Genetics and Signaling of Drug Resistance and Sensitivity in AML Cell Lines, Xenografts, and Primary Patient Samples

急性髓系白血病(AML)是一种高度致命性的白血病亚型，5年存活率为20%。使用 下一代测序的到来，一些特定的基因损伤驱动了AML并提供了 预后信息已经确定。我们小组以前的工作(共同PD/PI，Druker)提供了证据 靶向ABL激酶抑制剂伊马替尼阻断慢性髓系BCR-ABL融合蛋白活性 白血病(CML)，极大地提高了患者的生存率。类似的方法也被应用于AML，一种更 基因复杂的白血病，一些药物改善了结果，但没有一种药物如此成功 作为ABL抑制剂治疗慢性粒细胞白血病，由于反应不完全和耐药性的快速发展。项目1，共 这项名为《规避耐药性的药物组合》(D2CR)的博士学位论文将侧重于了解 药物敏感性或耐药性增强的内在机制，目的是设计新的治疗方法 战略。对于这个项目，我们的长期目标是提名能够增强前期药物的药物 用于将在临床上测试的联合方案的敏感性和/或规避耐药性 审判。我们的直接目标是确定重要的目标基因和途径 对特定药物的敏感性或耐药性，并使用细胞系、患者样本、 以及异种移植来源的细胞。这些目标是基于我们的中心假设，即异质 AML的遗传格局与复杂的信号反馈环共同作用于内在 药物敏感和耐药的机制。项目1将提供关键的临床前数据以推进 在项目3的初级患者样本和异种移植模型中测试的组合中使用的候选药物。 为了实现这些目标，提出了3个目标：1)确定药物敏感/耐药的遗传机制 通过必要基因和耐药性筛查在AML细胞系中-我们将在全基因组范围内进行 CRISPR/Cas必需基因和再敏化筛选以确定参与基因靶点和途径 药物活性增强或抗药性增强。这两个屏幕组合生成的数据将提供关键 对5种选定药物(crenolanib，quizartinib， Ruxolitinib、Trametinib和ventoclax)在具有不同遗传背景的AML细胞中。2)计算上 验证、提炼和告知与药物内在机制有关的候选途径和基因 敏感性或抵抗力--此迭代建模步骤将利用我们的 内部癌症目标知识库，并将在AIM 3.3中优先考虑目标以进行进一步验证) 在假设驱动、重点关注的CRISPR/CAS实验中验证新的基因靶点-我们将开发一种 靶向CRISPR/Cas sgRNA文库干扰推测与药物敏感性有关的基因或 细胞系模型、异种移植和原发患者样本中的耐药性。我们希望确定基因靶点 项目3药物联合检测中的进一步讯问。

Acute myeloid leukemia (AML), a highly lethal subtype of leukemia, has a 5-year survival rate of <20%. With the advent of next-generation sequencing, a number of specific genetic lesions that drive AML and provide prognostic information have been identified. Previous work by our group (co-PD/PI, Druker) provided proof that imatinib, a targeted ABL kinase inhibitor that blocks activity of the BCR-ABL fusion protein in chronic myeloid leukemia (CML), dramatically improves patient survival. Similar approaches have been applied to AML, a more genetically complex leukemia, and some drugs have improved outcomes, but none have been as successful as ABL inhibitors for CML due to incomplete responses and the rapid development of resistance. Project 1 of this DRSC Program, Drug Combinations to Circumvent Resistance (D2CR), will focus on understanding intrinsic mechanisms of enhanced drug sensitivity or resistance, with the goal of devising novel therapeutic strategies. For this Project, our long-term goals are to nominate drugs that enhance upfront drug sensitivity and/or circumvent resistance for use in combination strategies that will be tested in clinical trials. Our immediate goals are to identify essential target genes and pathways contributing to sensitivity or resistance to specific drugs and to validate their roles using cell lines, patient samples, and xenograft-derived cells. These goals are based on our central hypothesis that the heterogeneous genetic landscape of AML, in tandem with complex signaling feedback loops, contributes to intrinsic mechanisms of drug sensitivity and resistance. Project 1 will provide critical preclinical data to advance drug candidates for use in combinations tested in primary patient samples and xenograft models in Project 3. To accomplish these goals, 3 Aims are proposed: 1) Identify genetic mechanisms of drug sensitivity/resistance in AML cell lines through essential-gene and drug-resistance screens – We will perform genome-wide CRISPR/Cas essential gene and re-sensitization screens to identify gene targets and pathways contributing to enhanced drug activity or resistance. Data generated by the combination of these 2 screens will provide key insights into cell-intrinsic mechanisms of sensitivity or resistance to 5 select drugs (crenolanib, quizartinib, ruxolitinib, trametinib, and venetoclax) in AML cells with diverse genetic backgrounds. 2) Computationally validate, refine, and inform candidate pathways and genes contributing to intrinsic mechanisms of drug sensitivity or resistance – This iterative modeling step will leverage the intrinsic genetic factors identified by our in-house Cancer Targetome Knowledgebase and will prioritize targets for further validation in Aim 3. 3) Validate new gene targets in hypothesis-driven, focused CRISPR/Cas experiments – We will develop a targeted CRISPR/Cas sgRNA library to perturb genes hypothesized to contribute to drug sensitivity or resistance in cell line models, xenografts, and primary patient samples. We expect to identify gene targets for further interrogation in drug combination testing in Project 3.