Ctr9 as a Predictive Biomarker for EZH2 Inhibitor Sensitivity

项目总结 大约70%的乳腺癌(BC)是雌激素受体(ER)阳性的，也是以管腔为特征的 BC型；因此，要进行内分泌治疗(ET)。ER阳性腔细胞向ER阴性细胞的转变 干细胞样细胞(SLCs)是内毒素抵抗的主要机制；然而，潜在的机制仍然 很大程度上是未知的。我们发现，CTR9是PAFC转录激活因子复合体的一个亚单位，是一种决定因子。 不知道腔细胞的身份。使用可诱导和稳定的Ctr9基因敲除(KD)BC细胞系，实质上 观察到内质网稳定性降低和染色质上抑制性组蛋白标记H3K27me3的增加 当Ctr9耗尽时，表明腔内BC细胞向SLC细胞过渡。表现出的细胞可塑性 通过Ctr9结合两种下游效应蛋白，Jarid2和KDM6A，控制抑制水平 组蛋白标记，H3K27me3。Ctr9沉默导致PRC2亚单位Jarid2的减少，并触发PRC2 亚型从活性较低的PRC2.2切换到活性较高的H3K27me3的PRC2.1。除了增强 PRC2 H3K27me3沉积活性，Ctr9 KD导致KDM6A减少，KDM6A是 H3K27me3。此外，Ctr9的缺失会产生脆弱性，使BC细胞对EZH2高度敏感 抑制剂(EZHi)。EZH2在SLCs中高表达，用于干细胞的维持和扩增。最近， 他赛托斯特是一种EZH2 i，被FDA批准用于治疗滤泡性淋巴瘤。我们假设损失了 Ctr9导致了从表达Ctr9的管腔谱系向EZH2统治的SLC的转变，因此细胞 对乙型肝炎病毒敏感。我们将通过追求三个目标来验证我们的假设：(1)剖析Ctr9是如何耗尽的 结果：(2)确定Jarid2和KDM6A在细胞周期中的作用。 (3)确定Ctr9和H3K27me3是否具有预测性 体内EZH2敏感性的生物标志物及Ctr9水平与H3K27me3的负相关研究 人类标本。因为与ER阴性的BC相比，EZH2的表达水平更高 ER+BC、EZH2抑制在ER阴性BC中已被广泛研究。这项研究具有很大的影响力 因为我们的发现，Ctr9区分腔细胞中EZH2的活性和H3K27me3的水平开启了一种兴奋- 使用EZH2 i靶向Ctr9low/H3K27me3高ER+BC的可能性。成功完成这项研究将 一项临床试验证明使用Ctr9/H3K27me3作为独立的生物标志物来选择ER+BC患者进行治疗 与EZH2i。

PROJECT SUMMARY Approximately 70% of breast cancers (BC) are estrogen receptor (ER)-positive, also characterized as luminal- type BC; thus, are treated with endocrine therapy (ET). The transition from ER+ luminal cells to ER-negative ‘stem-like cells’ (SLCs) is a major mechanism of ET resistance; however, the underlying mechanism remains largely unknown. We discovered that Ctr9, a subunit of PAFc transcriptional activator complex, is a determi- nant of luminal cell identity. Using inducible and stable Ctr9 knockdown (KD) BC cell lines, a substantial decrease of ER stability and increase of H3K27me3, a repressive histone mark on chromatin, were observed upon Ctr9 depletion, indicating of a transition from luminal BC cells to SLCs. The cellular plasticity manifested by Ctr9 engages two downstream effector proteins, Jarid2 and KDM6A, that control the levels of repressive histone mark, H3K27me3. Ctr9 silencing leads to decreased Jarid2, a subunit of PRC2, and triggers a PRC2 subtype switch from less active PRC2.2 to PRC2.1 with higher H3K27me3 activity. In addition to enhancing PRC2 H3K27me3 deposition activity, Ctr9 KD results in a decrease of KDM6A, the ‘eraser’ enzyme for H3K27me3. Furthermore, Ctr9 depletion generates vulnerability that renders BC cells hypersensitive to EZH2 inhibitors (EZHi). EZH2 is highly expressed in SLCs for stem cell maintenance and expansion. Recently, tazemetostat, an EZH2i, is FDA-approved for the treatment of follicular lymphoma. We hypothesize that loss of Ctr9 leads to the transition from a Ctr9-expressing luminal lineage to an EZH2-governed SLC, thus the cells become sensitive to EZH2i. We will test our hypothesis by pursuing three aims: (1) Dissect how Ctr9 depletion results in transition from ER+ luminal cells to SLCs; (2) Determine the roles of Jarid2 and KDM6A during luminal to SLC transition and sensitivity to EZH2i; (3) Determine whether Ctr9 and H3K27me3 are predictive biomarkers for EZH2i sensitivity in vivo and study the inverse correlation of Ctr9 levels with H3K27me3 in human specimens. Because of the higher levels of expression of EZH2 in ER-negative BC as compared to ER+ BC, EZH2 inhibition has been extensively investigated in ER-negative BC. The study is of high impact because our findings that Ctr9 demarcates EZH2 activity and H3K27me3 levels in luminal cells opens an excit- ing possibility to target Ctr9low/H3K27me3high ER+ BC using EZH2i. Successful completion of this study will justify a clinical trial to use Ctr9/H3K27me3 as independent biomarker to select ER+ BC patients for treatment with EZH2i.