Jayaraman, Swaathi

Abstract The selective estrogen receptor (ER) modulator, tamoxifen, is the only endocrine agent with approvals for both the prevention and treatment of premenopausal and postmenopausal estrogen-receptor positive breast cancer as well as for the treatment of male breast cancer. Endoxifen, a secondary metabolite resulting from CYP2D6-dependent biotransformation of the primary tamoxifen metabolite, N-desmethyltamoxifen (NDT), is a more potent antiestrogen than either NDT or the parent drug, tamoxifen. However, endoxifen’s antitumor effects may be related to additional molecular mechanisms of action, apart from its effects on ER. In phase 1/2 clinical studies, the efficacy of Z-endoxifen, the active isomer of endoxifen, was evaluated in patients with endocrine-refractory metastatic breast cancer as well as in patients with gynecologic, desmoid, and hormone-receptor positive solid tumors, and demonstrated substantial oral bioavailability and promising antitumor activity. Apart from its potent anticancer effects, Z-endoxifen appears to result in similar or even greater bone agonistic effects while resulting in little or no endometrial proliferative effects compared with tamoxifen. In this review, we summarize the preclinical and clinical studies evaluating endoxifen in the context of breast and other solid tumors, the potential benefits of endoxifen in bone, as well as its emerging role as an antimanic agent in bipolar disorder. In total, the summarized body of literature provides compelling arguments for the ongoing development of Z-endoxifen as a novel drug for multiple indications.

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 Background: The tamoxifen (TAM) metabolite, ENDX, demonstrated promising antitumor activity in endocrine resistant breast cancer (BC) in both phase I and phase II settings. Furthermore, ENDX resulted in superior in vivo antitumor activity compared to TAM and letrozole in aromatase-expressing aromatase inhibitor-sensitive and resistant MCF7AC1 models. Recently, we identified protein kinase C beta 1 (PKCβ1), which regulates cell proliferation and tumorigenic transformation, as a novel target of ENDX. ENDX-bound PKCβ1 at concentrations achieved in phase I/II ENDX studies (100-300 nM). In contrast, TAM binding to PKCβ1 occurred at concentrations 7-10 folds higher (2 μM) than achievable with TAM 20 mg/day dosing. However, the clinical relevance of targeting PKCβ1 kinase activity is unclear, since drugs that target PKCβ1 (enzastaurin) have been ineffective in BC and other solid tumors. Therefore, we sought to understand how ENDX altered PKCβ1 and to further compare and contrast ENDXs effects to that of PKCβ1 kinase inhibition in ERα+ BC. Methods: The effects of PKCβ1 silencing and ENDX treatment on gene expression was analyzed by RNAseq in MCF7AC1 cells. The impact of PKCβ1-silencing on cell cycle was evaluated by flow cytometry. Protein expression of cell cycle regulators in PKCβ1 and ENDX-treated MCF7AC1 and T47D cells were compared to TAM and enzastaurin in vitro and to letrozole, TAM or control in vivo. The effects of PKCβ1 and drugs on growth were analyzed by cell proliferation assays. PRKCB gene amplification was assessed in primary tumors using TCGA data and in metastatic tumors using whole-exome sequencing data from patients enrolled in the PROMISE study (NCT 03281902). Results: RNAseq analysis revealed E2F targets and G2M checkpoints as the top hallmark genesets significantly downregulated in both PKCβ1-silenced and ENDX-treated MCF7AC1 cells. Flow cytometry demonstrated that PKCβ1 silencing increased G1 and reduced S phases of the cell cycle. Western blot analyses of PKCβ1-silenced MCF7AC1 and T47D cells displayed reduced protein levels of the cell cycle regulators Cyclin D1, Retinoblastoma (Rb), phospho-RbS807/811, CDK4, Chk1 and E2F1 that regulate G1/S transition. While short term ENDX (48 hours) treatment did not alter PKCβ1 levels, prolonged in vitro ENDX treatment profoundly reduced PKCβ1 protein levels and the aforementioned cell cycle regulators, faithfully replicating PKCβ1 silencing effects. In contrast, enzastaurin had no impact on proliferation or cell cycle proteins in either model. Consistent with this finding, ENDX, but not TAM or letrozole, reduced protein levels of ERα and cell cycle regulators in vivo. Overexpression of PKCβ1 induced TAM, but not ENDX, resistance and had little impact on responsiveness to enzastaurin. While PRKCB gene amplification was uncommon in newly diagnosed ERα+/HER2- BC (5%, TCGA), PRKCB was amplified in 40% of metastatic ERα+/HER2- BC (PROMISE study). Conclusion: We have confirmed the relevance of a new ENDX target, PKCβ1, in ERα+/HER2- BC. While targeting PKCβ1 kinase activity elicited no anticancer effects in ERα+ cells, PKCβ1 downregulation, either by siRNA or ENDX, resulted in profound ERα turnover, reduced protein levels of essential cell cycle mediators and profoundly inhibited cell proliferation. Furthermore, PKCβ1 protein expression is associated with TAM, but not ENDX, resistance, a finding whose clinical relevance is further magnified by identification of PRKCB amplification in metastatic ERα+ BC, confirming its potential importance in progression. Efforts are currently underway to elucidate the mechanistic basis for ENDX-induced PKCβ1 and ERα degradation and the contribution of these effects to the superior antitumor activity of ENDX in ERα+ BC. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Vera J Suman, Elizabeth S Bruinsma, Ciara C O'Sullivan, Liewei Wang, Richard Weinshilboum, James N Ingle, John R Hawse, Matthew P Goetz. Antitumor activity of Z-endoxifen (ENDX) is mediated via PKCβ1-dependent ERα loss and cell cycle arrest in ERα-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-04.

