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.

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). 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). 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. 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 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.

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.

Abstract Introduction: Small molecule therapeutics of estrogen receptor-positive/HER2-negative breast cancer remains an area of active investigation where novel agents are greatly needed for treatment of hormone therapy resistant metastatic disease. The biguanide hexyl-benzyl-biguanide (HBB) is a potent inhibitor of CYP3A4 arachidonic acid (AA) epoxygenase activity and inhibits breast cancer cell proliferation and MCF-7 breast cancer tumor growth in nude mice. To explore the impact of bioisosteric substitution of the benzyl moiety of HBB with a cubane moiety, we synthesized hexyl-(cuban-1-yl-methyl)-biguanide (HCB) and tested its potency for the inhibition of the cognate CYP3A4 target AA epoxygenase activity as well as breast cancer cell proliferation of hormone therapy sensitive and resistant cell lines. Results: HCB selectively inhibited CYP3A4-mediated biosynthesis of (±)-14,15-EET with an IC50 of 4.7±0.2 uM vs. 64.8±6.5 uM for 8,9-EET and 26.5±1.9 uM for 11,12-EET. At 24 hours, HCB inhibited proliferation of MCF-7 (ER+HER2-), BT474 (ER+HER2+) and MDA-MB-231 (ER-HER2-) cells at IC50 of 8.4±1.2, 11±1.3 and 15±0.9 uM, respectively. At 48 hours, HCB inhibited proliferation of aromatase inhibitor and fulvestrant resistant (LR,FR), and cyclin dependent kinase inhibitor (CDKi) palbociclib resistant (LR,FR,PR) MCF-7 cell lines; LR,FR MCF-7AC1 (IC50 =1.34±0.1 uM) and LR,FR,PR MCF-7AC1 (IC50 =1.64±0.2 uM). Addition of 14,15-EET (1 uM) partially rescues MCF-7 cells from HCB-mediated inhibition of proliferation. OXPHOS is promoted, in part, by EETs. HCB is a potent OXPHOS inhibitor and rapidly inhibits O2 consumption of the MCF-7 and ZR75 (ER+HER2-) cells in a dose-dependent fashion (P<0.05). HCB treatment (10 uM) reduces mitochondrial membrane potential to 57.4±15.3% (P<0.001) of vehicle control in MCF-7 cells. Treatment with HCB at 20 uM for 0.5 hour also causes mitochondrial swelling in MCF-7 cells. HCB (10 uM) activates AMPK within 0.5 hour and increases the level of phosphorylation from 2.4±0.3 to 25.1±6.0 folds in a time dependent fashion in MCF-7 cells from 0.5-24 hours. Conclusion: These results show that HCB inhibits proliferation of ER+HER2- breast cancer cells, in part through inhibition of OXPHOS and suppression of the CYP product 14,15-EET. This inhibition is highly active in hormonal therapy and CDKi resistant ER+HER2- breast cancer cells. These results suggest that HCB is a novel and potent biguanide that has potential to be developed for inhibition of hormone therapy resistant and CDKi resistant breast cancer. Citation Format: Zhijun Guo, Jianxun Lei, Kwon Ho Hong, Beverly Norris, Craig M. Flory, Swaathi Jayaraman, Connor McDermott, Elizabeth Ambrose, Irina Sevrioukova, Tom Poulos, Ilia Denisov, Stephen Sliga, Robert J. Schumacher, Gunda I. Georg, John R. Hawse, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB078.

