MDA-MB-468 cells treated with docetaxel or EZH2i+AKTi II

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy, however responses are typically short-lived1,2. Thus, there is an urgent need to develop more effective treatments. PI3K pathway components represent plausible therapeutic targets, as approximately 70% of TNBCs have PIK3CA/AKT1/PTEN alterations3–6. However, unlike hormone receptor-positive tumors, it is still unclear if or how PI3K pathway inhibitors will be effective in triple-negative disease7. Here we identify a promising AKT inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors potently synergize with agents that suppress the histone methyltransferase, EZH2, and promote robust tumor regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. Once differentiated, these agents kill TNBCs by hijacking signals that normally drive mammary gland involution. Finally, using machine learning approach, we developed a classifier that can be used for patient selection. Together these findings identify a promising therapeutic strategy for this highly aggressive tumor type and illustrate how deregulated epigenetic enzymes can insulate tumors from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit. HCC38, HCC1395, and HCC1937 cells were treated EZH2i (5 uM tazemetostat), AKTi (5 uM ipatasertib) or EZH2i+AKTi. On day -5, MDA-MB-468 cells were seeded at 40% confluency and treated with 5uM tazemetostat or DMSO. On day -3, cells were passaged again at 40% confluency and maintained in DMSO or tazemetostat. On day -1, cells were seeded into 6cm plates at 250,000 cells per plate and maintained in DMSO or tazemetostat. On day 0, cells were treated in media containing 2% FBS with AKTi (ipatasertib) and/or EZH2i (tazemetostat). Cells were collected at 24 hours, cells were isolated and then prepared for ATACseq.

三阴性乳腺癌（Triple negative breast cancer, TNBC）是最具侵袭性的乳腺癌亚型，复发率最高[1]。晚期三阴性乳腺癌的主流标准治疗方案为联合或不联合免疫治疗的全身化疗，但患者应答通常持续时间较短[1,2]。因此，亟需开发更为有效的治疗手段。PI3K通路（PI3K pathway）相关组分是颇具潜力的治疗靶点，因为约70%的三阴性乳腺癌存在PIK3CA/AKT1/PTEN基因突变[3-6]。然而，与激素受体阳性肿瘤不同，目前仍不明确PI3K通路抑制剂是否可在三阴性乳腺癌中发挥疗效，以及其发挥疗效的具体机制[7]。本研究鉴定出一种颇具前景的基于AKT抑制剂（AKT inhibitor）的三阴性乳腺癌治疗联合方案。具体而言，本研究证实AKT抑制剂可与抑制组蛋白甲基转移酶（histone methyltransferase）EZH2的药物产生强效协同作用，并在多种体内三阴性乳腺癌模型中促成显著的肿瘤消退。AKT抑制剂与EZH2抑制剂通过以下方式发挥上述作用：首先协同将基底样三阴性乳腺癌细胞诱导为分化程度更高的管腔样细胞状态，而单一药物无法有效实现这一过程。细胞完成分化后，这类药物可通过劫持正常驱动乳腺退化的信号通路来杀伤三阴性乳腺癌细胞。最后，本研究借助机器学习（machine learning）方法开发了一款可用于患者筛选的分类器（classifier）。综上，本研究的发现为这种高侵袭性肿瘤类型确立了一种极具前景的治疗策略，并阐明了失调的表观遗传酶（epigenetic enzymes）如何使肿瘤逃脱致癌脆弱性的靶向打击。本研究同时揭示了如何通过劫持发育过程中的组织特异性细胞死亡通路来实现治疗获益。HCC38、HCC1395及HCC1937细胞分别接受EZH2抑制剂（EZH2i，5μM他泽司他（tazemetostat））、AKT抑制剂（AKTi，5μM伊帕替尼（ipatasertib））或EZH2i联合AKTi处理。第-5天时，将MDA-MB-468细胞以40%汇合度接种，并用5μM他泽司他或二甲基亚砜（DMSO）处理；第-3天时，再次以40%汇合度传代细胞，并用DMSO或他泽司他维持培养；第-1天时，将细胞以每孔250,000个细胞的密度接种于6cm培养皿中，并用DMSO或他泽司他维持培养；第0天时，在含2%胎牛血清（FBS）的培养基中加入AKTi（伊帕替尼）和/或EZH2i（他泽司他）处理细胞；处理24小时后收集细胞，分离细胞并制备用于ATAC测序（ATACseq）的样品。