EVI1-phosphorylation at S436 regulates interactions with CtBP1 and DNMT3A and promotes self-renewal

The transcriptional regulator EVI1 has an essential role in early development and haematopoiesis. However, acute myeloid leukaemia (AML) driven by aberrantly high EVI1 expression has very poor prognosis. To investigate effects of posttranslational modifications on EVI1 function, we carried out a mass spectrometry (MS) analysis of EVI1 in AML and detected dynamic phosphorylation at serine 436 (S436). EVI1-WT (wild type), with S436 available for phosphorylation, but not EVI1-S436A, conferred haematopoietic progenitor cell self-renewal associated with significantly higher organised transcriptional patterns. In silico modelling of EVI1-S436 phosphorylation showed affinity reduction to CtBP1, and EVI1-WT showed reduced interaction with CtBP1 compared with EVI1-S463A. The target the motif harbouring S436 is targeted by CDK2 and CDK3 kinases, which both interacted with EVI1-WT. The methyltransferase DNMT3A bound preferentially to EVI1-WT compared to EVI1-S436A, and a hypo-methylated cell population associated with EVI1-WT expression is not maintained with EVI1-S436A. These data point to a role of the EVI1-S436 phosphorylation for directing functional protein interactions for haematopoietic self-renewal. Targeting EVI1-S436 phosphorylation may benefit therapy for EVI1-driven leukaemia. Poly-A RNA sequencing (RNAseq) analysis of Evi1-mediated modulation of gene expression RNA extracted from murine haematopoietic progenitor cells at 48 hrs after lentiviral transduction with Evi1-WT, Evi1-S436A or vector only and untransduced cells.