Truncated ASXL1 in Myelodysplastic Syndromes (MDS): Using CRISPR Cas9 to mimic common mutations found in MDS to examine how cell behavior changes.

Mutations in the Additional Sex Combs-Like 1 (ASXL1) gene frequently occur in myelodysplastic syndromes (MDS) and correlate with poor patient prognosis. While ASXL1 mutations are known to create truncated proteins, their precise role in disease progression remains unclear. This study investigated whether truncating mutations in ASXL1 confer a competitive advantage in hematopoietic cells. Using CRISPR-Cas9 genome editing, we introduced multiple ASXL1 mutations, including the common G646Wfs variant and other truncating mutations (Y591X, E635fs, and R693X), into mouse 32D cells and primary bone marrow cells. Through competitive co-culture experiments and competitive methylcellulose assays, we demonstrated that cells harboring ASXL1 truncating mutations consistently outcompeted both wild-type cells and those with ASXL1 knockout mutations. Notably, the G646Wfs mutation showed particular dominance in competition assays, even when initially present at low frequencies. We also examined these mutations in the context of U2af1 Q157R, a splicing factor mutation commonly co-occurring with ASXL1 mutations in MDS patients. The competitive advantage of ASXL1 truncating mutations was maintained across different genetic backgrounds, suggesting a fundamental role in driving clonal expansion. These findings indicate that ASXL1 truncating mutations are not simply loss-of-function variants but likely confer novel properties that promote cellular fitness in hematopoietic cells, aligning with recent work by Köhnke et al. (2024) showing enhanced PR-DUB complex activity in ASXL1-mutant cells. This work provides new insights into how ASXL1 mutations may contribute to clonal dominance in myeloid malignancies and suggests potential therapeutic implications for targeting cells harboring these mutations, particularly through approaches that might normalize aberrant histone modification patterns.