Abstract 3385: ETNK1 mutations promote ROS production and DNA damage through increased mitochondrial activity

Abstract

Abstract Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the myelodysplastic/myeloproliferative syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S, and G245V substitutions. We previously showed that ETNK1 mutations cause a decreased catalytic activity of the enzyme. Despite this evidence however, their oncogenic role remained largely unexplained. Since ETNK1 activity is essential for the synthesis of phosphatidylethanolamine (PE) and given that PE is one of the most abundant phospholipids in the inner membrane of mitochondria, we focused our attention on mitochondrial activity. In order to characterize the oncogenic effect of ETNK1 variants we generated CRISPR/Cas9 clones carrying heterozygous N244S mutation and homozygous ETNK1 deletion (KO cells) on the 293 Flp-In™ cell-line. Both N244S and KO cells showed a significant increase in mitochondrial activity (1.78 and 2.13 fold increase, respectively; p= 0.0096 and p=0.0050) compared to WT, as assessed by MitoTracker™ Red. In line with this finding, electron microscopy revealed a significant modification in mitochondria morphology for N244S and KO cells, changing from an elongated, tubular shape to a round, swollen one. ATP (1.67 and 1.68 fold; p<0.0001; ATPlite Luminescence Assay System) and ROS production (1.66 and 1.74 fold increase; p<0.0001; CellROX™ Green Reagent) were similarly increased. Histone H2AX phosphorylation (γ-H2AX) analysis revealed a higher number of foci in N244S and KO cells (2.60±0.22 and 2.89±0.27; p<0.0001) compared to WT (0.56±0.08). A similar increase in γ-H2AX (3.6 fold; p=0.0037) was present in primary samples from aCML patients carrying ETNK1 mutation compared to ETNK1-WT ones. In line with these data, a higher mutation rate was detected in N244S and KO cells (8.09*10-7±9.6*10-8 and 8.20*10-7±1.28*10-7; p=0.0060 and p=0.0264) compared to WT (2.98*10-7±8.2*10-8) by 6-thioguanine assay. The reconstruction of the hierarchy of somatic mutations in ETNK1-mutated aCML patients revealed that ETNK1 variants invariably occur very early in the evolution history of the aCML patients. Taken together, our results show that impairment of ETNK1 function causes an increase in mitochondrial activity, which in turn leads to increased ROS production driving the accumulation of DNA mutations. Since the characterization of aCML subclonal architecture indicates ETNK1 mutations as a very early event in the history of the disease, we hypothesize that ETNK1 could contribute to the onset of aCML through the activation of a mutant phenotype, which in turn would accelerate the accumulation of further oncogenic mutations. Citation Format: Diletta Fontana, Mario Mauri, Antonio Niro, Luca Massimino, Mayla Bertagna, Giovanni Zambrotta, Mario Bossi, Stefania Citterio, Barbara Crescenzi, Giovanni Signore, Vincenzo Piazza, Cristina Mecucci, Guido Cavaletti, Delphine Rea, Carlo Gambacorti-Passerini, Rocco Piazza. ETNK1 mutations promote ROS production and DNA damage through increased mitochondrial activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3385.