Molecular basis for microtubule-destabilization dependent MYB degradation

Dana-Farber Cancer Inst, Boston MA

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Abstract

Project 2 Abstract MYB is a master transcription factor in hematopoiesis, a recurrently translocated oncogene, and a selective dependency in leukemia. Following a report that microtubule destabilizing agent (MDA) mebendazole powerfully and selectively induces degradation of the MYB protein, we demonstrated the same effect in structurally diverse MDAs. Using proteome-wide mass spectrometry and genetic screens, we found that MDA-induced MYB degradation is fully rescued by proteasome or E1 ubiquitin ligase inhibitors, and identified MAP3K1 as the E3 ubiquitin ligase required for MDA-dependent MYB degradation. Strikingly, MAP3K1 is unique among E3 ubiquitin ligases in having a tubulin binding domain in addition to ubiquitin ligase and kinase domains. Our laboratory has identified several mechanisms of drug-induced degradation of hematopoietic transcription factors. We found that thalidomide derivatives act as molecular glues to induce degradation of IKZF1 and IKZF3 by the CRBNCRL4 E3 ubiquitin ligase, a finding that explains the activity of these drugs in multiple myeloma. 1-3 Subsequently, we described a mechanism of drug-induced polymerization and degradation of BCL6, enabling a novel therapeutic approach for lymphomas. 4 These mechanisms of drug-induced protein degradation rely on interactions that do not occur physiologically. MYB-induced degradation is different, as multiple biochemically distinct mechanisms of destabilizing microtubules induce MYB degradation. For this reason, elucidation of the mechanism linking microtubule polymerization with MYB stability has implications for both drug activity and the normal physiological link between microtubule destabilization and MYB activity. We hypothesize that the link between MYB stability and microtubule polymerization will reveal a novel mechanism of transcription factor regulation through microtubule-dependent regulation of MYB stability, cellular localization, protein-protein interactions, and transcriptional activity. We now propose to elucidate the mechanism and biology of MDA-dependent MYB degradation, which may have implications both for cancer drug development and for the regulation of hematopoiesis. In Specific Aim 1, we will dissect the activity of MAP3K1 activity, evaluating its domain structure, microtubule-dependent protein- protein interactions, and ubiquitin ligase activity. In Specific Aim 2, we propose to dissect MYB structure- function, elucidating the critical amino acids for degradation and leukemia-dependent cell killing, including mutations that cause resistance to MDA in MLL-AF9 leukemia models. In Specific Aim 3, we will investigate the biological connections between microtubule polymerization, cell cycle, and MYB activity. We will investigate the implications of these findings for the role of MDAs in normal hematopoietic progenitor cells and in MYB- dependent leukemia cells.