ASXL1 Directs Neutrophilic Differentiation via Modulation of MYC and RNA Polymerase II

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. An epigenetic regulator, ASXL1 canonically directs the deposition of H3K27me3 via the polycomb repressive complex 2. However, its precise role in myeloid lineage maturation is incompletely described. We utilized single cell RNA sequencing (scRNA-seq) on a murine model of hematopoietic-specific ASXL1 deletion and identified a specific role for ASXL1 in terminal granulocyte maturation. Terminal maturation is accompanied by down regulation of Myc expression and cell cycle exit. ASXL1 deletion leads to hyperactivation of Myc in granulocyte precursors and a quantitative decrease in neutrophil production. This failure of normal developmentally-associated Myc suppression is not accompanied by significant changes in the landscape of covalent histone modifications including H3K27me3. Examining the genome-wide localization of ASXL1 in myeloid progenitors revealed strong co-localization with RNA Polymerase II (RNAPII) at the promoters and spread across the gene bodies of transcriptionally active genes. ASXL1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription, consistent with the known role of ASXL1 as a mediator of RNAPII pause release. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator and highlighting a novel potential oncogenic mechanism for ASXL1 mutations in myeloid malignancies. ### Competing Interest Statement B.J.D. potential competing interests-- SAB: Aileron Therapeutics, Therapy Architects (ALLCRON), Cepheid, Vivid Biosciences, Celgene, RUNX1 Research Program, No-vartis, Gilead Sciences (inactive), Monojul (inactive); SAB & Stock: Aptose Bioscienc-es, Blueprint Medicines, EnLiven Therapeutics, Iterion Therapeutics, Third Coast Therapeutics, GRAIL (SAB inactive); Scientific Founder: MolecularMD (inactive, ac-quired by ICON); Board of Directors & Stock: Amgen; Board of Directors: Burroughs Wellcome Fund, CureOne; Joint Steering Committee: Beat AML LLS; Founder: VB Therapeutics; Research Funding: EnLiven Therapeutics; Clinical Trial Funding: Novar-tis, Bristol-Myers Squibb, Pfizer; Royalties from Patent 6958335 (Novartis exclusive license) and OHSU and Dana-Farber Cancer Institute (one Merck exclusive license and one CytoImage, Inc. exclusive license). The remaining authors have no conflicts to report.