Abstract Introduction: Cancer cell-intrinsic CYP monooxygenases promote tumor progression. In ER+HER2-, breast cancer cells, CYP3A4 promotes tumor growth, in part, through epoxyeicosatrienoic acid (EET) biosynthesis (1). The biguanide diabetes drug metformin is currently being studied in breast cancer clinical trials. We have discovered that metformin binds to the active site heme of CYP3A4, thereby inhibiting EET biosynthesis (1). Structure-based design led to the discovery of the 100-fold more potent metformin analog N1-hexyl-N5-benzyl-biguanide (HBB). HBB specifically inhibits CYP3A4 arachidonic acid (AA) epoxygenase activity in breast cancer cells, suppresses the growth of breast cancer cell lines (IC50=3-30 uM), and inhibits growth of the MCF-7 ER+ mammary tumor model, similar to CYP3A4 gene silencing. Although CYP3A4 synthesizes EETs, how EETs may promote tumor growth is unknown. Recently, we found that HBB inhibits nuclear transit of estrogen receptor alpha (ERα) and 70kD FITC-dextran, but the mechanism was unknown (2). Metformin was discovered to inhibit nuclear pore complex (NPC) function, but the mechanism was also unknown (3). We therefore hypothesized that CYP-derived EETs function as second messengers that regulate nuclear translocation of ERα in breast cancer by promoting permeability of the NPC. This hypothesis implies that biguanide drugs suppress nuclear translocation of the ERα through inhibition of CYP-derived EETs that serve as second messengers to open the NPC. Methods: Breast cancer cells were serum-starved for 16 hours and then treated with metformin, HBB, the (±)-14,15-EET regioisomer, or EET agonists, for varying times (30 min to 6 hours). Cells were fixed, permeabilized, and incubated with antibodies specific for ERα. After incubation with fluorescent secondary antibodies and Hoechst 33342 dye, cell images were acquired with a confocal microscope and the nuclear ERα fluorescence signal was quantified relative to the Hoechst signal. Quantitative PCR was used to measure the expression of ERα regulated genes. Results: (±) 14,15-EET (5 uM) treatment for 1 hour increased nuclear ERα by 66 ± 26% (n=9, p=0.031) in MCF-7 cells and 77 ± 24% (n=7, p=0.015) in ZR-75 cells. Treatment with EET agonists C22 (5 uM) and EET-A (5 uM) for 1 hour increased nuclear ERα by 32 ± 13% (n=7, p=0.047) and 39 ± 14% (n=7, p=0.02) in MCF-7 cells. Metformin (5 mM) treatment for 6 hours reduced nuclear ERα in MCF-7 by 35 ± 4.9 % (mean ± SEM, n=8, p=0.003). HBB (20 uM) treatment for 2 hours reduced the expression of three ERα regulated genes in MCF-7 cells: estrogen receptor alpha (ESR1) was reduced by 15 ± 1.7% (n=3, p=0.001), progesterone receptor (PGR) by 21 ± 0.6% (n=3, p=0.02) and L-type amino acid transporter 1 (SLC7A5) by 14 ± 0.2% (n=3, p=0.002). Conclusion: CYP epoxygenase activity in ER+HER2- breast cancer cells produces EETs, which function as second messengers that promote nuclear transit of ERα. Metformin and HBB inhibit nuclear ERα translocation, in part, through inhibition of CYP3A4 AA epoxygenase activity and this inhibition reduces ERα mediated gene expression, providing a novel mechanism for ERα regulation. 1. Cell Chem Biol. 2017 Oct 19; 24(10) 1259 - 1275. 2. Abstract of 109th AACR meeting (#LB-023), April 2019 3. Cell. 2016 Dec 15;167(7):1705-1718 Citation Format: Zhijun Guo, Jianxun Lei, Shaoping Wu, Julissa Molina-Vega, Paloma Cervantes, Aline R. Rom'Mand, Dylan C. Castillejo Mijangos, John R. Falck, Carol Lange, Jinhua Wang, David A. Potter. Metformin inhibits nuclear localization of estrogen receptor alpha in breast cancer cells, in part, by inhibition of CYP-mediated epoxyeicosatrienoic acid (EET) biosynthesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4372.

Introduction: Cancer cell-intrinsic CYP monooxygenases promote tumor progression. In ER+ breast cancer cells, CYP3A4 is required for tumor growth and localizes to mitochondria where it promotes electron transport chain and respiration, while suppressing autophagy, in part, through epoxyeicosatrienoic acid (EET) biosynthesis (1). The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 arachidonic acid epoxygenase activity, reduced oxygen consumption rates and suppressed growth of breast cancer cell lines (IC50=3-30 μM) and an established ER+ mammary tumor model. HBB inhibited activation of mTOR and suppressed S6Kinase phosphorylation. HBB also suppressed intratumoral mTOR. Recently, nuclear transit of RagC has been shown to promote mTORC1 function, a process sensitive to metformin (2), which inhibits the nuclear pore complex (NPC). Therefore, we hypothesized that HBB as a more potent biguanide may inhibit mTOR through reduction of nuclear RagC by regulating NPC. HBB may also regulate the localization of other nuclear proteins such as estrogen receptor alpha (ERα). Method: ER+HER2- breast cancer cells (MCF-7 and T47D) were seeded on chamber slides and treated with vehicle or HBB for varied periods of time. Cells were fixed, permeabilized, and incubated with specific antibodies against RagC and ERα. After incubation with fluorescence tagged secondary antibody, slides were stained with DAPI and analyzed by confocal microscopy. Levels of nuclear proteins of interest were normalized against DAPI staining. Results: CYP3A4 shRNA knockdown reduced nuclear RagC in MCF-7 cells by 61.2±12.3 % (n=3, p Conclusion: These data suggest that the biguanide sensitive enzyme CYP3A4 may play a role in nuclear localization of ERα and RagC, which work in tandem to promote the growth of ER+HER2- breast cancer cells. 1. Cell Chemical Biology. 2017 Oct 19; 24(10) 1259 – 1275. 2. Cell. 2016 Dec 15;167(7):1705-1718 Citation Format: Zhijun Guo, Shaoping Wu, Julissa Molina-Vega, Rafael Castillo, Jaime Barrera, Veronica Johnson, Carol Lange, David Potter. CYP monooxygenases regulate nuclear localization of ERa and mTORC1 component RagC in ER+HER2- breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-023.

