast cancer as well as regulatory T cell (Treg) proliferation. However, there are no known strategies to exploit this vulnerability. Metformin has shown promise but lacks sufficient potency and clinical utility. Through structure-based design, heme binding hexyl cubane derivatives of metformin were identified, which potently inhibited (±)14,15-EET biosynthesis. Fluorination of the hexyl moiety improved pharmacokinetics (PK) and reduced toxicity. Among the fluorinated derivatives, C5F2-HCB potently inhibited (±)14,15-EET biosynthesis (IC50=3.1 uM for C5F2-HCB vs. 5 mM for metformin). C5F2-HCB and metformin are candidate agents for TME activation from “cold” to “hot” due to their inhibition of EETs biosynthesis and EET-driven oxidative phosphorylation (OXPHOS) which promotes tumor hypoxia. We then tested whether C5F2-HCB or metformin could inhibit immune exclusion in the immunologically “cold” ovarian dependent STAT1 KO SSM2ucd mammary carcinoma model, in part, by reducing Tregs and/or increasing CD8 or CD4 tumor infiltrating T cells (TIL). We hypothesized that biguanides, by reducing EETs, would suppress Tregs and promote effector T cells in the TME. We also hypothesized that C5F2-HCB would be more effective than metformin in modulating the TME, based on greater potency for inhibition of (±)14,15-EET biosynthesis. Both biguanides were tested at their maximum tolerated dose (C5F2-HCB; 18 mg/kg daily and metformin; 50 mg/kg every other day). Results: In the SSM2ucd allograft, all combination treatment groups with biguanides reduced tumor growth relative to the IgG control at the endpoint (day 63) : metformin + IgG (51.5±9.8% of control), C5F2-HCB + IgG (53.3±12.8%), metformin + anti-PD-1 (50.4±11.5%), and C5F2-HCB + anti-PD-1 (49.6±12.8%) [(P < 0.05) for all comparisons]. In contrast, anti-PD-1 antibody alone failed to reduce tumor growth at the endpoint. Flow cytometry analysis of the TME revealed that only C5F2-HCB + IgG treatment increased CD8+ tumor-infiltrating lymphocytes (TILs) compared to all other treatment groups (>2-fold; P < 0.05). Additionally, only C5F2-HCB + anti-PD-1 reduced intratumoral Tregs (>3-fold; P < 0.05). C5F2-HCB in combination with IgG or anti-PD-1 reduced intratumoral macrophages by >50% (P < 0.001) and reduced the M2/M1 macrophage ratio by 32-61% (P < 0.05) relative to other groups. No effects on CD4+ T cells were observed. Furthermore, C5F2-HCB and IgG or anti-PD-1 increased intratumoral neutrophils by 75- 120% compared to the anti-PD-1 or IgG (P < 0.05). (Multiple comparisons tested by ANOVA). Conclusion: Relative to IgG alone, C5F2-HCB and metformin inhibited growth of the SSM2ucd allograft in combination with either anti-PD1 or IgG. In contrast to metformin, only C5F2-HCB modulated the TME, including increased CD8+ TILs with IgG, reduced Tregs with anti-PD-1, and decreased M2/M1 macrophage ratio with IgG and anti-PD-1. C5F2-HCB, in contrast tometformin, was effective in modulating an immune excluded TME and may contribute to a strategy for clinical translation. Citation Format: Zhijun Guo, Jianxun Lei, Joshua McCarra, Qing Cao, Michael J. Pryzbilla, Brenda Koniar, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John R. Hawse, Antonino B. D'Assoro, Ilia G. Denisov, Stephen G. Sligar, Kathryn L. Schwertfeger, Michael A. Farrar, Dipak Panigrahy, Hammock D. Bruce, Elizabeth A. Ambrose, Gunda I. Georg, Goetz P. Matthew, David A. Potter. C5F2-hexyl-(cuban-1-yl-methyl)-biguanide (C5F2-HCB) overcomes an immune excluded tumor microenvironment (TME) and suppresses tumor growth in the ovarian dependent ER+HER2- SSM2ucd mammary carcinoma allograft model [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 LB010.

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.

