Rapid Fire & Poster Presentation 41st Lorne Genome Conference 2020

Novel direct targets of JAK2-STAT3/5 signalling in myeloproliferative neoplasms determined by SLAM-seq and ChIP-seq (#202)

Charlene Lam 1 , Kevin Gillinder 1 2 , Graham Magor 1 , Helen Mitchell 3 , Andrew Perkins 1 3
  1. Monash University, Melbourne, VIC, Australia
  2. Ruby Red Foundation, Sydney, NSW, Australia
  3. The Alfred Hospital, Melbourne, VIC, Australia

Myeloproliferative neoplasms (MPNs) are a group of chronic blood cancers characterised by clonal proliferation of mature blood elements of myeloid lineages in the bone marrow. The three major subtypes of MPNs, polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are mainly driven by a somatic driver mutation (V617F) in the pseudo-kinase domain of JAK2 tyrosine kinase. This gain-of-function JAK2V617F mutation exerts its effect by constitutively activating its canonical downstream signalling molecules, STAT3 and STAT5 transcription factors, which ultimately leads to the activation of target genes that promotes unrestrained proliferation of the haematopoietic and progenitor cells.

In order to identify novel direct targets of JAK2-STAT3/5 signalling in MPNs, we undertook ChIP-seq for pSTAT3 and pSTAT5 in human JAK2V617F-positive MPN cell lines, HEL and SET-2.1 Our ChIP-seq data reveals similar pSTAT5 and pSTAT3-bound sites, suggesting commonality of functions, as well as a moderately high overlap between JAK-STAT target genes in human MPN and EPO-induced genes in mouse cell lines2. In addition, they bind as dimers to GAS elements, but with slightly different site preferences. Hence, to characterise JAK2-dependent gene transcription, we performed SLAM-seq, a method for direct quantification of newly synthesized mRNAs, in response to JAK2 inhibition. We found ~6000 differentially transcribed genes 90 minutes following ruxolitinib treatment in HEL cells. Moreover, gene ontology and pathway analysis suggest that JAK2 inhibition induces differential transcription of a myriad of genes that are involved in cell survival and down-regulation of cytokine signalling. We validated a number of candidate genes in vitro as well as in primary MPN bone marrow samples by qRT-PCR.

Collectively, we discovered novel direct JAK2-STAT3/5 target genes that are likely to be associated with the biology of MPNs. These genes provide new insights into the biology of JAK2V617F-driven MPNs, and a source of potential new biomarkers and drug targets.

  1. Mackinnon RN, Wall M, Zordan A, et al. Genome organization and the role of centromeres in evolution of the erythroleukaemia cell line HEL. Evol Med Public Health. 2013;2013(1):225-240.
  2. Gillinder KR, Tuckey H, Bell CC, et al. Direct targets of pStat5 signalling in erythropoiesis. PLoS ONE. 2017;12(7).