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

项目摘要 急性髓系白血病(AML)是一种致命的血癌，其特征是分化“障碍”，可防止 髓系祖细胞成熟导致不受控制的增殖。化疗，目前的护理标准， 通常是无效的，可能会导致细胞毒性和复发。迫切需要新的治疗方案 治疗急性髓系白血病预后不良。辨证治疗是一种旨在恢复潜伏期成熟的新方法。 编程并诱导细胞周期退出。这种治疗策略对早幼粒细胞(APL)AML亚型是有效的 但在其他AML中未得到充分开发。表观遗传因素有助于维持AML特有的分化障碍。 去甲基酶LSD1已成为分化治疗的一个有前景的靶点。药理抑制 LSD1(LSD1i)在许多AML亚型中诱导细胞分化。然而，分化的程度 不同的急性髓细胞白血病模型不同，在激进的急性髓细胞白血病模型中影响不大。因此，LSD1i不会诱导 作为单药治疗的终末分化。我们假设以额外的表观遗传学为靶点 与LSD1i同时存在的调节剂可能会诱导完全的、终末分化并导致疾病 减刑。为了确定LSD1i的增强子，我们进行了CRISPR分化增益筛选 在LSD1i共同处理或不联合处理的多个AML细胞模型中以染色质为主的sgRNA文库。这些屏幕 揭示了MEN1的KO与LSD1i结合时的协同诱导分化作用。我证实了 联合小分子抑制LSD1和MEN1诱导分化和抑制增殖作用最强 通常在MLL重排的AML中，在选定的MLL野生型AML中程度较小。这项建议具有 两个目标：首先，我将测试靶向MEN1和LSD1抑制剂在 原发患者样本的体外和患者来源的急性髓系白血病移植模型。患者样本将得到处理 将MEN1抑制剂与LSD1抑制剂结合使用，将对分化和增殖产生影响 量化的。其次，我将确定MEN1和LSD1抑制协同的表观基因组学机制 诱导终末分化，然后测试这些机制在原发患者中是否保守 样本。

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.