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 Introduction: Immune checkpoint blockade (ICB) has clinical activity in triple negative breast cancer (TNBC) but is less effective in the ER+HER2- signature, where there is a cold immune microenvironment (IM) and regulatory T cells (Tregs) may suppress effector T cells. Agents that activate the IM by turning cold tumors hot may support ICB. The biguanides hexyl-benzyl-biguanide (HBB) and its bioisostere hexyl-(cuban-1-yl-methyl)-biguanide (HCB) are candidate agents to activate the IM because they potently inhibit biosynthesis of immunosuppressive epoxyeicosatrienoic acids (EETs) and EET-driven oxidative phosphorylation (OXPHOS), while blocking N-glycosylation of immune checkpoint (IC) proteins. We hypothesized that reversal of hypoxia by biguanides in the ovarian dependent ER+HER2- STAT1 KO SSM2ucd mouse mammary carcinoma (MC) model would suppress Tregs and promote effector T cells in the tumor IM. While the SSM2ucd model did not express immune checkpoint protein PD-L1 (B7-H1), it did express related IC proteins B7-H3 and B7-H4. We hypothesized that by inhibiting OXPHOS and reducing N-glycosylation of immune checkpoint proteins, HBB and HCB may promote efficacy of ICB. We chose the SSM2ucd model to test impact of HCB on the ER+ MC IM. Results: SSM2ucd cells exhibited longer tumor latency (60 days) than the basal 4T1 (10 days) and 67NR (20 days) mouse MC models. SSM2ucd tumor reimplantation shortened latency by more than half, to 20 days. Immunohistochemistry showed that B7-H3 and B7-H4 protein levels were 1.2 (P=0.001) and 1.3-fold (P=0.04) higher in reimplanted tumors vs. control. In SSM2ucd cells, HCB inhibited N-glycosylation of B7-H3 (P=0.01) by 35% and B7-H4 (P=0.02) by 45% and suppressed TGFβ induction of B7-H3 by 21% (P=0.02) and B7-H4 by 79% (P=0.001) at 24 hours, while 14,15-EET promoted N-glycosylation of B7-H3 (1.2-fold; P=0.03) and B7-H4 (1.3-fold; P=0.04) at 4 hours. Effects of HBB and HCB on anti-CD3 and anti-CD28 stimulated mouse splenocytes were assayed. The proliferative effects of HBB on CD4+ and CD8+ cells peaked at 12 uM (p<0.001) and for HCB at 6.25 uM (p<0.001). Tregs decreased with HCB at a threshold of 12 uM (p <0.001) and with HBB at 25 uM (P < 0.001) while 14,15-EET increased Tregs (1.2 fold; P=0.02) and suppressed the CD8+:Treg ratio (0.79; P=0.02). In the SSM2ucd mammary allograft model, although HCB 12mg/kg daily did not inhibit tumor growth, it reduced intratumoral hypoxia by 20% (P=0.01), increased CD8+ TIL by 4.4-fold (P=0.04), decreased Treg:CD4+ TIL ratio by 76% (P=0.02), and decreased Treg:CD8+ TIL ratio by 88% (P=0.01) relative to control. Conclusion: B7-H3 and B7-H4 expression inversely correlated with latency of ER+ MC and may represent targets for immune checkpoint antibodies and their drug conjugates. HCB, an inhibitor of OXPHOS and EET biosynthesis, reduced intratumoral hypoxia, increased CD8+ TIL and reduced the Treg:CD8+ ratio, potentially supporting ICB therapy of ER+ MC by turning cold tumors hot. Supported by CDMRP BCRP Grant BC180596, Award Number W81XWH-19-1-0099 Citation Format: Zhijun Guo, Jianxun Lei, Hrishi Venkatesh, David Owen, Adam Bass, Christine Cannon, Joshua McCarra, Brenda Koniar, Craig Flory, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John Hawse, Emmanuel S. Antonarakis, Emanuel F. Petricoin, Julia Wulfkuhle, Robert D. Cardiff, Elizabeth A. Ambrose, Gunda I. Georg, Kaylee L. Schwertfeger, Michael A. Farrar, Brad St. Croix, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB102.