Abstract Background: The first in-human phase-I clinical trial of the tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in both aromatase inhibitor (AI) and tamoxifen resistant ER+ breast cancer patients (Goetz M.P., et al; JCO, 2017). By utilizing a letrozole-sensitive MCF7 aromatase expressing (MCF7AC1) cell line, and its corresponding letrozole-resistant (MCF7LR) cell line, we have shown superior antitumor and antiestrogenic activity of Z-endoxifen over tamoxifen in the AI-sensitive and resistant settings in vivo. This study also revealed that Z-endoxifen may target protein kinases in addition to estrogen receptor alpha. Follow-up studies demonstrated that Z-endoxifen, but not tamoxifen, binds to protein kinase C beta 1 (PKCβ1), a member of the serine/threonine-specific protein kinase family that regulates signaling pathways involved in cell proliferation and tumorigenic transformation (Guo C., et al; SABCS, 2017). We have further shown that PKCβ1 silencing, similar to Z-endoxifen, potently inhibits ER+ cell proliferation and additionally causes ERα turnover. Here, we report a potential mechanism by which PKCβ1 may mediate its anti-proliferative effects in ER+ breast cancer cells. Methods: The effects of siRNA-mediated PKCβ1-silencing on gene transcription were evaluated by RNAseq in MCF7AC1 cells. RNAseq results were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) in PKCβ1-silenced MCF7AC1 cells as well as in ER+ T47D cells. The effects of Z-endoxifen on the mRNA expression of genes altered by PKCβ1-silencing in MCF7AC1 cells were evaluated by qRT-PCR analysis. PKCβ1 silencing effects on protein expression in MCF7AC1 cells were evaluated by immunoblot analysis. Results: RNAseq analysis of PKCβ1-silenced MCF7AC1 cells compared to non-silenced cells revealed NF-κB signaling pathway as the top biological pathway significantly altered by PKCβ1 silencing (p=5.93e-08). Enhanced mRNA expression of multiple NF-κB downstream target genes including CXCL10, RELB, MMP1 and IL6 were observed following PKCβ1 silencing. qRT-PCR analyses validated increased mRNA expression of these genes in PKCβ1-silenced MCF7AC1 and T47D cells. Notably, Z-endoxifen treatment in MCF7AC1 cells mirrored PKCβ1 silencing effects, resulting in enhanced mRNA expression of the aforementioned genes. Evaluation of the protein expression of RelA and RelB, the canonical and noncanonical NF-κB transcription factors respectively, in PKCβ1-silenced MCF7AC1 cells revealed significant upregulation of RelA (p=0.0203) but not RelB (p=0.1360) expression. ERα silencing in MCF7AC1 cells did not affect RelA protein expression. Consistent with prior reports, treatment of MCF7 cells with TNFα inhibited cell proliferation. Finally, targeted siRNA-mediated silencing of RelA in PKCβ1-silenced MCF7AC1 cells significantly attenuated PKCβ1-mediated growth inhibition (p=0.0002). Conclusion: Our findings demonstrate that both Z-endoxifen treatment and PKCβ1-silencing commonly activates a NF-κB-mediated gene signature in ER+ breast cancer cells. Notably, PKCβ1-silencing induces the protein expression of the NF-κB transcription factor RelA in an ERα-independent manner, which appears to contribute to PKCβ1-mediated growth inhibition. Current studies are ongoing to evaluate the impact of RelA silencing on Z-endoxifen mediated growth inhibition. This information will foster our understanding of the estrogenic and non-estrogenic mechanisms by which Z-endoxifen mediates its antitumor activity in ER+ breast cancer. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Elizabeth S Bruinsma, John R Hawse, Matthew P Goetz. Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-06.