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

快速动力学方法定义 MYB 在白血病中的直接基因调控功能

• 批准号: 10644259
• 负责人: Maxim Pimkin
• 金额: $ 22.08万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644259
• 关键词: Acute Myelocytic Leukemia; Adult; Agonist; Allogeneic Bone Marrow Transplantation; Allogenic; Benchmarking; Binding; Biological Assay; Biology; Bone Marrow; Boston; Cell Cycle; Cell Cycle Kinetics; Cell Cycle Regulation; Cell Differentiation process; Cell Line; Chemicals; Childhood; Chromatin Structure; Complex; Coupled; Couples; Cyclin D1; Cyclins; Dana-Farber Cancer Institute; Data; Dependence; Development; Engineering; Environment; Etiology; Fostering; G1/S Transition; Gene Expression; Genes; Genetic Transcription; Goals; Hematopoietic Stem Cell Transplantation; Heterogeneity; Immune; Incidence; Kinetics; Laboratories; Lead; Link; Luminescent Measurements; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Mediating; Mentors; Methods; Modality; Modeling; Molecular Mechanisms of Action; Molecular Probes; Mus; Oncogenic; Oncology; Outcome; Output; Paint; Pediatric Hospitals; Phase; Phosphorylation; Phosphotransferases; Proliferating; Proteome; Proteomics; Proto-Oncogenes; Public Health; Publishing; Regulation; Regulator Genes; Reporter; Resolution; Resources; Structure-Activity Relationship; System; TAF1 gene; Testing; Therapeutic; Time; Tissues; Training; Transcription Initiation; Treatment Efficacy; Treatment outcome; Work; Xenograft Model; acute myeloid leukemia cell; antagonist; anticancer research; design; experimental study; human model; inhibitor; leukemia; novel; novel therapeutics; pharmacologic; pre-clinical; programs; protein degradation; protein protein interaction; safety testing; screening; small molecule; small molecule inhibitor; stem; stem cells; superresolution microscopy; targeted treatment; therapeutic development; therapeutic evaluation; therapeutic target; tool; transcription factor; transcriptomics; tumorigenesis

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.

项目摘要/摘要 急性髓样白血病（AML）仍然是治疗性挑战，伯恩伯恩（Burnen） 需要开发新的疗法。转录因子（TF）MYB是关键转录 在先前的工作中，骨髓和AML原型癌基因的茎博士和祖细胞。 Pimkin已将MYB确立为顶级选择性AML转录依赖性和AML的关键调节剂 免疫逃脱，使其成为进一步机械和治疗探索的重要目标。这 提案的重点是AML中MYB的机制和药物抑制，重点是 超快速动力学分辨率下转录动力学和染色质结构的稳态测量。 AIM 1将在细胞周期中表征MyB和CDK6：Cyclin d之间的功能关系， 测试MYB与CDK6：Cyclin d复合物之间的相互作用的假设链接增殖 AML的控制和分化停滞。 AIM 1中的实验将测试MYB之间的相互作用是否 和CDK6：细胞周期蛋白D在细胞周期的不同阶段调节MYB的转录输出， 这种相互作用是否取决于CDK6催化活性。 AIM 1中的关键方式将包括 靶向蛋白质降解，新生转录组学，超分辨率显微镜和分裂发光 在细胞环境中的蛋白质蛋白相互作用的测量。 AIM 1的结果将绘制 在细胞周期中MYB功能的全面机械图，并在概念上证明 致癌的TF夫妇如何通过细胞循环控制谱系转录。 AIM 2将定义分子 新型小分子MYB调节剂KI-TM1-001的作用机理和测试疗法效率， 测试Ki-TM1-001是反映功能异质性的混合MYB激动剂/拮抗剂的假设 MYB驱动的转录。 AIM 2中的实验将阐明Ki-TM1-001是否抑制了MYB-的子集 通过破坏MYB和TFIID之间的相互作用来调节基因，同时显示出独特的机制 在MYB调节的基因的另一个子集中起作用的作用，该基因起着激动剂的作用。 AIM 2中的关键方式将 包括针对Ki-TM1-001的公正目标参与分析的高级蛋白质组学方法 识别其直接交互者，包括光性/“点击”测定和蛋白质组积分稳定性更改 测定与高分辨率质谱法（PISA）结合。 Ki-TM1-001作为AML的效率 治疗性将在临床前AML模型中进行测试。这些实验将产生一个验证的化学物质 探测MYB功能和铅分子的进一步治疗发育。环境 提案包括HHMI的组合专业知识和资源，波士顿儿童医院，Dana-Farber 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。该提议是 旨在为Pimkin博士提供细胞周期控制和化学生物学的深入培训，包括小 分子开发和目标参与分析，促进了他向独立的过渡。