Validation KAT sgRNAs and supplemental KAT inhibitor response data.

Supplemental analysis of CUT&RUN data, and KAT inhibitor response.

Counts from BioID experiment identifying SETBP1-interactors.

Mutations in SET binding protein 1 ( SETBP1 ) are associated with an adverse prognosis in myeloid malignancies. These mutations stabilize SETBP1 protein, driving increased expression of a progenitor-associated gene expression program through incompletely described mechanisms. A proteomic screen revealed interactions between SETBP1 and members of the MYST acetyltransferase complexes, including the catalytic subunits-KAT6A and KAT7. Mutant SETBP1 increases the localization of MYST complexes at known SETBP1 target genes, including the Hoxa cluster, where it drives increased histone acetylation and gene expression. Treatment of SETBP1 D868N -expressing myeloid progenitors with MYST inhibitors reduced target gene expression. To establish the efficacy of MYST inhibition in vivo , we treated mice harboring a syngeneic SETBP1 -mutant leukemia with the clinical-grade MYST inhibitor-PF-9363. This resulted in complete hematologic control and increased survival. MYST inhibition was also highly effective against a SETBP1 -mutant PDX model. These studies identify MYST acetyltransferases as promising therapeutic targets in SETBP1 -mutant malignancies. Statement of Significance:SETBP1 mutations are markers of high-risk myeloid malignancies, but we lack any targeted therapies to improve outcomes. In this study, we identify MYST acetyltransferases as key drivers of mutant SETBP1-driven transcription. MYST inhibitors are highly effective against SETBP1-mutant leukemia and represent a promising avenue for clinical translation.

SETBP1 expression and chromatin localization in the inducible K562 model.

Abstract Mutations in SETBP1 are associated with adverse prognosis in myeloid malignancies. These mutations stabilize the SETBP1 protein, driving increased expression of a progenitor-associated gene expression program through incompletely described mechanisms. A proteomic screen revealed interactions between SETBP1 and MYST acetyltransferase complexes, including the catalytic subunits—KAT6A and KAT7. In cell line and primary hematopoietic models, mutant SETBP1 increased the localization of MYST complexes at known SETBP1 target genes, including the HOXA cluster, in which they were shown to drive increased histone acetylation and gene expression. Treatment of SETBP1D868N-expressing myeloid progenitors with MYST inhibitors reduced target gene expression. To establish the efficacy of MYST inhibition in vivo, we treated mice harboring a syngeneic SETBP1-mutant leukemia with the clinical-grade MYST inhibitor—PF-9363. This resulted in hematologic control and increased survival. MYST inhibition was also highly effective against a SETBP1-mutant patient-derived xenograft model. These studies identify MYST acetyltransferases as promising therapeutic targets in SETBP1-mutant malignancies. Significance: SETBP1 mutations are markers of high-risk myeloid malignancies, but targeted therapies to improve outcomes in these patient populations are limited. In this study, we identified MYST acetyltransferases as key mediators of mutant SETBP1-driven transcription and showed that MYST inhibitors are highly effective against SETBP1-mutant leukemia models. See related commentary by Soto and Gritsman, p. 506

Acute myeloid leukemia (AML) exhibits substantial transcriptional heterogeneity across differentiation states that influences therapeutic response to BCL2 inhibition with venetoclax. While hematopoietic stem cell (HSC)-like AMLs show high sensitivity to venetoclax and monocytic-like AMLs demonstrate resistance, the therapeutic behavior of leukemias harboring both transcriptional programs remains poorly defined. Analysis of a large AML cohort reveals a distinct patient population exhibiting concurrent HSC- and monocyte-like transcriptional signatures, which we term stem-monocytic AML. Ex vivo drug sensitivity profiling demonstrates that stem-monocytic AMLs exhibit venetoclax resistance comparable to pure monocytic disease, despite expressing HSC-like transcriptional features. Using a leukemia cell line model that recapitulates stem-monocytic AML characteristics, we show through immunophenotyping and single-cell lineage tracing that venetoclax preferentially depletes immature blasts while sparing differentiated monocytic populations. Single-cell transcriptomic and chromatin accessibility analyses identify enrichment of myeloid differentiation transcription factors, particularly PU.1, in resistant populations. A targeted CRISPR knockout screen confirms that PU.1 disruption induces differentiation arrest and enhances venetoclax sensitivity primarily in the immature immunophenotypic compartments. Pharmacologic PU.1 inhibition with the small molecule DB2313 synergizes with venetoclax in both cell line models and primary patient samples. These findings establish stem-monocytic AML as a transcriptionally and functionally distinct subtype and nominate combined PU.1 and BCL2 inhibition as a rational therapeutic strategy for improving venetoclax response in this patient population.

Glycerol gradient sedimentation analysis of SETBP1 complexes.

Mutations in the epigenetic regulator ASXL1 are common in myeloid malignancies and portend a near-universally poor prognosis. While multiple mechanisms for mutant ASXL1-dependent oncogenesis have been proposed, none have been functionally validated in the context of the human hematopoietic stem cell, where these mutations almost certainly arise. Here, we extensively characterized a CRISPR-engineered human hematopoietic stem and progenitor cell model of ASXL1 mutations. In this context, mutant ASXL1 expression decreases differentiation, increases clonogenicity in serial replating experiments, and improves engraftment in immunodeficient mice. We also show that endogenous truncating mutations in ASXL1 drive protein stabilization and confirm that mutant ASXL1 is resistant to proteasomal degradation. At the transcriptional level, these phenotypes are driven by significant repression of the stress-response genes and by increased expression of bromodomain and extra-terminal family protein targets. Using protein-interaction screens, genomic and functional approaches, we link the positive transcriptional changes in ASXL1 -mutant cells to BRD4-dependent RNA polymerase II pause release and identify a mechanism for transcriptional repression via a previously uncharacterized interaction with the transcription factor MECOM. Finally, we demonstrate that ASXL1 -mutant AML exhibits increased MECOM activity consistent with our gene-editing models. Collectively, these studies highlight a highly reproducible model of mutant ASXL1 in the appropriate cell context. Further, they are the first to functionally describe the mutant ASXL1 interactome in the context of the human HSC, identifying new dependencies with therapeutic potential.

SPI1 knockdown does not disrupt dual FLT3/LSD1 inhibition synergy.

Regulon Enrichment Analysis of Primary AML Blast Bulk RNA-seq from 681 Patient Samples

Efficacy of dual FLT3/LSD1 inhibition in MOLM13 cells.

Transcription Factor Enrichment Analysis of Primary AML Bulk RNA-Seq (24 Hours After Drug Treatment)

Supplementary Materials and Methods

Excess over Bliss of Primary AML Blasts from 72 Patient Samples

Drug synergy between FLT3 and LSD1 inhibitors in FLT3-ITD and FLT3-wildtype cell lines.

Super-Enhancer-Type Analysis of STAT5 ChIP-Seq Peaks (24 Hours After Drug Treatment).

Correlation of Regulon Enrichment Signatures with MYC Gene Expression in Primary AML

GFI1 knockdown weakens the effects of dual FLT3/LSD1 inhibition.

Log2 Normalized Counts of Differentially Expressed Genes from MOLM13 Bulk RNA-seq (24 Hours After Drug Treatment)