Abstract B009: DNA-proximal MYB phosphorylation is required for oncogenic transcription in acute myeloid leukemia

Abstract

Dysregulation of gene expression is an essential feature of most human cancers, including acute myeloid leukemia (AML), yet genetically distinct AML subtypes often share pathologic dependencies on transcription factors and gene-regulatory mechanisms. In particular, MYB is aberrantly activated in most studied forms of AML, where it is required for blockade of differentiation and self-renewal of leukemia cells. MYB is rarely mutated and generally not over-expressed in most human myeloid leukemias, presenting a key question concerning mechanisms of its activation that should be targeted therapeutically. To elucidate potential mechanisms of MYB activation, we investigated post-translational modifications (PTMs) of endogenous MYB proteins purified from human AML cells. Using comprehensive bottom-up high-resolution mass spectrometry, we achieved 95% MYB peptide sequence coverage and identified 10 unique MYB amino acid residues containing phosphorylated, acetylated and methylated PTMs. This included phosphorylation of S11 and S12 in the N-terminal domain (NTD) proximal to the DNA-binding domain of MYB, as validated using phospho-specific MYB antibody, mutagenesis, and dephosphorylation experiments. Notably, S11/S12 phosphorylation was preferentially detected across multiple AML cell lines and patient specimens from diverse molecular subtypes as compared to healthy human blood progenitor and stem cells. We found that MYB is required for the growth and survival of leukemia cells, which can be rescued by the expression of wild-type MYB but not MYB mutants that cannot be phosphorylated at S11/S12 in its NTD. Using label-free quantitative mass spectrometry combined with affinity purifications, we defined specific protein co-factors that are selectively assembled with phosphorylated as compared to non-phosphorylated MYB in the nuclei of AML cells. We found that S11-phosphorylated MYB preferentially assembles with other hematopoietic transcription factors including LMO2, LYL1, JUN, and TCF12. Co-assembled MYB transcription factors were associated with cooperative chromatin complexes regulating genes controlling leukemia cell differentiation, growth and survival. To nominate upstream therapeutic vulnerabilities, we identified candidate kinases regulating MYB N-terminal phosphorylation using diverse approaches including recombinant kinase screen and motif-based prediction. Dual inhibition of CK2 and PIM3 reduced MYB N-terminal phosphorylation, suggesting that kinase-dependent control of MYB phosphorylation may provide a pharmacologically actionable strategy to disrupt MYB-driven transcriptional circuitry. In all, these studies identify DNA-proximal phosphorylation of MYB as a critical regulatory mechanism required for MYB oncogenesis through cooperative organization of transcription factors. This work reveals a shared, mutation-independent mechanism of MYB activation across AML subtypes and nominates MYB N-terminal phosphorylation and its upstream kinases as candidate therapeutic targets for MYB-dependent leukemia.