Function of ASXL1 Mutations in Chronic Neutrophilic Leukemia

Mutations in Additional Sex Combs Like 1 are frequent in myeloid malignancy and are associated with poor prognosis. We find ASXL1 mutations to be nearly ubiquitous in patients with CSF3R mutated chronic neutrophilic leukemia (CNL). A member of the polycomb repressive complex 2 (PRC2), ASXL1 represses gene expression through the deposition of inhibitory H3K27me3 marks. Mutations in ASXL1 act both as a loss of function and also potentially possess neomorphic function. Previous studies demonstrate that the impact of ASXL1 mutations is highly dependent on the pattern of co-occuring mutations. When present in isolation, ASXL1 mutations block myeloid differentiation and produce a dysplastic phenotype through decreased repression of Hox genes. When co-expressed with Runx1 mutations, a highly proliferative immature leukemia develops, consistent with the co-occurrence of these mutations in AML. CNL is defined by the absence of dysplasia arguing that the phenotype produced by ASXL1 mutations is modulated by the presence of activating mutations in CSF3R. In this study we find that CSF3R^T618I confers cytokine independent growth in mouse bone marrow colony forming assay. ASXL1^MT2 (Exon 12 truncation mutation) alone does not produce cytokine independent growth when expressed alone and does not modify colony formation when co-expressed with CSF3R^T618I. However, cells expressing both CSF3R^T618I and ASXL1^MT2 display increased replating capacity relative to cells only expressing CSF3R^T618I, demonstrating that mutant ASXL1, in this context, supports increased self-renewal. ASXL1 mutations have been previously reported to block differentiation when expressed in isolation. To explore the impact of ASXL1^MT2 on CSF3R^T618I-induced differentiation, we utilized murine bone marrow cells immortalized through transduction with a HoxB8-ER fusion gene (HoxB8 cells). These cells differentiate down the neutrophilic lineage after estrogen withdrawal. We find that expression of CSF3R^T618I alone increases differentiation in HoxB8 cells. In contrast with prior studies, expression of ASXL1^MT2 alone does not alter HoxB cell differentiation after estrogen withdrawal. Surprisingly, when CSF3R^T618I and ASXL1^MT2 are expressed together, differentiation is markedly increased. These data suggest that the impact of mutation in this global chromatin modifier are highly context specific and can augment the oncogenic program produced by co-occurring partner mutations. Furthermore, they provide explanation for the finding that CSF3R and ASXL1 mutant CNL displays no evidence of differentiation block or dysplasia. Further studies will explore the mechanism of ASXL1-mediated potentiation of myeloid differentiation in the context of CSF3R^T618I, extend these findings to in vivo models, and establish the impact of ASXL1 mutations on drug sensitivity in CSF3R mutant CNL.

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