SMARCC1 loss impairs differentiation and enhances self-renewal in ASXL1-mutant hematopoietic cells Free

Additional sex combs-like 1 (ASXL1) is a histone modifier regulating hematopoiesis. Its mutations are common in secondary acute myeloid leukemia (AML), linked to poor prognosis. Its mutations are also frequently observed in clonal hematopoiesis and are associated with increased risk of developing leukemia. However, the mechanisms driving leukemogenesis and specific therapeutic targets remain elusive.

Here, we performed a CRISPR-Cas9 knockout screen using a heterozygous knock-in mouse model harboring an Asxl1 mutation (G646WfsX12) homologous to human ASXL1 mutations, to identify genes whose loss contributes to leukemogenesis in ASXL1-mutant hematopoietic stem/progenitor cells (HSPCs). We generated a custom knockout single-guide RNA (sgRNA) library targeting putative tumor suppressor genes and transduced it into c-kit-positive, lineage-negative HSPCs isolated from bone marrow of Cas9-expressing Asxl1-mutant or wild-type mice. Self-renewal was assessed via serial colony-forming assays in semi-solid methylcellulose media over five generations. While wild-type cells did not exhibit serial replating capacity, Asxl1-mutant cells retained serial replating potential. DNA was extracted from cells of the third and fifth generation colonies, and next generation sequencing was performed to identify sgRNAs enriched relative to baseline. Several candidate genes were selected, and individual knockout validation experiments confirmed that loss of Smarcc1, a core subunit of the SWI/SNF chromatin remodeling complex, enhanced serial replating capacity, with greater effect in Asxl1-mutant cells.

Flow cytometry analysis of colonies revealed that Gr-1 positivity was reduced in Smarcc1 knockout cells, more pronouncedly in Asxl1-mutant cells. These findings led us to hypothesize that Smarcc1 deficiency cooperates with Asxl1 mutations to inhibit myeloid differentiation and sustain an undifferentiated, self-renewing state. To validate this, we first used mouse myeloid progenitor cell line 32Dcl3 stably expressing either wild-type or mutant human ASXL1, and conducted Smarcc1 knockout. Upon G-CSF-induced differentiation, Gr-1 positivity was consistently lower in Smarcc1-knockout cells, with a greater reduction in ASXL1-mutant cells. Second, using human chronic myeloid leukemia cell line K562, which harbors a heterozygous ASXL1^Y591X mutation, and its CRISPR-edited ASXL1-wild-type counterpart, we performed SMARCC1 knockout. SMARCC1 knockout increased c-kit positivity in both cell lines, with a larger increase in ASXL1-mutant cells. Together, these data suggest that SMARCC1 deficiency cooperates with ASXL1 mutations to impair differentiation and maintain an undifferentiated state. Moreover, analysis of public databases of AML clinical samples revealed no SMARCC1 genomic mutations, but SMARCC1 expression was significantly reduced in ASXL1-mutant AML compared to ASXL1 wild-type AML.Collectively, our findings suggest that SMARCC1 loss collaborates with ASXL1 mutation–induced histone modification changes to activate transcription of self-renewal genes or repress differentiation genes. This likely occurs through altered SWI/SNF complex binding profiles or transcription factor binding motifs at regulatory regions. This is the first report demonstrating that SMARCC1 loss enhances self-renewal in ASXL1-mutant hematopoietic cells. Elucidation of this molecular mechanisms is expected to provide novel insights into the epigenetic landscape of ASXL1-mutant leukemia and contribute to the development of targeted therapeutic strategies. Further validation using mouse models and clinical specimens is warranted.