Introduction: Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We and others have reported that loss of ASXL1 impairs hematopoietic stem/progenitor cell functions (HSC/HPCs) and leads to myeloid dysplastic syndrome (MDS) in mice. It remains controversial whether truncating mutations in ASXL1 result in gain- or loss-of-function, or whether they confer dominant-negative activity in vivo . Answering this question is critical for determining how to target these mutations therapeutically.

Methods: We generated a transgenic mouse model using HS321/45-vav vector that expresses the Asxl1 Y588X cDNA under the control of the Vav1 promoter. The transgene contains the entire coding region of mouse Asxl1 (full-length) with a stop codon mutation at the Y588 site (Asxl1 Y588X), leading to a production of truncating protein ASXL1aa1-587 in the hematopoietic system only. Utilizing the transgenic model, we performed a serial hematopoietic analyses, including peripheral blood (PB) counts, histological characterization on blood smear, tissue sections of femur and spleen, and flow cytometric analysis. Bone marrow(BM) transplantation was performed to identify the effects of truncating ASXL1aa1-587 on HSC/HPCs functions. RNA-seq and ATAC-seq analyses were performed to survey the differentially expressed genes in HSC/HPCs of WT and Asxl1 Y588X Tg mice. Liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation (IP) experiments were performed to identify the specific protein binding partner of ASXL1aa1-587. An epigenetic drug screening was used to identify specific targets to block the gain-of-function protein, ASXL1aa1-587, in the pathogenesis of myeloid malignancies in vivo.

Results: The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patientswith ASXL1 mutations. Competitive transplantation of WT and Asxl1Y588XTg BM cells revealed that expression of A SXL 1 aa587 was associated with a higher percentage of CD45.2+ cells in the PB of the recipients, indicating that A SXL11-587aa expression increases HSC self-renewal capacity in vitro and in vivo. ATAC- and RNA-Seq analyses revealed that the ASXL11-587aa truncating protein expression results in more open chromatin in cKit+ cells compared to wildtype cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. LC-MS) and co-IP experiments showed that ASXL11-587aa acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hyper-sensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors.

Conclusion: This study demonstrates that ASXL11-587aa plays a gain-of-function role in promoting myeloid malignancies and that BET bromodomain inhibitors significantly reduced the colony forming capacity of ASXL1Y588XTgHSPCs. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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