Differentiation-focused CRISPR Screen identifies LSD1 and Menin as combination therapy targets that induce terminal differentiation in AML

项目摘要 急性髓细胞白血病（AML）是一种致命的血液癌症，其特征是分化“阻滞”， 骨髓祖细胞成熟导致不受控制的增殖。化疗，目前的标准治疗， 通常无效并可导致细胞毒性和复发。迫切需要新的治疗方案， 治疗预后不良的AML。分化治疗是一种旨在重新激活潜在成熟的新方法 程序并诱导细胞周期退出。这种治疗策略在早幼粒细胞（APL）AML亚型中具有治愈性 但在其他AML中研究不足。表观遗传因素有助于维持AML分化阻滞的特征。 脱甲基酶LSD 1已成为分化治疗的有希望的靶点。药理学抑制 LSD1（LSD1i）在许多AML亚型中诱导细胞分化。然而，分化的程度 在AML模型之间存在差异，在侵袭性AML模型中具有适度的影响。因此，LSD1i不会诱导 终末分化作为单药治疗。我们假设，针对额外的表观遗传 同时与LSD1i调节剂可能会诱导完全，终末分化，并导致疾病 缓解。为了鉴定LSD1i的增强剂，我们用CRISPR进行了分化获得性筛选。 在有或没有LSD1i共处理的多个AML细胞模型中的染色质聚焦的sgRNA文库。这些屏幕 揭示了当MEN1的KO与LSD1i组合时的分化的协同诱导。我确认 LSD 1和MEN 1的组合小分子抑制最能诱导分化并减少增殖 通常在MLL重排的AML中，在较小程度上在选定的MLL野生型AML中。这一建议 两个目标：首先，我将测试靶向MEN1与LSD1抑制剂组合的治疗潜力， 体外和患者来源的AML移植模型中的原代患者样品。将对患者样本进行处理 MEN1抑制剂与LSD1抑制剂组合，并且对分化和增殖的作用将是 量化。其次，我将确定MEN1和LSD1抑制协同作用的表观基因组机制 诱导终末分化，然后测试这些机制是否在原发性患者中保守， 样品

Project Summary Acute myeloid leukemia (AML) is a lethal blood cancer characterized by a differentiation “block” that prevents myeloid progenitor maturation resulting in uncontrolled proliferation. Chemotherapy, the current standard of care, is often ineffective and can result in cytotoxicity and relapse. New treatment options are desperately needed to treat AML’s poor prognosis. Differentiation therapy is a novel method that aims to reactivate latent maturation programs and induce cell cycle exit. This therapeutic strategy is curative in the promyelocytic (APL) AML subtype but underexplored in other AMLs. Epigenetic factors help sustain the characteristic AML differentiation block. The demethylase LSD1 has emerged as a promising target for differentiation therapy. Pharmacologic inhibition of LSD1 (LSD1i) induces cellular differentiation in many AML subtypes. However, the extent of differentiation varies between AML models, with a modest effect in aggressive AML models. Therefore, LSD1i will not induce terminal differentiation as a monoagent treatment. We hypothesized that targeting additional epigenetic regulators simultaneously with LSD1i may induce complete, terminal differentiation and lead to disease remission. To identify potentiators of LSD1i, we conducted CRISPR gain-of-differentiation screens with a chromatin-focused sgRNA library in multiple AML cell models with or without LSD1i co-treatment. These screens unveiled a synergistic induction of differentiation when KO of MEN1 is combined with LSD1i. I confirmed that combinatorial small molecule inhibition of LSD1 and MEN1 induces differentiation and reduces proliferation most commonly in MLL-rearranged AMLs, and to a lesser extent in selected MLL-wild type AMLs. This proposal has two aims: First, I will test the therapeutic potential of targeting MEN1 in combination with LSD1 inhibitors in primary patient samples ex vivo and in patient-derived AML transplant models. Patient samples will be treated with a MEN1 inhibitor in combination with LSD1 inhibitors, and effects on differentiation and proliferation will be quantified. Secondly, I will determine the epigenomic mechanisms by which MEN1 and LSD1 inhibition synergize to induce terminal differentiation, and then test whether these mechanisms are conserved in primary patient samples.