Protein levels of isocitrate dehydrogenase 1 and 2 are prognostic and independent of mutation status in acute myelogenous leukemia

Abstract INTRODUCTION: Mutations in the Isocitrate Dehydrogenase 1 and 2 (IDH1/2) occur in ~20% of Acute Myelogenous Leukemia (AML) cases and leads to the gain of a novel enzymatic function, which produces the oncometabolite D-2-Hydroxyglutarate (2HG). This inactivates TET2, which generates a block in histone demethylation, resulting in a global increase of methyl groups in histone tails and DNA, which contributes to tumorigenesis. However, the normal product of IDH1/2, αKetoglutarate (αKG), must also bind to TET2 for it to function, so we asked whether IDH1/2 protein levels could be prognostic in AML, possibly due to effects on αKG availability. METHODS: We measured the levels of 438 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 derived CD34+ cells. IDH MUT status, treatment, and outcome data were known for 592 patients, of which 141 (24%) were MUT (IDH1 only=57; IDH2 only=79; both=5) and 451 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; 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: The cohort was divided according to IDH1 levels into tertiles and regrouped into lower 1/3rd, named Low (WT=172, MUT=20) and upper 2/3rds, termed High (WT=350, MUT=42). IDH1-Low cases had higher frequency of unfavorable cytogenetics, complex karyotype (CK), -5/5q- and -7/7q-, but lower rates of Inv16 and mutations in CEBPA, DNMT3A, FLT3-ITD, JAK2 and NPM1 (P<0.001, <0.001, <0.001, <0.001, 0.04, 0.04, 0.003, 0.03, 0.03, 0.001). We also used IDH2 levels to split the AML cohort into tertiles, but this time regrouping into upper 1/3rd, named High (WT=157, MUT=39) and lower 2/3rds, termed Low (WT=345, MUT=45). IDH2-Low cases had higher frequency of unfavorable cytogenetics, CK, -5/5q-, -7/7q- and TP53 MUT, but lower rates of mutations in CEBPA, JAK2 and KIT (P=0.007, 0.003, <0.001, 0.05, 0.002, <0.001, 0.008, 0.002). IDH1-MUT patients had an Overall Survival (OS) more similar to IDH1-WT-Low than to WT-High (5ys OS: MUT-Low=5%, MUT-High=16%, WT-Low=18%, WT-High=28%; P=0.007). Notably, IDH1 WT-High and Low expression conferred distinct prognosis depending on the therapy: WT-High performed better with AraC-based intensive chemotherapy (IC) compared to Venetoclax plus Hypomethylating agent (VH), but for WT-Low therapy did not change outcome (5ys OS: IC WT-High=41%, IC WT-Low=29%, VH WT-High=7.7%, VH WT-Low=24%; P<0.001). Meanwhile, IDH2 MUT patients had an Overall Survival (OS) more similar to WT-High than to WT-Low, which was better than WT-Low (5ys OS: MUT-Low=27%, MUT-High=30%, WT-Low=21%, WT-High=24%; P=0.01). Importantly, OS of IDH2 WT-High and Low also differed according to therapy, with WT-High doing better with IC compared to VH, and WT-Low performing similarly with either (5ys OS: IC WT-High=40%, IC WT-Low=30%, VH WT-High=5.9%, VH WT-Low=18%; P=0.002). Similar patterns were observed for Remission Duration (RD) in WT patients for both IDH1 and 2. In the CoxPH UV model of OS, IDH1 levels were prognostic, independent of mutation status, with Low levels being harmful, along with other clinical, cytogenetic and molecular features (e.g. age, CK, CEBPA MUT, etc.). In the MV model, IDH1 levels retained significance, as well as age and 2nd AML. In contrast, levels of IDH2 were not prognostic in UV/MV models. Differential expression (DE) analysis between IDH1 WT-Low and High yielded 8 proteins (up: CD4, ERN1, GAB2.pY452, LEF1, ZAP70; down: EZH2, IDH1, SYK), which are mostly related to immune response (e.g. mast cell activation, PDL1, ZAP70 and CSF3 pathways) and FLT3 signaling, while in IDH2 WT-Low vs High, 4 proteins were DE (up: AKT1_2_3.pT308, CD4; down: IDH2 and KIT). CONCLUSIONS: IDH1 and 2 levels were predictive for outcomes, with High expression of the WT protein being more favorable in both cases. Notably, those with WT-High responded poorly to VH compared to IC. Considering the DE analyses, high expression of CD4 in both IDH1/2 WT-Low patients could be used as a potential target for CAR-T cell therapy. Finally, proteomics could be leveraged to prospectively identify IDH1/2 WT- Low cases that could benefit from future therapy.