The intersection of proteogenomic approaches identifies PA2G4 as a new target in 3q26 AML.

Introduction. Despite advancements in next-generation technologies, progresses in treating subtypes of acute myeloid leukemia (AML) driven by aberrantly activated transcription factors (TFs) have been limited. However, the integration of orthogonal omics led to the identification of drugs for “undruggable” TFs. Here, we sought to discover modulators of EVI1/MECOM gene, the most lethal oncogenic TF, hyper expressed in AML carrying chromosome 3q26 abnormalities. Methods. We intersected phenotypic, gene expression-based, proteomic approaches to identify modulators of EVI1. We screened and scored 5294 compounds on their ability to suppress proliferation of 3q26 AML models (EVI1high). From EVI1null transcriptional signatures we defined an EVI1 “on” and “off” state and used Connectivity Map to identify inducers of an EVI1 “off” status. To investigate the EVI1 co-transcriptional complex, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). Then, target hits were validated in vitro, in vivo with studies in 3q26 patientderived AML xenografts models and in humans through an N-of-1 clinical trial. Results. Histone deacetylase inhibitors (HDAC[/i] emerged as the top class of compounds able to repress leukemia proliferation by suppressing EVI1. HDACi induced apoptosis and reduced EVI1 in AML models, while the EVI1 expression sensitizes cells to HDACi. We translated these results in a clinical trial proposing entinostat in association with azacytidine to 3q26 AML patients. EVI1 expression decreased in blasts 6 hours after entinostat administration and EVI1 depletion paralleled clinical improvements. To exclude for a contribution of azacytidine in our trial, we completed an in vivo study and we demonstrated a reduction of AML blasts in mice treated with entinostat but not with azacytidine. Longitudinal single-cell RNA sequencing of 3q26 AML blasts of patients treated with azacytidine-entinostat revealed an inhibitory effect on Myc target genes as observed in cell lines treated with HADCi suggesting that multiple HDACi regulates the Evi1-Myc axis in 3q26 AML. HDACi-mediated or genetic silencing of EVI1 led to a direct decrement of MYC. To dissect the EVI1 co-transcriptional complex and explain the modulation of Myc signaling, we performed a RIME. Eleven EVI1 interactors were targets of Myc signaling, including PA2G4, a protein modulated by HDACi. PA2G4 silencing or inhibition (WS6) depleted MYC protein levels recapitulating the effect of HDACi and WS6 reduced leukemic growth in vivo by suppressing an EV1-MYC signature suggesting that PA2G4is a druggable mediator of EVI1 complex. Conclusions. In conclusion, we propose a clinical treatment strategy that capitalizes on the EVI1-suppressing capacity of HDACis in 3q26 AML patients who lack biomarker-directed treatment approaches. Furthermore, based on the mechanism driving efficacy upon HDAC inhibition, our work positions PA2G4 as a druggable target for 3q26 AML patients.