Myeloid Lineage Enhancers Drive Oncogene Synergy in CEBPA/CSF3R Mutant Acute Myeloid Leukemia

Abstract

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. This finding of mutation-synergy is broadly applicable other mutations that activate the JAK/STAT pathway or disrupt CEBPA function (i.e. activating mutations in JAK3 and Core Binding Factor translocations). The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation, confirming predictions from clinical sequencing data. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity. B.J.D.

Combined inhibition of JAK/STAT pathway and lysine-specific demethylase 1 as a therapeutic strategy in CSF3R/CEBPA mutant acute myeloid leukemia

“…We previously reported that retroviral expression of mutant CSF3R (CSF3R T618I ) and C-terminal mutant CEBPA (CEBPA V314VW ) in mouse bone marrow is sufficient to permit indefinite culture in cytokine-free media (17). To identify therapies active against this subtype of AML, we performed a functional screen utilizing a library of compounds for which we have accumulated sensitivity data on a large number of primary patient samples ( Fig.…”

“…During normal blood development, LSD1 is present in numerous chromatin-remodeling complexes that decommission enhancers, leading to gene silencing (19). As mutant CEBPA produces a unique epigenetic landscape in AML and enhancer activation is crucial to the oncogenic activity of CEBPA mutant AML (17,20), we investigated the efficacy of multiple targeted epigenetic inhibitors against our CEBPA/ CSF3R mutant cell line. We assessed the in vitro cytotoxicity of inhibitors of LSD1 (GSK2979552 and GSK-LSD1), Jumonji histone demethylase (KDM4C, JHDM inhibitor VIII), disruptor of telomeric silencing 1-like (DOT1L, EPZ004777), enhancer of zeste 2 (EZH2, UNC1999), histone deacetylases (HDAC, panobinostat), and DNA methyl transferases (DNMT, azacitidine) ( Fig.…”

“…We next compared genes that were differentially expressed in response to LSD1 inhibition with RNA-seq on mouse bone marrow expressing mutant CSF3R alone or mutant CSF3R and mutant CEBPA ( Fig. 2C) (17). LSD1 inhibition up-regulated numerous genes that were expressed in the setting of CSF3R T618I alone but suppressed when CEBPA V314VW was coexpressed.…”

“…Mutant CSF3R activates both proliferative and differentiative transcriptional programs driving the proliferation of neutrophil precursors and the overproduction of mature neutrophils that are the hallmark of this disease (15,16). However, when a C-terminal CEBPA mutation is present, differentiation downstream of CSF3R is blocked, but proliferation is unabated (17). In mouse models, only the combination of mutations leads to a completely penetrant, rapidly lethal AML, while single mutations produce long latency disease in only a fraction of animals (17).…”

“…However, when a C-terminal CEBPA mutation is present, differentiation downstream of CSF3R is blocked, but proliferation is unabated (17). In mouse models, only the combination of mutations leads to a completely penetrant, rapidly lethal AML, while single mutations produce long latency disease in only a fraction of animals (17). Mutant CEBPA blocks the activation of a key subset of enhancers that are crucial for myeloid differentiation, driving AML initiation.…”