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

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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.