Abstract Introduction: Solid tumors are often resistant to immunotherapy due to a hostile microenvironment for immune cells, characterized by hypoxia and low nutrients. Metformin has been shown to potentiate PD-1 blockade and improve intratumoral T-cell function and tumor response through reduction of hypoxia (1). A novel biguanide (neo-biguanide) that potentiates immunotherapy similar to metformin, but is more potent, would be more ideal for combination with immunotherapy. Recently, we have discovered that metformin binds to the mitochondria associated enzyme CYP3A4 and thereby suppresses the electron transport chain (ETC) and oxygen consumption rates (OCR) (2). Structural biology approaches led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which binds to the CYP3A4 heme with higher affinity than metformin and much more potently inhibits the ETC and OCR (2). HBB potently and specifically inhibits CYP3A4 arachidonic acid (AA) epoxygenase activity, causing OCR and growth inhibition of breast cancer cell lines and established mouse mammary tumors (IC50=3-30 uM). Because lymphocytes express relatively lower levels of the CYP3A4 target of HBB (3), we hypothesized that HBB may permit T lymphocyte growth at concentrations that inhibit tumor epithelia. Therefore, effects of HBB on T cell proliferation and activation were tested with mouse splenocytes. Methods: HBB effects on T cell growth were tested across a range of concentrations (0 to 50 uM). C57BL/6J mouse splenocytes were incubated with or without biguanides in the presence of anti-mouse CD3 and anti-mouse CD28 antibodies. After 24 hours, cells were labeled with fluorescently tagged antibodies to mouse Thy-1, CD4, CD8, CD25, CD44, CD62L and CD73 surface markers and T cell subsets were analyzed by flow cytometry. Results: At low concentrations (0.09 to 1.5 uM) HBB did not inhibit T cell growth, but at higher concentrations (3 to 25 uM) HBB caused proliferation of CD4+ and CD8+ T lymphocytes with optimum concentrations of 6 to 12 uM (1.9-fold for CD4+ and 1.6-fold for CD8+; P<0.05 for both), conditions that inhibited breast cancer cells. Notably, CD4+FoxP3+ regulatory T cells were not increased by HBB. Metformin treatment caused an increase of CD4+ and CD8+ T lymphocytes only at concentrations >5 mM. Conclusions: HBB potently increases the proliferation of CD4+ and CD8+ mouse T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies at concentrations that suppress human and mouse mammary carcinoma cells. In contrast, metformin failed to show proliferation-inducing activity for T cells at concentrations below 5 mM. HBB therefore is a potent T lymphocyte activator, while inhibiting breast cancer epithelial cells, supporting further study of HBB to facilitate immune therapy in animal models. 1. Cancer Immunol Res. 2017 Jan 1; 5 (1) 9-16. 2. Cell Chemical Biology. 2017 Oct 19; 24(10) 1259 - 1275. 3. Drug Metab Dispos. 2008 Jan;36(1):182-9. Citation Format: Zhijun Guo, David Owen, Pamela Rosato, David Masopust, Michael A. Farrar, David A. Potter. N1-hexyl-N5-benzyl-biguanide promotes proliferation of CD4+ and CD8+ T lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3525.

While cytochrome P450 (CYP)-mediated biosynthesis of arachidonic acid (AA) epoxides promotes tumor growth by driving angiogenesis, cancer cell intrinsic functions of CYPs are less understood. CYP-derived AA epoxides, called epoxyeicosatrienoic acids (EETs), also promote the growth of tumor epithelia. In cancer cells, CYP AA epoxygenase enzymes are associated with STAT3 and mTOR signaling, but also localize in mitochondria, where they promote the electron transport chain (ETC). Recently, the diabetes drug metformin was found to inhibit CYP AA epoxygenase activity, allowing the design of more potent biguanides to target tumor growth. Biguanide inhibition of EET synthesis suppresses STAT3 and mTOR pathways, as well as the ETC. Convergence of biguanide activity and eicosanoid biology in cancer has shown a new pathway to attack cancer metabolism and provides hope for improved treatments that target this vulnerability. Inhibition of EET-mediated cancer metabolism and angiogenesis therefore provides a dual approach for targeted cancer therapeutics.

