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The Distinct Biological Implications of Asxl1 Mutation and Its Roles In Hsu et al. Journal of Hematology & Oncology (2017) 10:139 DOI 10.1186/s13045-017-0508-x RESEARCH Open Access The distinct biological implications of Asxl1 mutation and its roles in leukemogenesis revealed by a knock-in mouse model Yueh-Chwen Hsu1, Yu-Chiao Chiu2, Chien-Chin Lin1,3, Yuan-Yeh Kuo4, Hsin-An Hou5, Yi-Shiuan Tzeng4, Chein-Jun Kao3, Po-Han Chuang3, Mei-Hsuan Tseng5, Tzu-Hung Hsiao2, Wen-Chien Chou1,3,5* and Hwei-Fang Tien5* Abstract Background: Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia- like disease in mice. However, actual effects of a “physiological” dose of mutant ASXL1 remain unexplored. Methods: We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system. Results: Heterozygotes (Asxl1tm/+) marrow cells had higher in vitro proliferation capacities as shown by more colonies in cobblestone-area forming assays and by serial re-plating assays. On the other hand, donor hematopoietic cells from Asxl1tm/+ mice declined faster in recipients during transplantation assays, suggesting compromised long-term in vivo repopulation abilities. There were no obvious blood diseases in mutant mice throughout their life-span, indicating Asxl1 mutation alone was not sufficient for leukemogenesis. However, this mutation facilitated engraftment of bone marrow cell overexpressing MN1. Analyses of global gene expression profiles of ASXL1-mutated versus wild-type human leukemia cells as well as heterozygote versus wild-type mouse marrow precursor cells, with or without MN1 overexpression, highlighted the association of in vivo Asxl1 mutation to the expression of hypoxia, multipotent progenitors, hematopoietic stem cells, KRAS, and MEK gene sets. ChIP-Seq analysis revealed global patterns of Asxl1 mutation-modulated H3K27 tri-methylation in hematopoietic precursors. Conclusions: We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies. Keywords: Asxl1, MN1, Hematopoietic stem cell, Engraftment Background developmental abnormalities [5]. ASXL1 binds a deubi- Additional sex combs-like 1 (ASXL1) is the human quitinase BAP1 to form a critical complex for H2A K119 homolog of Drosophila additional sex combs (Asx) [1], deubiquitination through the catalysis of polycomb frequently mutated in acute myeloid leukemia (AML) repressive complex 1 [6, 7]. The deubiquitination activity and other myeloid malignancies [2–4]. Germline hetero- is enhanced when BAP1 is complexed with truncated zygous nonsense mutation of ASXL1 results in Bohring- form of ASXL1 [8]. BAP1 deletion produces phenotypes Opitz syndrome, a congenital disease with multi-system mimicking human chronic myelomonocytic leukemia in mice [9]. Thus, it is likely that ASXL1-BAP1 axis is * Correspondence: [email protected]; [email protected] important to prevent leukemogenesis [9]. 1Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan We previously analyzed the clinical implications of 5Division of Hematology, Department of Internal Medicine, National Taiwan ASXL1 mutation in a large cohort of patients and found University Hospital, No. 7, Chung-Shan S Rd, Taipei 10002, Taiwan that this mutation occurred in 10.8% (54/501) of de Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hsu et al. Journal of Hematology & Oncology (2017) 10:139 Page 2 of 15 novo AML patients and predicted a shorter survival marrow hematopoietic cells could lead to MDS-like dis- [10]. Several studies also showed that ASXL1 mutation ease [19], our mice bearing a “physiological dose” of mu- was a poor prognostic factor in myeloid malignancies tant Asxl1 did not show obvious trend of developing [10–14]. blood diseases throughout their life span. However, with Since the discovery of ASXL1 mutation in myeloid overexpression of MN1, mutant Asxl1 hematopoietic malignancies in 2009 [15], many studies about its patho- stem cells and progenitors (HSPCs) were more likely to physiology have been reported. However, controversies engraft in recipient mice than wild-type HSPCs, suggest- exist among these reports. For example, in vivo deletion ing that Asxl1 mutation could lower the threshold of of Asxl1 was shown to result in subtle phenotypes engraftment driven by MN1 overexpression. Global including defects in