Pryzbilla, Michael J.

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.