Abstract Introduction: Silencing of the human cytochrome P450 (CYP) monooxygenase enzyme CYP3A4 in the ER+ MCF-7 tumor cell line inhibits tumor growth in the mammary fat pad, but the relative roles of tumor cell intrinsic CYPs vs. vascular/microenvironment CYPs remain to be determined and a syngeneic animal model is needed to answer this question. The SSM2ucd ovarian dependent mouse mammary carcinoma cell line forms a transplantable estrogen-receptor positive (ER+) mouse mammary tumor in which growth dependence on cell intrinsic CYPs may be tested in vitro and in vivo. The SSM2ucd cell line is derived from 129SvJ: homozygous Stat1-null female mice, which develop ER+ mammary tumors (1,2). Expression profiling of spontaneously arising mouse mammary carcinomas in a TP53 KO model revealed that the CYP3A4 ortholog Cyp3a11 is up-regulated in these tumors. Methods: Cyp3a11 bactosomes were used to assay for NADPH dependent biosynthesis of EETs from arachidonic acid using an LC-MS/MS method. siRNA silencing and CRISPR/Cas9 knock out of the Cyp3a11 exons 2 and 3 was performed and the cell lines were characterized for EET dependence using an MTT assay. The highly potent inhibitor and chemical probe of CYP epoxygenase activity, hexyl-benzyl-biguanide (HBB), was used to test dependence of SSM2ucd cells on CYP epoxygenase activity. Total cellular EETs in SSM2ucd cells exposed to HBB at the IC50were measured by the LC-MS/MS method. Results: Cyp3a11 was shown to have robust NADPH dependent epoxygenase activity, while SSM2ucd cells were EET dependent for growth. Silencing of Cyp3a11 was demonstrated to inhibit proliferation of the SSM2ucd cell line, which was partly abrogated by EETs. CRISPR/Cas9 knock out of the Cyp3a11 in the SSM2ucd cell line was performed and exon 2 and exon 3 deleted cell lines were derived. SSM2ucd Cyp3a11 knock out cell lines were more sensitive to EET-induced proliferation under serum free condition than control lines. HBB potently inhibits the SSM2ucd cell line in vitro (IC50=25 uM) and reduces total cellular EET levels in this cell line at IC50. These results suggest that we may see inhibitory activity of HBB in a syngeneic 129/SvJ tumor transplantation model. Conclusions: The Cyp3a11 gene can be silenced in the SSM2ucd cell line, without disrupting cell viability, allowing further study of the roles of EETs in mouse mammary carcinoma proliferation, survival and clonogenicity. The Cyp3a11 gene has been knocked out in the SSM2 ucd cell line, allowing in vitro and in vivo studies of the roles of Cyp3a11 in the growth of ovarian dependent mouse mammary carcinoma. 1. Breast Cancer Res. 2012 Jan 20;14(1):R16 2. PLoS One. 2015 Jun 15;10(6):e0129895] Citation Format: Zhijun Guo, Patrick McGrarrah, Ted Bebi, Ashley Mooneyham, Alejandra Osorio, Cesar Herrera, Victor Arrieta, Sebastian Mohar, Irwin Hernandez, Robert Klink, Robert Cardiff, David A. Potter. Ovarian-dependent SSM2ucd mouse mammary carcinoma cells depend on Cyp3a11 epoxygenase activity for proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 963. doi:10.1158/1538-7445.AM2017-963

