Date of Award
Doctor of Philosophy (PhD)
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. Asxl1 mutation knock-in mouse models present with a relatively modest phenotype, following a long latency period and ASXL1 mutations have not been modeled in combination with JAK2 V617F. The field lacks human model systems that focus on how combined JAK2 V617F and ASXL1 mutations affect myelofibrosis pathogenesis.
The studies presented here describe two complementary pluripotent stem cell models utilized to study how JAK2 V617F and ASXL1 mutations influence MPN disease biology. Induced pluripotent stem cells were derived from a patient harboring both JAK2 and ASXL1 mutations. In parallel, the ASXL1 mutation present in the patient derived iPSC lines as well as the most common ASXL1 mutation were both introduced in isolation and in combination with JAK2 V617F into H1 human embryonic stem cells. Following hematopoietic differentiation, colony forming assays and RNA sequencing were performed utilizing differentiated hematopoietic progenitors. The JAK2 mutant lines generated significantly more erythroid colonies than WT, ASXL1, and double mutant lines. The ASXL1 mutation resulted in almost exclusively myeloid colony growth and the double mutant line presented with an intermediate erythroid colony growth phenotype. When expression of genes involved in erythropoiesis were assessed, the double mutant progenitor expression profiles were more similar to ASXL1 mutant profiles than gene expression patterns found in JAK2 mutant progenitors. This data suggested that the ASXL1 mutation blunted the erythroid proliferation phenotype associated with JAK2 V617F.
The observed phenotypes were validated utilizing myelofibrosis patient cells sorted from patients harboring JAK2 V617F and double mutant patients harboring both JAK2 V617F as well as an ASXL1 mutation. JAK2 mutant patient cells generated more erythroid colonies than JAK2/ASXL1 double mutant cells, while the double mutant cells differentiated into predominately myeloid lineage colonies. This study has demonstrated that JAK2 V617F induces an expansion of the erythroid lineage, while ASXL1 impairs erythroid colony growth and skews differentiation toward the myeloid lineage utilizing both human pluripotent cells as well as primary myelofibrosis patient samples. Future studies include utilizing the mutant pluripotent stem cell lines to assess potential mechanisms as to how JAK2 and ASXL1 mutations influence differentiation via signaling mass cytometry and manipulation of gene targets of interest revealed by the RNA sequencing data.
Chair and Committee
Daniel Link, Matthew Walter, Luis Batista, Jorge Di Paola,
Collins, Taylor Brost, "Functional Interrogation of JAK2 and ASXL1 Mutations in Myeloproliferative Neoplasms" (2021). Arts & Sciences Electronic Theses and Dissertations. 2361.