Project 3 - Novel Therapies for Spliceosomal-Mutant MDS.

项目摘要 目前骨髓增生异常综合征（MDS）和急性髓性白血病（AML）的治疗是不令人满意的。 我们和其他人发现，前体mRNA剪接复合物的成分在细胞中反复突变， 并诱导RNA剪接的改变。这种小分子调节剂 复合物（即，剪接调节剂）已经由我们的合作者和其他人开发。我们最近 报道了突变剪接因子的表达或剪接的药理学干扰 调节剂增加了R环的丰度，R环是含有DNA：RNA杂合体的结构， 置换的单链DNA R环触发ATR依赖的DNA损伤检查点反应， 介导R环的分解以保护细胞免受基因组不稳定性和细胞死亡。我们的初步 临床前数据表明剪接体突变细胞比野生型细胞对ATR抑制更敏感。 我们假设剪接因子突变产生了ATR抑制（ATRi）的脆弱性， 当单独使用或与剪接结合使用时， 调节剂药物。我们将在一项ATRi治疗髓系白血病患者的II期临床试验中检验这一假设。 恶性肿瘤和使用明确定义的体外和体内模型。在具体目标1中，我们将进行第二阶段 在复发性/难治性MDS和慢性粒单核细胞白血病（CMML）中进行ATR抑制试验， 确定这种新疗法的敏感性和耐药性的分子基础。我们预测， 与没有剪接体突变的患者相比，剪接体突变对ATRi的应答率增加。 因子突变相关研究将评估药效学终点，并检查 药物敏感性和耐药性，如果它发生。在具体目标2中，我们将测试ATRi与 剪接体突变型MDS模型中的剪接调节剂。初步证据显示 剪接因子突变体表达造血细胞对剪接体的小分子调节剂 加剧R环形成并增加细胞对ATRi的敏感性。我们将使用基因定义的 在体外和体内暴露于多种剪接体调节剂单独或组合的细胞和小鼠模型， 与ATRi的组合。最后，我们将进行全基因组CRISPR/Cas9筛选，以识别基因， 可以使剪接体突变细胞对ATRi的细胞毒性作用敏感的途径， 在未来的临床试验中。该项目通过首次在人类中进行 ATRi在骨髓恶性肿瘤患者中的临床试验。我们将确定遗传生物标志物， 预测对ATRi的反应，并建立提名联合治疗所需的临床前数据 这可用于开发第一种新的：用于治疗MDS和AML的新型药物组合， 剪接因子突变。

Project Summary Current therapies for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are unsatisfactory. We and others discovered that components of the pre-mRNA splicing complex are recurrently mutated in patients with MDS and AML and induce alterations in RNA splicing. Small molecule modulators of this complex (i.e., splicing modulators) have been developed by our collaborators and others. We recently reported that expression of mutant splicing factors or pharmacologic perturbation of splicing with splicing modulators increase the abundance of R loops, which are structures containing DNA:RNA hybrids and displaced single-strand DNA. R loops trigger an ATR-dependent DNA damage checkpoint response that mediates resolution of the R loop to protect cells from genomic instability and cell death. Our preliminary preclinical data suggest that spliceosome mutant cells are more sensitive to ATR inhibition than wild-type cells. We hypothesize that splicing factor mutations create a vulnerability to ATR inhibition (ATRi) that can be exploited for the development of novel therapeutic strategies when used alone or in combination with splicing modulator drugs. We will test this hypothesis in a phase II clinical trial of an ATRi in patients with myeloid malignancies and using well-defined in vitro and in vivo models. In Specific Aim 1, we will conduct a phase II trial of ATR inhibition in relapsed/refractory MDS and chronic myelomonocytic leukemia (CMML) and determine the molecular basis of sensitivity and resistance to this novel therapy. We predict that patients with spliceosome mutations will have an increased response rate to ATRi compared to patients without a splicing factor mutation. Correlative studies will assess pharmacodynamic endpoints and examine the mechanisms of drug sensitivity and resistance, if it occurs. In Specific Aim 2, we will test the efficacy of ATRi in combination with splicing modulators in models of spliceosome-mutant MDS. Preliminary evidence suggests that exposure of splicing factor mutant expressing hematopoietic cells to small molecule modulators of the spliceosome exacerbates R loop formation and increases sensitivity of cells to ATRi. We will use genetically-defined cellular and mouse models exposed in vitro and in vivo to a variety of spliceosome modulators alone or in combination with ATRi. Finally, we will perform a genome-wide CRISPR/Cas9 screen to identify genes and pathways that could sensitize spliceosome-mutant cells to the cytotoxic effects of ATRi that could be pursued in future clinical trials. This project addresses SPORE translational endpoints by conducting a first in human clinical trial of an ATRi in patients with myeloid malignancies. We will identify genetic biomarkers that are predictive of response to ATRi and establish the preclinical data necessary to nominate combination therapies that could be used to develop the first novel:novel combination of drugs for the treatment of MDS and AML with splicing factor mutations.