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

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.