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

Cytochrome P450 3A4 (CYP3A4) promotes ER+HER2- breast cancer cell proliferation and survival, in part, by biosynthesis of epoxyeicosatrienoic acids (EETs). EETs are known to regulate mitochondrial function in non-transformed cells, but the roles of CYP3A4 and EETs in regulation of breast cancer bioenergetics are unknown. Hexyl-benzyl-biguanide (HBB) is useful probe of CYP3A4 epoxygenase activity and selectively inhibits EET biosynthesis (IC50 = 9 uM vs. IC50 = 50 uM for CYP2C8). HBB caused depolarization of mitochondria in MCF-7 cells, while (±)-14,15-EET provided partial protection. The soluble epoxide hydrolase (sEH) inhibitor t-AUCB ameliorated inhibition of oxygen consumption rates (OCR) by HBB (20 uM), while there was no effect on extracellular acidification rate (ECAR), indicating that the primary effect of HBB is on OCR. At 30 minutes, HBB added to MCF-7 cells transiently suppressed phosphorylation of pyruvate kinase muscle isozyme 2 (PKM2) on Tyr-105, which has been reported to favor enzymatically inactive dimer over active tetramer. Suppression of phosphorylated PKM2 correlated with subsequent PKM2 tetramer formation and increase of intracellular pyruvate and extracellular lactate at 1 hour. The (±)-14,15-EET regioisomer reduced the pro-glycolytic PKM2 tetramer at 1 hour, suggesting that HBB may promote PKM2 tetramer, in part, through reduction of EET. Prolonged exposure to HBB (20 uM) in cultured cells activated phosphorylation of PKM2 on Tyr-105, but there was increased cellular necrosis correlating with reduced mitochondrial respiration and reduction of ATP stores, indicating that loss of respiration was the dominant effect. HBB inhibited the ER+HER2- MCF-7 xenograft, similar to CYP3A4 silencing. HBB promoted phosphorylation of intratumoral PKM2 on Tyr-105, consistent with long-term exposure to HBB in cultured MCF-7 cells. Notably, MCF-7 tumor response to HBB did not correlate with phosphorylation of AMPK-alpha on Thr-172, a marker of AMPK activation. Metformin (5 mM) exhibited no effect on PKM2 or its phosphorylation in cultured MCF-7 cells. Together, these results indicate that part of the inhibitory effect of HBB on ER+HER2- breast cancer is mediated through inhibition of respiration. Significance: These results establish HBB as a useful chemical probe of respiration, with indirect effects on PKM2 regulation. HBB may also be useful as a potential therapeutic candidate for ER+HER2- breast cancer. Citation Format: Zhijun Guo, Irina Sevrioukova, Eric Hanse, Xia Zhang, Ilia Denisov, Ting-Lan Chiu, Rebecca Cuellar, Christian Torres, Julia Wulfkuhle, Emanuel Petricoin, Qing Cao, Haitao Chu, Beverly Norris, Robert Schumacher, Ameeta Kelekar, Ian Blair, Jorge Capdevila, John Falck, Thomas Poulos, Steven Sligar, Gunda Georg, Elizabeth Amin, David A. Potter. Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 44.

e11563 Background: Epoxides of arachidonic acid called epoxyeicosatrienoic acids (EETs) have been implicated in the progression of estrogen receptor positive (ER+) breast cancer. EETs are synthesized by cytochrome P450 enzymes in breast cancer cells. Elevated (±)14,15-EET in breast tumors has been associated with increased expression of CYP enzymes that exhibit epoxygenase activity and decreased soluble epoxide hydrolase. We tested the hypothesis that EETs synthesized by breast tumors are detectible in the plasma of breast cancer patients before surgery and are reduced after lumpectomy or mastectomy. Methods: Patients with newly diagnosed ER+ HER2- breast cancer at the Masonic Cancer Center, University of Minnesota and the Sidney Kimmel Cancer Center at Thomas Jefferson University who were eligible for lumpectomy or mastectomy were eligible for the study. Plasma was obtained up to two weeks before and 14 to 21 days after lumpectomy or mastectomy. Following extraction, EETs were separated and measured by ESI-LC-MS/MS. Data were analyzed with a linear mixed effects model and P values were adjusted for multiple comparisons. Results: There were 11 evaluable patients in this study. After surgery (±)-8,9-EET was reduced to 0.75 (P = 0.0034) and 0.68-fold pre-surgery levels (P = 0.0054) in two subjects, and (±)-14,15-EET was reduced to 0.61 (P = 0.043) in a third subject. In one subject (±)-8,9-EET was increased to 1.64-fold pre-surgical level (P = 0.0067), (±)-11,12-EET to 1.55-fold (P = 0.018) and (±)-14,15-EET to 1.85-fold (P = 0.0042). Three additional subjects had reductions of EET regioisomers after surgery, but these changes were not significant after correction for multiple comparisons. The remaining 4 patients had no change in EET levels. Conclusions: This study demonstrates that measurement of plasma EETs is feasible in breast cancer patients. There were changes of EET regioisomer levels after surgery in 4 of 11 patients with newly diagnosed breast cancer. These results indicate that lumpectomy or mastectomy can be associated with altered plasma EET levels in a subset of ER+HER2- patients, indicating feasibility of a larger study to test this hypothesis. Clinical trial information: NCT01009437.