Dissecting and targeting canonical and non-canonical oncogenic functions of EZH2 in cancer

项目概要/摘要 携带MLL1重排的白血病患者通常表现出非常差的预后，需要新的治疗 治疗策略。多项独立研究已鉴定出 EZH2（一种组蛋白甲基转移酶）和 Polycomb 抑制复合物 2 (PRC2) 的催化亚基，作为 MLL1 重排中有吸引力的药物靶点 白血病。然而，目前EZH2的催化抑制剂的抗肿瘤作用有限。我们的初步 研究表明，EZH2 还结合 c-MYC，并在癌基因（例如 MLL1 重排白血病中的 Cyclin E1），这与众所周知的 PRC2:EZH2 驱动的规范不同 与基因抑制相关的功能。为了通过以下方式针对典型和非典型致癌作用 EZH2，我们使用蛋白水解靶向嵌合体 (PROTAC) 技术来生成新型 EZH2 小分子 分子降解剂，包括 MS177。 Our extensive preliminary studies have demonstrated that MS177 有效降解 PRC2:EZH2 和 EZH2 的非 PRC2 伴侣（例如 c-MYC，它将 EZH2 结合到 激活致癌基因），从而抑制 EZH2 的典型和非典型致癌活性 肿瘤。重要的是，我们的初步结果还表明 MS177 优于所有酶抑制剂 EZH2 治疗 MLL1 重排白血病。因此，我们假设：（1）EZH2 具有较少研究的、非 canonical oncogenic function, which acts in parallel with the PRC2:EZH2-dependent one to produce more MLL1 重排白血病中出现侵袭性肿瘤表型； (2) 针对规范和非规范 PROTACs 对 EZH2 的典型活性代表了一种新颖且优于抑制 EZH2 的治疗策略 EZH2 单独的酶活性。 Dissection of the mechanisms underlying EZH2-mediated oncogenesis and development of an optimal EZH2 PROTAC as a drug candidate will have significant impact on improving MLL1重排白血病患者的治疗。为了实现这一目标，我们将进一步描述这种新的 EZH2 在 MLL1 重排白血病中的非典型致癌作用（目标 1a）并定义 EZH2 的作用 PROTAC 抑制 EZH2 的典型和非典型致癌活性（目标 1b）。我们会 还通过采用多个独立的方法确定 EZH2 PROTAC 的体外和体内治疗效果 MLL1 重排白血病模型，包括人/鼠癌细胞和患者来源的异种移植物 (PDX) （目标 2）。最后，我们将优化 EZH2 PROTAC 线索，使其成为候选药物（目标 3）。完成 拟议的研究不仅将为 MLL1 重排白血病提供新的机制理解 开发，但也将验证创新的治疗策略并提供有前途的治疗候选药物 用于治疗受影响的癌症患者。

PROJECT SUMMARY/ABSTRACT Leukemia patients bearing MLL1 rearrangement generally display very poor prognosis, demanding new treatment strategies. Multiple independent studies have identified EZH2, a histone methyltransferase and catalytic subunit of Polycomb Repressive Complex 2 (PRC2), as an attractive drug target in MLL1-rearranged leukemias. However, the current catalytic inhibitors of EZH2 have limited anti-tumor effect. Our preliminary studies show that EZH2 also binds c-MYC and has a non-canonical function in activation of oncogenes (such as Cyclin E1) in MLL1-rearranged leukemias, which differs from the well-known PRC2:EZH2-driven canonical function related to gene repression. In order to target both canonical and non-canonical oncogenic actions by EZH2, we used the Proteolysis Targeting Chimera (PROTAC) technology to generate novel EZH2 small- molecule degraders including MS177. Our extensive preliminary studies have demonstrated that MS177 effectively degrades both PRC2:EZH2 and non-PRC2 partners of EZH2 (e.g., c-MYC, which binds EZH2 to activate oncogenes), thus suppressing both canonical and non-canonical oncogenic activities of EZH2 in tumor. Importantly, our preliminary results also show that MS177 is superior to all of enzymatic inhibitors of EZH2 in treating MLL1-rearranged leukemias. Thus, we hypothesize that: (1) EZH2 has a less-studied, non- canonical oncogenic function, which acts in parallel with the PRC2:EZH2-dependent one to produce more aggressive tumor phenotypes seen in MLL1-rearranged leukemias; and (2) targeting both canonical and non- canonical activities of EZH2 by PROTACs represents a novel and superior therapeutic strategy to inhibition of EZH2’s enzymatic activity alone. Dissection of the mechanisms underlying EZH2-mediated oncogenesis and development of an optimal EZH2 PROTAC as a drug candidate will have significant impact on improving treatments for MLL1-rearranged leukemia patients. Towards this goal, we will further characterize such a new non-canonical oncogenic role of EZH2 in MLL1-rearranged leukemias (Aim 1a) and define effects of EZH2 PROTACs on suppressing both canonical and non-canonical oncogenic activities of EZH2 (Aim 1b). We will also determine in vitro and vivo therapeutic effects of EZH2 PROTACs by employing multiple independent MLL1-rearranged leukemia models including human/murine cancer cells and patient-derived xenografts (PDX) (Aim 2). Lastly, we will optimize our EZH2 PROTAC leads into a drug candidate (Aim 3). Completion of the proposed research will not only provide novel mechanistic understanding of how MLL1-rearranged leukemias develop, but will also validate an innovative therapeutic strategy and deliver a promising therapeutic candidate for the treatment of affected cancer patients.