Orthogonal Proteogenomic Approaches Identify the Druggable PA2G4-MYC Axis in 3q26 AML

Despite next-generation technologies improved our understanding of the complexity of high-risk acute myeloid leukemia (AML), effective targeted therapies are delayed due to the prevalence of major oncogenic disease drivers such as RAS, RUNX1, TP53. In some cases, the integration of orthogonal omics led to the identification or repurposing of drugs for "undruggable" transcription factors (TFs), prospecting a benefit for patients with the most aggressive leukemia subtypes.

Here, we sought to discover how to target the Ecotropic Viral Integration Site-1 gene (EVI1/MECOM), the most lethal oncogenic TF in AML carrying chromosome 3q26 abnormalities. To this end, we intersected phenotypic, gene expression-based and proteomic high-throughput approaches for small molecule and target identification. We screened a collection of 5294 compounds enriched for Food and Drug Administration (FDA) approved or bioactive drugs at 1 µM and identified those that caused more than 95% reduction of cell viability compared to vehicle control in MOLM1 and UCSD/AML1 cell lines carrying 3q26 rearrangments (EVI1^high). We then selected a set of sentinel genes that defined an EVI1 "on" and "off" transcriptional signature based on the TF1 EVI1^high cell line treated with an EVI1-directed siRNA or control, and used the Connectivity Map to identify inducers of an EVI1 "off" state. Histone deacetylase inhibitors (HDACi) scored as the top class of compounds able to repress leukemia proliferation by suppressing the EVI1 signature. We focused on the compounds with pan (AR-42, belinostat) or selective (entinostat) HDAC inhibitory properties. These molecules induced apoptosis and decreased the levels of EVI1 in AML cell lines and in primary EVI1^high blast cells. Interestingly, the expression of EVI1 sensitizes cells to HDAC inhibition both in an inducible EVI1 cell model and in EVI1^high compared to EVI1^low AML cell lines. Primary EVI1^high blasts were also more sensitive to HDAC inhibition compared to those from other AML subtypes, while equally resistant to chemotherapy agents.

We translated these results in a "N-of-1" clinical trial proposing entinostat in association with azacytidine to patients with relapse/refractory 3q26 AML based on a compassionate use program for this regimen. Pharmacodynamic modulation of EVI1 was assessed at 6 hours (T6) after entinostat administration in patients and in matched patient-derived AML xenografts (PDLX). EVI1 expression markedly decreased in circulating blasts collected at T6 compared to T0 (before treatment), and EVI1 depletion paralleled clinical and hematologic improvements at follow-up evaluations. This effect was not observed in patients treated with cytarabine. Longitudinal single-cell RNA sequencing (scRNASeq) of bone marrow AML cells collected before and after patients' treatment cycle revealed an inhibitory effect on Myc target genes in the leukemic clone. This effect was also observed in cell lines treated with AR-42, belinostat and entinostat suggesting that different HDACi regulates the Evi1-Myc axis in 3q26 AML. Not surprisingly, HDACi mediated or genetic silencing of EVI1 led to a decrement of MYC in EVI1^high AML models.

To dissect the EVI1 co-transcriptional complex and explain for the modulation of Myc signaling in EVI1^high AML, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). Sixty-nine proteins co-immunoprecipitated with endogenous EVI1 both in cell lines and in 3q26 PDLX models. Eleven of these EVI1 interactors were targets of Myc signaling, including PA2G4, a RNA-binding protein that was markedly modulated by HDACi. Importantly, PA2G4 silencing or inhibition (WS6) depletes MYC at transcriptional and protein levels recapitulating the phenotypic effect seen with HDACi in EVI1^high cell models. Consequently, WS6 treatment reduced the growth of AML cells in PDLX models in vivo by suppressing an EV1-MYC signature at single-cell leukemia level without inducing widespread toxicities. In contrast, PA2G4 overexpression rescued EVI^high AML cell lines from the effects induced by HDACi inhibition. These data support the evidence that PA2G4 is a mediator of Evi1-Myc axis in AML.

In conclusion, our work positions PA2G4 at the crosstalk of the Evi1-Myc leukemogenic signal for developing new therapeutics and, at this interesting time, urges upfront HDACi-based combination therapies in patients with 3q26 AML.

Mecucci:Abbvie: Speakers Bureau; GSK: Speakers Bureau; Novartis: Speakers Bureau. Colla:Amgen: Research Funding.