Proteolytic Control and Therapeutic Targeting of Oncogenic Gene Expression in Acute Myeloid Leukemia
Authors: Uni Masahiro , Steinberg Asher Preska , Slavit Alicia , Rha Sophia , Cheng Shuyuan , Schmalbrock Laura K. , Su Jiayu , Ma Jing , McPherson Andrew , Mullighan Charles G. , Haferlach Torsten , Iacobucci Ilaria , Klco Jeffery M , Rabadan Raul , Kentsis Alex 
Published: December 2024

Dysregulated gene expression is one of the most prevalent features in human cancers, which is particularly pronounced in acute myeloid leukemias (AML). Recently, oncogenic gene expression in most subtypes of AML was found to be caused by aberrant assembly of the transcription factor MYB with its coactivator CBP/P300, as revealed using functional proteomics and peptide interference with the selective peptidomimetic inhibitors MYBMIM and CRYBMIM. However, MYB has key functions in normal hematopoiesis, and mutations of MYB and its cofactors are rare in AML. Thus, understanding of mechanisms by which MYB is selectively required for leukemia but not normal cells is a major need to develop selective therapeutic strategies to target MYB.

To elucidate potential epigenetic mechanisms of MYB dysregulation, we used long-read nanopore sequencing to define dominant isoforms aberrantly expressed in AML cell lines as compared to healthy CD34+ blood progenitor cells. In addition to overexpression of the canonical MYB isoform, we identified several C-terminal truncated MYB isoforms that are specifically and aberrantly expressed, as validated by cDNA cloning with anchored PCR, high-resolution protein analysis and mass spectrometry proteomics. Importantly, using short-read transcriptomics we found that these leukemia-specific isoforms are expressed in the majority of patients in diverse cohorts of pediatric and adult AML and MDS (n=887 and 42, respectively).

Using structure-function studies, we found that C-terminal truncated MYB isoforms exhibit reduced proteolysis and increased protein stability. We found that the C-terminal MYB degron is required for the proteolysis required for therapeutic effect of peptidomimetic MYB inhibitors, which lead to dissolution and chromatin eviction of oncogenic MYB enhanceosomes and suppression of oncogenic gene expression. This effect can be blocked by specific proteasome-selective inhibitors but not by overexpression of BCL2 which impairs proteolysis associated with apoptosis and myeloid differentiation. Remarkably, C-terminal MYB degron-dependent proteasomal proteolysis does not involve ubiquitination, as it is unaffected using specific E1 ubiquitin transfer inhibitors, has no measurable biochemical ubiquitination, and does not require ubiquitin transfer-related factors using genome-wide and ubiquitin-focused CRISPR screens. Importantly, ubiquitin-independent proteasomal degradation (UbInPD) was found in multiple diverse AML cell lines, but not in healthy CD34+ cells.

These studies establish UbInPD and epigenetic dysregulation of MYB C-terminal degron as an unanticipated mechanism of oncogene activation in AML. This also provides a potential general mechanism of oncogenic transcription factor protein dysregulation, as similar degrons exist in a variety of other oncogenic transcription factors, with implications for many hematologic malignancies and the development of pharmacologic strategies for their control, such as the recent direct MYB inhibitors.