the frequencies of myeloid and expression profiling in mutant versus wild-type Asxl1 lymphoid cells in blood, marrow or other hematopoietic mouse cells as well as in ASXL1-mutated versus wild- organs in mice but not myelodysplastic syndrome type human AML cells, with or without concurrent (MDS) or leukemia [16]. However, in other studies, MN1 overexpression, disclosed pathophysiological path- knockout of Asxl1 led to systemic developmental defects ways involved in Asxl1 mutation. ChIP-Seq experiments including MDS-like presentation, with alteration of the showed global Asxl1 mutation-modulated H3K27me3 self-renewal and repopulation capacities of the mutant patterns in HSPCs. hematopoietic stem/progenitor cells and global reduc- tion of H3K27 tri-methylation (H3K27me3) [17, 18]. Methods The pathophysiological effect of ASXL1 truncation Generation of Asxl1 mutation knock-in mice mutations in human myeloid malignancies is another The cognate mouse mutation is predicted to be matter of debate. For example, it was suggested that c.1925dupG; p.G643WfsX12, encoding 654 amino acids ASXL1 mutation was a loss-of-function mutation mimicking the most common form of human mutant because of failure in detecting mutant protein in human ASXL1 protein, compared to 1514 residues in wild-type leukemia cells [14]. However, the findings that overex- Asxl1 protein. Potential chimeras were crossed with pression of truncating mutation in hematopoietic cells of wild-type C57BL/6 mice to facilitate the confirmation of mice displayed human MDS features with de-repression germ-line transmission, their offspring who harbored of Hoxa9 in another study [19] and detectability of trun- Asxl1 mutation were backcrossed with C57BL/6 to gen- cating proteins in human cell lines bearing ASXL1 trun- erate inbred strains then maintained at C57BL/6 back- cating mutations argued for gain-of-function or ground. Heterozygous mice were mated with wild-type dominant negative effects of ASXL1 mutations [19, 20]. mice to get heterozygous mice and littermate control These controversies are likely due to different methods mice. Heterozygous mice were mated with each other to of genetic engineering of the animals or forced overex- get homozygous mice. Mice between 2- to 6-month age pression of the mutation. Overall, the pathophysiological were used for experiment except those were assigned to alterations in human acute myeloid leukemia (AML) long-term observation cohort. All animals were housed cells bearing ASXL1 mutations have not been explored in specific pathogen-free animal facility and all proce- systematically. dures were approved by IACUC of the National Taiwan To overcome these problems, we generated and ana- University College of Medicine. lyzed a mouse model bearing human-like Asxl1 muta- tion followed by extensive phenotypic and molecular Chromatin immunoprecipitation sequencing (ChIP-Seq) characterizations on this mouse model. In our model, We used Lin- bone marrow cells as a surrogate to iden- the Asxl1 mutation was knocked in to the endogenous tify genome-wide histone modification affected by Asxl1 Asxl1 allele, thus the mice have “physiological dose” of mutations. Chromatin lysate was harvested and soni- mutation, as we see in the patients. For translating to cated with a sonicator (Bioruptor®Pico) to shear the clinical situations, we also investigated the global expres- DNA into a length ~200 bp, then it was hybridized with sion profiles of our large AML cohort to delineate the anti-H3K27me3 (Millipore, Germany). Immunoprecipi- pathophysiology related to ASXL1 mutations. We found tated DNA was sent to the National Center for Genome that bone marrow cells from Asxl1 heterozygotes formed Medicine and sequenced by Illumina HiSeq 2000 more colonies in cobblestone-area-forming assays and sequencer with 100 × 2 bp paired-end sequencing. the ability to form colonies persisted longer in serial colony-forming cell assays. On the other hand, in vivo ChIP-Seq data analysis transplantation assays showed that donor bone marrow Sequencing reads were aligned to the mm10 mouse ref- cells from Asxl1 mutant mice declined faster in their erence genome by Burrows-Wheeler Alignment tool recipients than those from the wild-type mice. While (BWA; version
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