NEW DIRECT TARGETS OF PSTAT3 AND PSTAT5 IN HUMAN ERYTHROID AND MEGAKARYOCYTIC CELLS

Myeloproliferative neoplasms (MPNs) are characterised by excess production of mature blood cells accompanied by an increased risk of thrombosis and progression to marrow fibrosis and AML. Most are driven by a mutation (V617F) in the pseudo-kinase domain of JAK2, which leads to unrestrained cell proliferation. To find direct targets of JAK2-STAT signalling in MPNs, we undertook ChIP-Seq for pSTAT5 and pSTAT3 in cell lines, HEL and SET2. HEL cells have 13-16 copies of JAK2-V617F as determined by FISH and SNP arrays 1. They have high levels of pSTAT5 and pSTAT3 by phosphoflow and Western blotting. SET-2 cells were derived from a patient with JAK2-V617F+ ET; they also have high levels of pSTATs. We found ∼690 pSTAT5-occupied sites and >10,000 pSTAT3-occupied sites in HEL cells. The majority reside in distal enhancers. We found new enhancers in well-known target genes such as BCL2L1, which encodes the pro-survival protein, BCL-X. The human enhancer is in a similar relative position to the recently described mouse EPO-dependent enhancer 2. We found new direct target genes that encode for proteins with interesting predicted functions. SET-2 cells have similar pSTAT5 and pSTAT3-bound sites suggesting commonality of functions. We show pSTAT3 and pSTAT5 bind as dimers in vivo to typical GAS elements, but with slightly different site preferences. This has implications for differential target gene regulation. We undertook expression profiling using SLAM-seq following treatment with ruxolitinib and validated novel target genes by qRT-PCR. In short, we have discovered hundreds of direct JAK-STAT target genes involved in cell survival, proliferation, down regulation of cytokine signalling, and novel functions. The genes provide new insights into the biology of MPNs, and a source of potential new biomarkers and drug targets. We have validated some in primary MPN samples.