High protein expression of MDS1 and EVI1 complex locus (MECOM) is prognostically adverse and independent of chromosome 3 rearrangements in acute myelogenous leukemia

Abstract

Abstract INTRODUCTION: The MDS1 and EVI1 Complex Locus (MECOM), located at chromosome 3q26.2 is rearranged (MECOM-R) in ~1.5% of Acute Myelogenous Leukemia (AML) cases, and confers an adverse prognosis. MECOM-R causes overexpression of Ecotropic viral integration site 1 (EVI1), but this has also been observed without chromosome 3q26.2 changes. We hypothesized that measuring the protein levels of EVI1 could enhance AML stratification and prognostication, and identify common and targetable proteins hubs. METHODS: Reverse Phase Protein Arrays measured the level of 434 proteins (344 total, 90 post-translational modified), in 806 newly diagnosed, fresh, pre-treatment AML patient samples. Protein expression was normalized to normal bone marrow (NBM)-derived CD34+ cells. Chromosome 3 alteration (Ch3-alt) status, treatment and outcome data were known for 658 patients: 20 had MECOM-R, 54 had other Ch3-alt and 584 were Ch3 wild-type (Ch3-WT). LogRank tests compared outcomes; Fisher's Exact, Pearson's Chi-squared or Wilcoxon tests compared variables; Pearson's test correlated proteins (p<0.05 and R>0.2); 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: Patients were stratified by EVI1 levels: above maximum expression of NBM (High, N=30), greater than, or below, median expression of NBM (Up, N=54; Low, N=214) or below the minimum of NBM (Below, N=387). Most MECOM-R cases had high EVI1 levels (High=9, Up=6), but there were cases with low expression, (Low=4, Below=1; P<0.001), contradicting the idea that MECOM-R always causes EVI1 overexpression. Ch3-alt cases were not biased towards high EVI1 expression (P=0.21). High and Up cases (EVI-High) were associated with secondary (2nd) AML and unfavorable cytogenetics, while Low and Below cases (EVI-Low) showed the opposite trend (P<0.001, =0.018). Notably, complex karyotype (CK) was not biased towards EVI-High, but t(11q23), t(9;11) and -7/7q- were (P=0.85, <0.001, <0.001, <0.001). EVI-High was directly associated with mutations in EZH2, JAK2, PTPN11, RAS and WT1, but inversely correlated with IDH2 and NPM1 mutants (P=0.02, <0.001, <0.001, 0.001, 0.01, 0.03, 0.04). The Overall (OS) was similar in EVI-High (5ys OS: High=0%, Up=14%; P=non-significant (NS)) and in EVI-Low (5ys OS: Low=22% and Below=26%, P=NS), but markedly different between the High and Low groups (P<0.0001). Importantly, OS of EVI-High, regardless of Ch3 status, were significantly inferior to EVI-Low cases with Ch3-WT (5ys OS: MECOM-R High=0% and Up=0% vs Ch3-alt High=0% and Up=0% vs Ch3-WT High=0% and Up=19% vs Ch3-WT Low=23% and Below=27%; P<0.0001). In the CoxPH UV model of OS, both EVI-High and EVI-Low were prognostic, independent of Ch3 status, along with other clinical, cytogenetic and molecular features (e.g. age, CK, inv16, 2nd AML mutations). In the MV model, EVI-High retained significance, regardless of Ch3 status, as did age, CK, inv16 and 2nd AML. Since EVI-High cases have a heterogeneous genetic background (e.g. MECOM-R vs Ch3-WT), their underlying pathophysiology might be distinct. To verify this, we performed Western Blotting comparing MECOM-R and Ch3-WT (N=3, each). MECOM-R cases only showed EVI1 expression, but Ch3-WT had both MDS1/EVI1 and EVI1 isoforms. We did not find any differentially expressed proteins between MECOM-R and Ch3-WT but both shared correlation between EVI1 level and 11 proteins (out of 433 in our database) positively associated with both groups and related to: TGFβ cascade, SMAD signaling, mitochondrial biogenesis, and cellular response to unfolded protein. This demonstrates a similarity of protein expression signatures between MECOM-R and Ch3-WT-High. CONCLUSION: High EVI1 protein expression occurred in 13.7% of AML cases, mostly Ch3-WT (88%), showing poor OS and prognostic independence in CoxPH UV and MV models, regardless of Ch3 status. Despite differences in expressed EVI1 isoforms in MECOM-R and Ch3-WT, EVI-High patients didn't show differentially expressed proteins and shared similarly correlated proteins, which could be targeted regardless of Ch3 status. Our findings suggest that novel therapies for MECOM-R AML may be useful for all EVI-High cases, thus increasing their utility in 12% of cases, instead of just the known 1.5% classic MECOM-R. Finally, proteomics could be leveraged to identify the EVI-High AML cases that may benefit from MECOM-R-directed therapy.