Abstract Background: In phase I/II clinical trials, the tamoxifen metabolite Z-endoxifen (ENDX) demonstrated substantial oral bioavailability and promising antitumor activity in endocrine-refractory estrogen-receptor positive breast cancer (ER+ BC) and other solid tumors, with ENDX plasma concentrations reaching as high as 5 µM, a concentration that far exceeds the ERα-targeting nanomolar concentrations. We previously identified protein kinase C beta 1 (PKCβ1), an oncogenic signaling kinase which regulates cell proliferation and tumorigenic transformation, as a novel ENDX receptor. ENDX-bound PKCβ1 (KD: 100 nM) and inhibited PKCβ1 kinase activity (IC50: 357 nM) in vitro, at concentrations achieved in phase I/II ENDX studies. Given the observations that ENDX induces antitumor activity in breast cancers resistant to endocrine therapies, including AIs, tamoxifen and fulvestrant, we postulated that ENDX may impact additional signaling pathways. Therefore, in this study we performed unbiased mass spectrometry to analyze the effects of ENDX on the phosphoproteome and total proteome. Methods: In estrogen unstimulated aromatase-expressing ER+/human epidermal growth factor 2 receptor negative (HER2-) MCF7AC1 breast cancer cells, the effects of ENDX on the phosphoproteome and total proteome were analyzed using ENDX concentrations achieved in tamoxifen treated patients (0.01 and 0.1 µM) and concentrations observed in the ENDX phase I/II trials (5 µM). NetWorKIN and RoKAI prediction tools were utilized to identify the upstream kinases of ENDX downregulated phosphoproteins. In estrogen unstimulated MCF7AC1 as well as in the ER+/HER2- T47D breast cancer cells, the ability of ENDX, tamoxifen, fulvestrant and PKC kinase inhibitor enzastaurin to block insulin or PKC agonist phorbol 12-myristate 13-acetate (PMA)-stimulated AKTSer473 phosphorylation was analyzed by immunoblot (IB) assay. Additionally, ENDX effects on AKTSer473 phosphorylation in the MCF7AC1 xenograft model in vivo was analyzed by IB assay. Further, the effects of PKCβ1 silencing in MCF7AC1 cells and the effects of AKT inhibitor MK-2206 treatment on growth and AKTSer473 phosphorylation in the MCF7AC1 as well as long-term estrogen deprived T47D (T47D-LTED) cell lines were analyzed by cell proliferation and IB assays. Results: In MCF7AC1 cells unstimulated with estrogen, ENDX at 5 µM but not at 0.01 and 0.1 µM concentrations inhibited growth, and induced apoptosis, suggesting an ERα-independent effect. Further, ENDX at 5 µM displayed three-fold greater effects in downregulating the phosphoproteome compared to the other concentrations, with minimal impact on the total proteome. Protein kinase C beta (PKCβ) and AKT1 were identified as the prevalent upstream kinases for ENDX downregulated protein phosphorylations. Notably, in estrogen unstimulated MCF7AC1 and T47D cells, ENDX at 5 µM attenuated phosphorylation of AKTSer473 and AKT substrates in vitro in the presence of insulin and in vivo. In MCF7AC1 cells, PMA-induced phosphorylation of PKCβ1 as well as AKTSer473 and AKT substrate phosphorylations were blocked by ENDX at 5 µM, with ENDX inducing PKCβ1 protein degradation both in the presence of insulin and PMA. Further, ENDX effects on growth and AKTSer473 phosphorylation were phenocopied by siRNA-mediated PKCβ1 knockdown as well as treatment with the pan-AKT inhibitor, MK-2206. Notably, the potent PKCβ1 kinase inhibitor, enzastaurin, had no effects either on PKCβ1 protein degradation nor on downstream AKT signaling. Conclusion: Taken together, these findings suggest that ENDX may exert anticancer activity via dual effects on blocking ERα as well as by targeting PKCβ1 for degradation, thus suppressing AKT signaling and induction of apoptosis. These preclinical findings will be studied in a planned neoadjuvant trial comparing ENDX with exemestane and ovarian function suppression (EVANGELINE) that will activate in the Fall of 2022. Citation Format: Swaathi Jayaraman, Xinyan Wu, Krishna R. Kalari, Xiaojia Tang, Mary Kuffel, Elizabeth S. Bruinsma, Santosh Renuse, James N. Ingle, Joel Reid, Matthew Schellenberg, John Hawse, Akhilesh Pandey, Matthew P. Goetz. Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-04.

