Mutant ASXL1 exacerbates CALR-mutant myeloproliferative neoplasms by enhancing a distinct megakaryocyte differentiation program

ASXL1 mutations confer a poor prognosis in myeloid neoplasms, including in CALR-mutated myeloproliferative neoplasms (MPN). We generated Calr-Asxl1 co-mutant mice and found more severe thrombocytosis and increased splenomegaly, compared with Calr single-mutant animals. Calr-Asxl 1 co-mutant megakaryocytes displayed more pronounced morphological abnormalities and enhanced emperipolesis characteristic of inflammation. Concordant with this, a differentiation trajectory directly to megakaryocytes from hematopoietic stem cells (HSC) was more prominent in Calr-Asxl1 co-mutant mice. In this non-canonical trajectory, where megakaryocyte-biased HSCs directly differentiate into megakaryocyte progenitors, we found upregulation of Mef2c , a key transcriptional regulator of megakaryocyte lineage commitment. Mature Calr-Asxl1 co-mutant megakaryocytes displayed an enhanced proinflammatory transcriptional state, whereas the transcriptional signature of Calr-Asxl1 co-mutant HSCs was indicative of inflammation-resistance. Genotyping of transcriptomes (GoT) confirmed an activated megakaryocytic differentiation program in CALR-ASXL 1 co-mutant HSCs in primary human MPN bone marrow. These findings link the enhanced severity of CALR-ASXL1 co-mutant MPN to altered megakaryocyte differentiation and help explain some of the biological basis for the more severe MPN phenotype observed in patients.