Fast-kinetics approaches to define direct gene-regulatory functions of MYB in leukemia

项目摘要/摘要 急性髓系白血病(AML)仍然是一个治疗挑战和重大的公共卫生负担， 有必要开发新的治疗方法。转录因子(Tf)myb是一个关键的转录因子。 骨髓干细胞和祖细胞的调节者和急性髓系白血病原癌基因。在之前的工作中，Dr。 Pimkin已经将MYB确立为AML的顶级选择性转录依赖和AML的关键调节因子 免疫逃逸，使其成为进一步机制和治疗探索的重要靶点。这 建议侧重于MYB在AML中的作用机制和药物抑制，重点是Pre-MYB。 在超快动态分辨率下转录动力学和染色质结构的稳态测量。 目的1描述MYB和CDK6之间的功能关系：细胞周期中的细胞周期蛋白D， MYB与CDK6：Cyclin D复合体相互作用导致细胞增殖的假设检验 AML的控制和分化停滞。目标1中的实验将测试MYB之间的相互作用 CDK6：细胞周期蛋白D调节MYB在细胞周期不同时相的转录输出 这种相互作用是否依赖于CDK6的催化活性。AIM 1中的关键医疗模式将包括 靶向蛋白质降解、新生转录、超分辨显微镜和分裂发光 在细胞环境中测量蛋白质-蛋白质的相互作用。目标1的结果将绘制一个 细胞周期中MYB功能的全面机制图和概念性演示 致癌转移因子如何将谱系转录与细胞周期控制结合起来。目标2将定义分子 新型小分子MYB调节剂KI-TM1-001的作用机理及疗效测试 检验Ki-TM1-001是反映功能异质性的MYB激动剂/拮抗剂混合的假设 由MYB驱动的转录。Aim 2中的实验将阐明Ki-TM1-001是否抑制MYB-B的一个子集- 通过破坏MYB和TFIID之间的相互作用来调控基因，同时显示出不同的机制 在MYB调节的基因的另一个子集上发挥作用，在那里它作为激动剂。AIM 2中的关键医疗模式将 包括旨在无偏见地对Ki-TM1-001进行靶向接合分析高级蛋白质组学方法 鉴定其直接相互作用因子，包括光亲和/“点击”试验和蛋白质组整体稳定性改变 分析结合高分辨率质谱仪(PISA)。KI-TM1-001作为急性髓系白血病的疗效观察 治疗方法将在临床前AML模型中进行测试。这些实验将创造出一种经过充分验证的化学物质 MYB功能的探针和进一步治疗开发的先导分子。这是一种 提案包括HHMI、波士顿儿童医院、Dana-Farber的综合专业知识和资源 麻省理工学院癌症研究所、布罗德研究所和科赫综合癌症研究中心。该提案是 旨在为皮姆金博士提供细胞周期控制和化学生物学方面的强化培训，包括 分子开发和目标参与简档，促进他向独立的过渡。

Project Summary/Abstract Acute myeloid leukemia (AML) remains a therapeutic challenge and a significant public health burden, necessitating development of novel therapies. The transcription factor (TF) MYB is a key transcriptional regulator of stem and progenitor cells in the bone marrow and an AML proto-oncogene. In prior work Dr. Pimkin has established MYB as a top selective AML transcriptional dependency and a critical regulator of AML immune escape, making it an important target for further mechanistic and therapeutic exploration. This proposal is focused on the mechanism and pharmacologic inhibition of MYB in AML, with an emphasis on pre- steady state measurements of transcriptional dynamics and chromatin structure at ultra-fast kinetic resolution. Aim 1 will characterize the functional relationship between MYB and CDK6:Cyclin D during the cell cycle, testing the hypothesis that an interaction between MYB and the CDK6:Cyclin D complex links proliferation control and differentiation arrest in AML. Experiments in Aim 1 will test whether the interaction between MYB and CDK6:Cyclin D modulates the transcriptional output of MYB in different phases of the cell cycle and whether this interaction is dependent of the CDK6 catalytic activity. Critical modalities in Aim 1 will include targeted protein degradation, nascent transcriptomics, super-resolution microscopy and split-luminescence measurements of protein-protein interactions in the cellular context. Results of Aim 1 will paint a comprehensive mechanistic picture of the MYB function during the cell cycle and conceptually demonstrate how an oncogenic TF couples lineage transcription with cell cycle control. Aim 2 will define the molecular mechanism of action and test therapeutic efficacy of a novel small-molecule MYB modulator KI-TM1-001, testing the hypothesis that KI-TM1-001 is a mixed MYB agonist/antagonist reflecting functional heterogeneity of MYB-driven transcription. Experiments in Aim 2 will elucidate whether KI-TM1-001 inhibits a subset of MYB- regulated genes by disrupting the interaction between MYB and TFIID, while displaying a distinct mechanism of action at another subset of MYB-regulated genes where it acts as an agonist. Critical modalities in Aim 2 will include methods of advanced proteomics aimed at an unbiased target engagement profiling of KI-TM1-001 to identify its direct interactors, including photoaffinity/”click” assays and proteome integral stability alteration assay coupled with high-resolution mass spectrometry (PISA). The efficacy of KI-TM1-001 as an AML therapeutic will be tested in pre-clinical AML models. These experiments will create a well-validated chemical probe for MYB function and a lead molecule for further therapeutic development. The environment for the proposal includes combined expertise and resources of HHMI, Boston Children’s Hospital, Dana-Farber Cancer Institute, Broad Institute and Koch Center for Integrative Cancer Research at MIT. The proposal is designed to provide Dr. Pimkin with intensive training in cell cycle control and chemical biology, including small molecule development and target engagement profiling, fostering his transition to independence.