SETBP1 Regulates Myst Acetyltransferase Complexes to Drive a Leukemogenic Gene Expression Program

Mutations in set binding protein 1 (SETBP1) are frequent in non-classical myeloproliferative disorders and are strongly associated with adverse prognosis. SETBP1 mutations are found in ~45% of chronic neutrophilic leukemia, 24% of atypical chronic myeloid leukemia, 4-15% of chronic myelomonocytic leukemia, and ~30% of juvenile myelomonocytic leukemia cases. Despite strong prognostic significance, we lack a comprehensive understanding of the oncogenic mechanism of SETBP1 mutations and have no therapies that target them.

SETBP1 is a nuclear protein that interacts with chromatin. SETBP1 mutations occur within its degradation motif, with the D868N point mutation being the most prevalent. These point mutations disrupt ubiquitination and degradation, leading to the accumulation of mutant SETBP1 protein. Work by several groups, including our own, has shown that overexpression of SETBP1 protein causes transcriptional dysregulation of gene regulatory programs that drive myeloid progenitor expansion and proliferation. Indeed, we find that expression of mutant SETBP1 increases the growth of murine and human stem and progenitor populations ex vivo and enhances their serial replating potential.

To understand SETBP1’s role in gene regulation in greater depth, we mapped the SETBP1 interactome using a proximity-dependent biotinylation mass spectrometry assay. SETBP1 was fused to a bacterial biotin ligase (BirA), tagging proteins in proximity to SETBP1 with biotin. Biotinylated proteins were purified on a streptavidin column and identified by mass spectrometry. This assay revealed that SETBP1^WT and SETBP1^D868N have very similar interaction profiles. Several epigenetic modulators with known roles in hematopoietic development, gene regulation, and myeloid malignancies were identified. Notably, members of MYST acetyltransferase complexes, including BRPF1, HBO1 (aka KAT7), and KAT6A, were identified as interacting with both wild-type and mutant SETBP1. These putative interactions with SETBP1 were validated by biotin pulldown followed by immunoblot as well as by co-immunoprecipitation.

Through genome-wide profiling by CUT&RUN in mouse primary hematopoietic stem and human K562 cells, we uncovered extensive overlap in genomic occupancy between SETBP1 and HBO1. This included genes known to be activated by mutant SETBP1, including Hoxa9, Mecom, Myc, Meis1, and a large group of ribosomal protein genes. At the biochemical level, we find that mutant SETBP1 increases the interaction of HBO1 with BRPF1, a subunit that enhances the catalytic activity of the complex. To confirm these results, we showed that the expression of mutant SETBP1 increased the deposition of H3K14 acetylation—the histone mark deposited by HBO1. Finally, treatment of SETBP1^D868N-expressing myeloid progenitors with inhibitors of HBO1 reduced the expression of SETBP1 target genes and normalized SETBP1-associated progenitor expansion. Thus, the HBO1 complex plays a critical role in SETBP1-induced oncogenesis and represents a promising target for treating SETBP1-mutant myeloid neoplasms.

Collectively, these studies highlight the important role of SETBP1 in regulating the activity of histone-modifying complexes at leukemia-associated genes. Furthermore, this study represents the first comprehensive map of the SETBP1-interactome and provides a roadmap for future therapeutic development for patients with difficult-to-treat SETBP1-mutant leukemias.