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Abstract While epoxyeicosatrienoic acids (EETs) have been implicated in breast cancer growth and progression, less is known about their effects on oncogene transcription. We have previously found that the oncogenic regioisomer (±)14,15-EET drives mitochondrial respiration, ATP synthesis, and proliferation of ER+/HER2- breast cancer cells [Cell Chem Biol. 2017 Oct 19;24(10):1259-1275]. RNAseq analysis was performed (5 replicates per condition) on serum starved (16 hours) MCF-7 cells which were then treated with (±)14,15-EET or vehicle (2 hours; serum and phenol red free medium without estradiol). Using gene set enrichment analysis (GSEA), we found that (±)14,15-EET activated an estrogen receptor alpha (ER) hallmark early response gene set and synchronously activated a MYC hallmark gene set. With activation of the MYC hallmark gene set, c-Myc gene expression was also induced at 2 hours (3.99-fold; P=2.3 x 10-17; FDR=2.8 x 10-14). These data suggest an alternative pathway for activation of estrogen and MYC regulated genes in the absence of estradiol. The 15 genes most transcriptionally activated by (±)14,15-EET at 2 hours were: VMP1, ZFP36, JUNB, FOS, IER3, EGR1, IER5L, ELF3, JUN, NR4A1, HES1, DUSP1, MYC, TOB1, and CITED2 [fold change range: 3.25 (CITED2) to 90.5 (FOS); all P< 2.86 x 10-17; all FDR < 3.03 x 10-14]. The ER regulated genes most transcriptionally activated (>1.5 fold) were: IER3, TOB1, AREG, CISH, KCNMB3, and PDK4 (fold change range: 1.77 to 4.35; P= 1.74 x 10-18 to 1.35 x 10-5; FDR=5.54 x 10-15 to 6.4 x 10-4). The MYC regulated genes most transcriptionally activated (> 1.5-fold change) were EIF4A1 (fold change= 2.37; P=1.0 x 10-7; FDR=1.33 x 10-5), IRF9 (fold change=1.58; P=0.0044; FDR= 0.026), and FOSL1 (fold change=1.51; P=0.008; FDR=0.04). Supporting the hypothesis of (±)14,15-EET activation of ER-regulated transcription, (±)14,15-EET promoted nuclear translocation of ER at 1 hour measured by DAPI normalized immunofluorescence [MCF-7 nuclear ER increase of 1.66-fold (P=0.031); ZR75-1 nuclear ER increase of 1.77-fold (P=0.015)]. Supporting the hypothesis of (±)14,15-EET activation of MYC-regulated transcription, (±)14,15-EET treatment promoted nuclear translocation of c-Myc with MCF-7 cells exhibiting a 1.22-fold increase at 2 hours (P=0.002). (±)14,15-EET also promoted nuclear translocation of FITC-70 kDa dextran with MCF-7 cells exhibiting an increase of 1.35-fold at 1 hour (P=0.029). These data suggest that (±)14,15-EET can induce an estradiol-like immediate early gene response in ER+/HER2- breast cancer cells correlating with c-Myc activation. In summary, while the effect of (±)14,15-EET on nuclear translocation may be partially cargo agnostic, (±)14,15-EET promotes ER and c-Myc nuclear translocation and associated transcription, mimicking a tandem hormonal and growth factor response. Citation Format: Jianxun Lei, Zhijun Guo, Julissa Molina-Vega, Paloma Cervantes, Swaathi Jayaraman, John R. Hawse, Carlos Perez, Juan Abrahante, Xiaojia Tang, Krishna Kalari, Jinhua Wang, John R. Falck, Carol Lange, Matthew P. Goetz, David Potter. (±) 14,15-epoxyeicosatrienoic acid induces hallmark ER and MYC gene expression and associated ER and c-Myc nuclear translocation in ER+/HER2- breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB564.