JAK2 V617F is the most frequent mutation found in myeloproliferative neoplasms (MPNs), with 50-60% of myelofibrosis (MF) patients harboring this mutation. Mutations in ASXL1 often co-occur with JAK2 V617F and are associated with decreased survival and increased risk of transformation to secondary acute myeloid leukemia. How mutant ASXL1 contributes to the MPN disease phenotype and confers poor prognosis is not fully understood. Controversy remains as to whether ASXL1 mutations found in patients confer a loss of function, gain of function, and/or dominant negative phenotype. Additionally, Asxl1 mutation knock in mouse models present with a relatively modest phenotype, following a very long latency period. A human model system has the potential to provide a useful tool to understand how these mutations affect ASXL1 function. This project therefore seeks to uncover how expression of mutant JAK2 in conjunction with mutant ASXL1 influences hematopoietic output and proliferation, utilizing human pluripotent stem cells.
Two complementary approaches have been utilized to study how ASXL1 mutations influence MPN pathogenicity. CD34+ cells from an MF patient harboring JAK2 V617F and an ASXL1 mutation (P920Tfs*4) were FACS-sorted and reprogrammed to generate distinct iPSC clones. Because the majority of the patient-derived iPSC clones were double mutant, CRISPR gene editing was utilized to revert the JAK2 and ASXL1 mutations to generate all four possible genotypes. In parallel, two different ASXL1 mutations (the most common G646Wfs*12 mutation as well as the P920Tfs*4 mutation) were introduced into H1 human embryonic stem cells, each in isolation and in combination with JAK2 V617F, resulting in the generation of single and double mutant lines.
These genetically engineered pluripotent stem cell lines were then differentiated specifically into definitive hematopoietic lineages, through a stepwise program regulated by Wnt signaling. This allowed us to analyze the role of ASXL1 and JAK2 mutations during the development of the various lineages of the hematopoietic system known to be dysregulated in MPNs, such as the myeloid and erythroid lineages. Following hematopoietic differentiation, colony forming assays were performed. The JAK2 mutant line produced four-fold more colonies than WT line, with a significant bias towards the generation of erythroid colonies. In contrast, the ASXL1 mutant line produced substantially fewer colonies than the WT line, with the majority of these colonies resembling myeloid colonies. The double mutant line (ASXL1/JAK2) generated more colonies than the WT and ASXL1 mutant lines, but fewer colonies as compared to the JAK2 mutant line. Of note, the JAK2 V617F mutation generated much larger erythroid colonies compared to the WT H1 control line or the double mutant line.
Our in vitro differentiation experiments also allowed us to sort equal numbers of CD45+CD34+ cells from the different genotypes, and plate them in methylcellulose. These experiments allow the ability to assess how mutations impact differentiation on a per cell basis. Similar trends were observed (i.e. more erythroid colonies with JAK2 and fewer and mostly myeloid colonies with ASXL1) with this approach, suggesting that the phenotypes observed can be attributed at least in part to progenitor cell output on a per cell basis and not solely reflecting the total number of progenitors generated through the pluripotent stem cell differentiation protocol.
In summary, with his human pluripotent stem cell model system, we have observed that JAK2 V617F induced an increase in total colony production with a marked expansion of the erythroid lineage, while mutant ASXL1 impaired colony production overall with substantial myeloid skewing. Cells expressing both mutations presented with an intermediate phenotype. Ongoing efforts include gene expression analysis to understand how JAK2 and ASXL1 mutations direct these observed functional differences.
Oh:Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.
Asterisk with author names denotes non-ASH members.