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Progressive hematopoietic defects in ASXL2 KO mice
than previous reports from Western populations.5,7-9,40,43 We obtained a mutational rate of 6% for NRAS and 2% for KRAS compared to 13-26% for NRAS and 4-7% for KRAS observed in previous studies.5,7-9,43 Similarly, ZBTB7A mutations that are frequent in Caucasian t(8;21) AML (9- 23%)9,40,43 were observed in only 3% of our Asian patient cohort. Although this might reflect a true difference between genetic backgrounds, this requires further confir- mation in independent t(8;21) AML cohorts from differ- ent genetic backgrounds.
In this study, we focused on functional characterization of Asxl2 in hematopoietic differentiation using a mouse model of Asxl2 deficiency. Mice used in this study were C57BL/6 x 129Sv F1 as the animals in either C57BL/6 or 129Sv background die perinatally,23 which precludes inves- tigation of Asxl2 knockout on an inbred genetic back- ground. Moreover, this mouse model exhibits constitutive loss of Asxl2, which is distinct from the truncating somatic mutations of ASXL2 observed in t(8;21) AML that may possibly generate a C-terminal truncated protein. Despite these limitations, our study strengthens the findings con- cerning the key role of ASXL2 in hematopoiesis reported in two recent studies.27,28 Furthermore, our study reports several additional features of Asxl2 deficient mice, which had not been described previously. While the significance of ASXL2 in maintaining normal hematopoiesis is evi- denced from these studies, we performed a parallel in- depth analysis in young (8-14 weeks old) versus old (>1- year old) mice, which helped establish the progressive phenotype associated with Asxl2 deficiency, including uncovering the crucial function of ASXL2 in development of the lymphoid lineage. Age-dependent defects in hematopoiesis in the knockout mice were characterized by an increased proportion of LSK cells and reduced fre- quencies of CMP and GMP cells in the BM of >1-year old KO mice. Asxl2-deficient mice also exhibited a progressive myeloproliferative phenotype, accompanied by increased peripheral WBC counts, splenomegaly and extramedullary hematopoiesis, indicating perturbed hematopoiesis. In addition, erythroid maturation was impaired in the old mice lacking ASXL2, signifying age- dependent defects in erythroid development. Both previ- ous studies showed that ASXL2 deficiency led to poor reconstitution ability of HSCs in transplantation models, consistent with our findings. We observed that Asxl2-defi- cient LSK cells exhibited poor reconstitution ability of the lymphoid lineage and flow cytometric analyses of >1-year old mice demonstrated defects in both T-cell maturation in the thymus and B-cell development in the BM of the old KO mice. Apart from the intrinsic effect of ASXL2 on lym- phoid differentiation, chronic overproduction of myeloid cells and accompanying inflammatory signals in the mice lacking ASXL2 may also negatively affect the lymphoid output. These findings establish ASXL2 as an essential
component of hematopoietic development in mice.
Gene expression analysis of sorted LSK cells from WT and Asxl2 KO BM identified several key regulators of hematopoiesis as downstream targets of ASXL2 in HSCs. Prominently, GSEA analysis revealed that the expression of genes regulated by RUNX1-RUNX1T1 correlated with the expression of ASXL2, indicating ASXL2 is possibly required for transcriptional activity of RUNX1-RUNX1T1. An identical finding was described recently for Asxl2-defi- cient LSK cells in an independent mouse model. Here the authors also detected binding of ASXL2 to the genomic loci occupied by RUNX1 and RUNX1-RUNX1T1, although no direct interaction between ASXL2 and either RUNX1 or RUNX1-RUNX1T1 was observed.27 Further studies are needed to investigate whether ASXL2 acts as a likely co-regulator of RUNX1-RUNX1T1 during leukemo- genesis. GSEA also indicated a correlation between Asxl2 deficiency and the expression levels of reported targets of BMI1 (a PRC1 member)44 and SUZ12 (a PRC2 member)45 (Online Supplementary Figure S15E and F), consistent with a role for ASXL2 in transcriptional regulation mediated
through the PRC complexes.12
In summary, the current study identified frequent
ASXL2 mutations in t(8;21) AML and characterized its role in hematopoietic development in mice. This work demon- strates that ASXL2 plays a critical role in multi-lineage dif- ferentiation and highlights how its loss leads to progres- sive hematopoietic defects and promotes myeloid expan- sion, thereby advancing our understanding of epigenetic machinery that regulates hematopoiesis.
Acknowledgments
We would like to thank the staff of Comparative Medicine, NUS for their support in mice maintenance and experiments. We would also like to acknowledge expert help and support from the FACS facility at CSI, Singapore. We also appreciate the help of Dr. Motomi Osato, CSI, Singapore, for providing reagents and Dr. Maya Jeitany for critical reading of the manuscript and useful discussions. We thank the Melamed Family and Reuben Yeroushalmi for their generous support.
Funding
This work was funded by the Leukemia and Lymphoma Society, the Singapore Ministry of Health’s National Medical Research Council (NMRC) under its Singapore Translational Research (STaR) Investigator Award to HPK (NMRC/STaR/0021/2014), Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2013-T2-2-150), the NMRC Centre Grant awarded to National University Cancer Institute of Singapore (NMRC/CG/012/2013) and the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiatives. The study was supported by grant MOHW104-TDU-B-212- 124-006, OMRPG3C0021 awarded to L-YS.
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