Low levels of enhancer of zeste homolog 2 (EZH2) protein are prognostically adverse and independent of mutation status in acute myelogenous leukemia.

Abstract INTRODUCTION: Mutations in the Enhancer of Zeste Homolog 2(EZH2) occur in ~ 4% of Acute Myelogenous Leukemia (AML) cases, and confer an adverse prognosis. Most of these mutations lead to loss of enzymatic function, causing a genome wide reduction in the deposition of the H3K27me3 repressive histone mark. EZH2 mutations (MUT) that cause low expression of the protein are associated with chemoresistance in AML. We hypothesized that low levels of wild-type (WT) EZH2 protein would produce a similar phenotype and bad prognosis. METHODS: We measured the levels of EZH2 and 433 other proteins using Reverse Phase Protein Arrays in 806 newly diagnosed, fresh, pre-treatment AML samples. Protein expression was normalized to non-G-CSF treated, normal bone marrow (NBM)-derived CD34+ cells. EZH2 mutation status, treatment, and outcome data were known for 529 patients, of which 24 (4.5%) had EZH2 mutation (MUT) and 505 were WT. LogRank tests were used to compared outcomes; Fisher's Exact, Pearson's Chi-squared or Wilcoxon tests for comparing variables; Pearson's correlation for protein correlation (p<0.01 and correlation coefficient >0.3); Wilcoxon tests adjusted by FDR for differential expression (p<0.05 and LFC>0.5); and Cox proportional hazards models (CoxPH) for Uni-(UV) and Multi-variate (MV) analysis. RESULTS: The cohort was divided into tertiles and regrouped into upper 1/3rd, named High (WT N=178, MUT N=5) and lower 2/3rds, termed Low (WT N=327, MUT N=19). Notably, 95% of AML cases had lower EZH2 protein compared to the median of NBM-derived CD34+ cells. Low EZH2 cases were older, had lower WBC count and blast percentage, and a higher frequency of secondary AML, unfavorable cytogenetics, -5/5q-, -7/7q-, and mutations in ASXL1 and RUNX1, but lower rates of inv16, t(8;21), 11q13 and mutations in FLT3, KIT, NPM1 and RAS (P<0.001, <0.001, <0.001, 0.002, 0.003, 0.02, <0.001, 0.001, 0.003, 0.009, <0.001, 0.007, <0.001, 0.04, <0.001, 0.007). EZH2 MUT patients showed an Overall Survival (OS) similar to WT-Low, but inferior to WT-High (5ys OS: MUT-Low=11%, MUT-High=20%, WT-Low=18%, WT-High=34%; P=0.007). Moreover, High and Low protein expression conferred opposite prognostic impact depending on whether patients received Ara-C based combinations (AraC) or Venetoclax plus Hypomethylating agent (VH) in EZH2-WT patients. Notably, the OS of WT-Low was bad, independent of therapy, but superior compared to WT-High treated with VH (5ys OS: AraC WT-High=48%, AraC WT-Low=29%, VH WT-High=0%, VH WT-Low=17%; P<0.001). EZH2 levels did not affect Remission Duration (RD) for WT patients treated with Ara-C (P=NS), but RD was significantly longer in WT-Low treated with VH, compared to WT-High with the same therapy (5ys RD: AraC WT-High=64%, AraC WT-Low=59%, VH WT-High=0%, VH WT-Low=39%; P<0.001). In the CoxPH UV model of OS, EZH2 levels were prognostic, independent of mutation status, along with other clinical, cytogenetic and molecular features (e.g. age, complex karyotype, 2nd AML, mutations in CEBPA, IDH1/2, etc.). In the MV model, EZH2 levels retained significance, as well as age, 2nd AML and some genetic features (e.g. -5/5q-, NPM1 MUT and others). We compared the protein signature of WT- Low patients with EZH2-MUT to verify whether they share a ‘mutant-like’ behavior. The differential expression (DE) analysis, did not yield any proteins, but 9 proteins related to cell cycle transitions (including mitosis), DNA Damage Response, and p53 signaling were positively correlated with both groups, suggesting commonality. In contrast, 48 proteins were DE between WT-Low and WT-High demonstrating functional differences due to loss of protein expression, with VEGF signaling and negative regulation of apoptosis enriched in WT-Low. CONCLUSIONS: Similar to EZH2 MUT cases, patients with Low-WT had poor outcomes, regardless of therapy, and the proteomic signature of EZH2 Low-WT was similar to EZH2 MUT, suggesting a similar pathophysiology in response to low EZH2 protein expression, regardless of cause. As therapies against EZH2 are being developed we postulate that these may be applicable to a much larger AML population, considering both EZH2 MUT (4%) and Low EZH2 WT (62%), greatly increasing their utility. Notably, those with WT-High responded poorly to VH, suggesting that these cases should not receive that regimen. Finally, proteomics could be leveraged to prospectively identify Low EZH2 WT cases that benefit from EZH2-directed therapy.