DNA Methylation Profiling Reveals Multiple Hits in MAPK Pathway in Triple Negative MDS/MPN with SF3B1 Mutation and Thrombocytosis

Myelodysplastic/myeloproliferative neoplasm with SF3B1 mutation and thrombocytosis (MDS/MPN-SF3B1-T) is a clonal hematopoietic stem cell disorder with overlapping features of myelodysplastic neoplasms (MDS) and myeloproliferative neoplasms (MPN). JAK2V617Fmutations are found in only 45-50% of cases (Broseus J, et al. Clinical features and course of refractory anemia with ring sideroblasts associated with marked thrombocytosis. Haematologica. juill 2012;97(7):1036‑41). CALR or MPL mutations are found in less than 5% of cases. Platelet’s proliferation remains then unexplained in the residual 50%, leading to diagnosis difficulties for these triple negative MDS/MPN-SF3B1-T (TN MDS/MPN-SF3B1-T). In order to understand the molecular mechanism underlying thrombocytosis, we explored DNA methylation in TN MDS/MPN-SF3B1-T. Here, we developed a novel approach of DNA methylation profiling from the data obtained with comparative reduced representation bisulfite sequencing (RRBS) on a cohort composed of 7 JAK2-mutated MDS/MPN-SF3B1-T (JAK2V617F MDS/MPN-SF3B1-T), 9 TN MDS/MPN-SF3B1-T, 8 myelodysplastic neoplasms with SF3B1 mutation (MDS-SF3B1), 10 JAK2V617F essential thrombocytemia (JAK2V617F TE) and 4 normal bone marrow (BM) samples. Comparative methylation analysis was initially performed by using the R package MethylKit (Akalin A, et al. methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 3 oct 2012;13(10):R87) and thereafter with a new designed approach integrating CpG position in differentially methylated regions (DMRs). This method identified 259 significant hypomethylated DMRs and 285 significant hypermethylated DMRs between JAK2V617F MDS/MPN-SF3B1-T and TN MDS/MPN-SF3B1-T. The filtering of significant DMRs relied on a clinical pertinence score, devised for selecting DMRs associated with JAK2V617F-unrelated thrombocytosis. This approach enabled the identification of 46 significant DMRs associated with 33 genes overrepresenting the mitogen-activated protein kinases (MAPK) and related pathways. Compared to JAK2V617F MDS/MPN-SF3B1-T, regulating DNA sequences of 4 of those genes were hypomethylated in TN MDS/MPN-SF3B1-T, and one of them was hypermethylated. This specific hypermethylation was reported to lead to upregulation of messenger RNA in previous works. Corresponding transcripts were quantified by quantitative polymerase chain reaction (qPCR) in TN MDS/MPN-SF3B1-T, JAK2V617F MDS/MPN-SF3B1-T, MDS-SF3B1 and normal BM samples. Consistently, qPCR confirmed overexpression of all candidate transcripts in TN MDS/MPN-SF3B1-T as compared to JAK2V617F MDS/MPN-SF3B1-T, MDS-SF3B1 and normal BM. In conclusion, we devised an innovative profiling method from RRBS measurements for selecting DMRs potentially associated with thrombocytosis but JAK2V617F-independent in TN MDS/MPN-SF3B1-T. We highlighted the implication of MAPK pathway in MDS/MPN-SF3B1-T and especially in TN MDS/MPN-SF3B1-T. The upregulation of MAPKs in MDS/MPN-SF3B1-T paves the way to specific therapeutic and diagnostic approaches in MDS/MPN-SF3B1-T and more generally in JAK2/CALR/MPL unmutated MPNs.