Development of Novel PROTACs Targeting the ENL YEATS Domain for Treating MLL-rearranged Leukemias

项目总结 混合系白血病(MLL)基因重排约占婴儿急性白血病的80% 淋巴细胞性白血病(ALL)和35-50%的婴儿急性髓系白血病(AML)。患者生育 MLL基因重排与预后不良有关。到目前为止，还没有有效的疗法 被批准用于治疗这种致命的疾病。分子上，MLL的染色体间易位导致In MLL的N末端与各种融合伙伴的C末端的框架融合，称为 “司机”的病变的疾病。在70多个MLL融合伙伴中，一小部分融合占 大多数白血病病例。总体而言，超过90%的MLL重排只涉及四个融合伙伴：AFF1，AF9， En1和AF10，所有这些都是超伸长复合体(SEC)或 组蛋白H3K79甲基转移酶DOT1L。据认为，这些MLL融合通过以下方式共享共同的途径 劫持SEC或DOT1L复合体以促进MLL融合靶基因的异常激活，导致 与白血病的发病机制有关。我们和其他人最近演示了ENL，它是 SEC和DOT1L复合体，对MLL融合的致癌功能至关重要。ENL包含一个进化上的 保守的叶芝结构域，我们发现它是组蛋白乙酰化的阅读器。我们还发现叶芝夫妇 对MLL重排的白血病细胞的生长和存活至关重要的是ENL结构域，而不是AF9。这些 主要研究结果有力地表明，ENL是MLL重排白血病的一个有前途的治疗靶点。然而， 而最近报道的一种小分子抑制剂有效地阻断了叶芝结构域之间的相互作用 对于ENL/AF9和乙酰化组蛋白H3，这种ENL抑制剂对依赖ENL的MLL-1的杀伤作用很小或没有作用。 白血病细胞重排，未能复制enl基因敲除的细胞杀伤效应。我们假设 ENL的药理学降解，而不是依赖于占用驱动的药理学的ENL抑制， 将为治疗MLL重排白血病提供一种新的有效的治疗策略。为了测试这一点 假设，我们建议开发一流的enl小分子降解物作为体内化学探针，使用 蛋白水解靶向嵌合体(PROTAC)技术及其在MLL重排白血病细胞中的应用 和老鼠模型。我们已经产生了有希望的初步结果，表明拟议的研究是 可行。本项目中产生的ENL降解器体内化学探针不仅有助于我们测试和验证 我们的治疗假设，但也可以在未来进一步优化为候选药物，并最终 为MLL重排白血病患者的临床翻译。

PROJECT SUMMARY The mixed-lineage leukemia (MLL) gene rearrangements account for approximately 80% of infant acute lymphoblastic leukemia (ALL) and 35-50% of infant acute myeloid leukemia (AML). Patients bearing rearrangements of the MLL gene are associated with dismal prognosis. To date, no effective therapies have been approved for treating the fatal diseases. Molecularly, inter-chromosomal translocations of MLL lead to in frame fusions of the N-terminus of MLL to the C-terminus of various fusion partners, which are known as the “driver” lesions of the diseases. Among more than 70 MLL fusion partners, a small subset of fusions account for most leukemogenic cases. In ALL, over 90% MLL rearrangements involve only four fusion partners: AFF1, AF9, ENL, and AF10, all of which are components of the super elongation complex (SEC) or the complex of the histone H3K79 methyltransferase DOT1L. It is believed that these MLL fusions share a common pathway by hijacking the SEC or DOT1L complex to promote aberrant activation of the target genes of MLL fusions, leading to the pathogenesis of leukemias. We and others have recently demonstrated that ENL, a component of the SEC and DOT1L complex, is critical for the oncogenic function of the MLL-fusions. ENL contains an evolutionally conserved YEATS domain that we identified as a reader of histone acetylation. We also found that the YEATS domain of ENL, but not AF9, is essential for growth and survival of the MLL-rearranged leukemic cells. These key findings strongly suggest that ENL is a promising therapeutic target for MLL-rearranged leukemias. However, while a recently reported small-molecule inhibitor effectively blocks the interactions between the YEATS domain of ENL/AF9 and acetylated histone H3, this ENL inhibitor has little or no effect on killing ENL-dependent MLL- rearranged leukemia cells, failing to phenocopy the cell killing effect of ENL knockout. We hypothesize that pharmacological degradation of ENL, instead of ENL inhibition that relies on occupancy-driven pharmacology, will provide a novel and effective therapeutic strategy for treating MLL-rearranged leukemias. To test this hypothesis, we propose to develop first-in-class ENL small-molecule degraders as in vivo chemical probes using the proteolysis targeting chimera (PROTAC) technology and evaluate them in MLL-rearranged leukemia cells and mouse models. We have generated promising preliminary results, suggesting that the proposed research is feasible. The ENL degrader in vivo chemical probes generated in this project will not only help us test and validate our therapeutic hypothesis, but can also be further optimized into drug candidates in the future and ultimately translated in the clinic for MLL-rearranged leukemia patients.