LBPO.ET02 · 实验与分子治疗 · Late-Breaking

氟化cuban-1-yl双胍类化合物克服对标准治疗fulvestrant和palbociclib的激素治疗耐药

Fluorinated cuban-1-yl biguanides overcome hormone therapy resistance to standard of care fulvestrant, and palbociclib

编号 LB196 展板 18 时间 4/20 02:00–05:00 区域 Section 53 主讲 Zhijun Guo, PhD

分会场 Late-Breaking Research: Experimental and Molecular Therapeutics 2

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作者与单位 Authors & Affiliations

Zhijun Guo¹, Jianxun Lei¹, Christian T. Wells¹, Allison M. Makovec¹, Andrew C. Yates¹, Swaathi Jayaraman², John R. Hawse², Joshua A. McCarra¹, Qing Cao¹, Michael J. Pryzbilla¹, Brenda L. Koniar¹, Beverly J. Norris¹, Craig M. Florey¹, Robert J. Schumacher¹, Michael A. Farrar¹, Kaylee L. Schwertfeger¹, Elizabeth A. Ambrose¹, Henry Wong¹, Gunda I. Georg¹, Antonino D'Assoro², Matthew P. Goetz², David A. Potter¹

¹University of Minnesota, Minneapolis, MN,²Mayo Clinic, Rochester, MN

摘要 Abstract

中文摘要

引言：环氧脂肪酸（EpFA）在ER+HER2-乳腺癌（BC）激素治疗耐药（HTR）中的机制尚不明确。EETs是一类通过促进氧化磷酸化（OXPHOS）促成BC进展和HTR的EpFA。然而，EETs如何促成HTR仍不清楚。在本研究中，我们从letrozole耐药（LR）的MCF-7AC1细胞系中分离出两个fulvestrant耐药（FR）亚克隆（称为LR/FR），它们偶然地对CDK抑制剂palbociclib（PR）耐药（称为MCF-7AC1 LR/FR/PR或多重耐药——MCF-7AC1 MR）。这些HTR细胞系表现出CYP3A4上调（2.3至4.3倍）和细胞内(±)14,15-EET水平升高，这些均可被双胍类治疗抑制（P<0.05）。构效关系研究促成了己基cuban-1-yl双胍（HCB）以及氟化HCB衍生物C5F2-HCB和C6F3-HCB的设计，它们可抑制CYP3A4介导的EET生物合成和HTR细胞系的生长。HCB氟化改善了药代动力学（PK）和毒性。方法：测试HCB和氟化HCB对EET生物合成、OXPHOS、PK、生长抑制以及MCF-7AC1 MR肿瘤模型中缺氧逆转的影响。结果：fulvestrant对MCF-7AC1 MR细胞系的选择导致cyclin D1和雌激素受体（ERalpha）表达无法检测，而cyclin E1、CDK4、CDK6、CYP3A4和c-MYC上调（P<0.001）。与亲本MCF-7细胞相比，MCF-7 MR细胞的(±)8,9、(±)11,12和(±)14,15-EET水平分别高出1.8、1.4和1.2倍（P<0.05）。最强效的氟化HCB即C6F3-HCB将细胞内(±)8,9-EET、(±)11,12-EET和(±)14,15-EET水平抑制了53-60%（P<0.029）。与MCF-7细胞系相比，MCF-7AC1 MR细胞被HCB及氟化衍生物更强效地抑制。比较MCF-7 MR细胞系与MCF-7，HCB的IC50值为4.1对7.0 uM，C5F2-HCB为10对25 uM，C6F3-HCB为5.4对12 uM（P值均<0.05）。C5F2-HCB表现出最佳的PK，对MCF-7AC1 MR异种移植模型的治疗显示出缺氧逆转（P<0.05）和C5F2-HCB的短暂抑制（第20天P=0.025）。P/F也短暂抑制该模型（第20天P=0.006）。C5F2-HCB和P/F在体外显示协同作用（CI=0.46），由于三联疗法在小鼠中具有可接受的毒性，从而得以在体内测试C5F2-HCB/P/F。经非参数统计检验（Mann Whitney；P=0.0389；Kolmogorov-Smirnov；P=0.0473），该联合方案相比标准治疗palbociclib/fulvestrant在体内显示协同作用，为该联合方案可能的临床开发提供了途径。结论：MR BC细胞系表现出CYP3A4上调和EETs的生物合成，表明EETs在HTR中的作用。C5F2-HCB/P/F对高度耐药的MCF-7 MR异种移植瘤表现出活性且毒性可接受，表明了一条临床开发的途径。

查看英文原文 English 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 C5F 2 -HCB and C6F 3 -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 (ERalpha) 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, C6F 3 -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 IC 50 values of 4.1 vs. 7.0 uM, C5F 2 -HCB 10 vs. 25 uM, and C6F 3 -HCB 5.4 vs. 12 uM (P values all <0.05). C5F 2 -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 C5F 2 -HCB (P=0.025 at 20 days). P/F also transiently inhibited this model (P=0.006 at 20 days). C5F 2 -HCB and P/F showed synergy in vitro (CI=0.46) leading to testing of C5F 2 -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 C5F 2 -HCB/P/F exhibits activity against the highly resistant MCF-7 MR xenograft with acceptable toxicity indicating an avenue for clinical development.

利益披露 Disclosure

Z. Guo, None.. J. Lei, None.. C. T. Wells, None.. A. M. Makovec, None.. A. C. Yates, None.. S. Jayaraman, None.. J. R. Hawse, None.. J. A. McCarra, None.. Q. Cao, None.. M. J. Pryzbilla, None.. B. L. Koniar, None.. B. J. Norris, None.. C. M. Florey, None.. R. J. Schumacher, None.. M. A. Farrar, None.. K. L. Schwertfeger, None.. E. A. Ambrose, None.. H. Wong, None.. G. I. Georg, None.. A. D'Assoro, None.. M. P. Goetz, None.. D. A. Potter, None.

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