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Progressive hematopoietic defects in ASXL2 KO mice
approach has identified additional mutagenic events involving ASXL2, ASXL1 and DHX15 genes in t(8;21) AML.5-10 Several secondary genetic events that co-operate with RUNX1-RUNX1T1 fusion in inducing leukemia have been demonstrated using mouse models.2-4,11
ASXL1, ASXL2 and ASXL3 are human homologs of Drosophila Asx (Additional sex combs) and function as epigenetic regulators through recruitment of Polycomb group repressor complexes (PRC) and Trithorax group activator complexes.12,13 ASXL proteins can interact with BAP1, NCOA1, EZH2, WTIP and nuclear receptors, which suggests diverse functions of ASXL family members in epigenetic and transcriptional regulation.12,13 Somatic mutations of ASXL1 occur in a range of hematologic dis- orders,14-20 and its deletion in mouse hematopoietic cells results in multilineage cytopenias and dysplasia, which are associated with global reduction of H3K27 trimethyla- tion.21 In contrast, somatic mutations of ASXL2 are observed exclusively in t(8;21) AML,5,8 while mutations of ASXL3 are not reported in hematologic malignancies.13 Silencing of ASXL2 is partially embryonic lethal depend- ing on the genetic background, and leads to congenital heart defects. Surviving homozygous Asxl2 mutant mice are smaller than wild-type littermates, demonstrate skele- tal homeotic transformations consistent with disruption of Polycomb/Trithorax complex functions, develop car- diac dysfunction, and exhibit decreased bone mineral den- sity.22-26 More recently, its role in normal hematopoietic development and leukemic development has been investi- gated.27,28
In the present study, we used exome sequencing to identify somatic mutations co-operating with RUNX1- RUNX1T1 rearrangement in AML. Frequent truncating mutations of ASXL2 were identified in our t(8;21) AML cohort. Using an Asxl2-deficient mouse model, we aimed to clarify the function of ASXL2 in hematopoiesis. We demonstrate that ASXL2 is crucial to maintain hematopoi- etic stem cell (HSC) number and function. Loss of ASXL2 leads to myeloproliferation and extramedullary hematopoiesis, as well as to progressive defects in multi- lineage differentiation. These results establish ASXL2 as a key component of the epigenetic machinery involved in maintaining hematopoietic development.
Methods
Acute myelogenous leukemia samples
t(8;21) AML was diagnosed by karyotypic analysis and/or reverse transcriptase PCR assay for the detection of fusion tran- script of RUNX1-RUNX1T1. Samples were collected with informed consent at diagnosis, during complete remission, and at time of frank relapse in accordance with the Declaration of Helsinki. Bone marrow (BM) mononuclear cells were obtained by Ficoll-Hypaque density gradient centrifugation (1.077 g/mL; Amersham Pharmacia, Sweden). The study was approved by the institutional review boards of the respective institutes.
Exome sequencing and somatic variant discovery
DNA was sheared using a Covaris instrument and assessed on a 2100 Bioanalyzer. Library preparation and exome sequencing were performed as described previously.29 For whole exome sequencing, DNA were captured using SureSelect Human All Exon 50Mb kit (Agilent), according to the manufacturer’s instruc- tions. For targeted capture library, Agilent’s SureSelect XT2 Target
Enrichment System for Illumina Multiplexed Sequencing was used. RNA baits were designed to capture exons of 530 genes (Online Supplementary Table S1) and libraries were sequenced on HiSeq 2000 (Illumina).
100 bp paired-end reads were aligned to human reference genome (reference build: hg19) using bwa-mem aligner with default parameters. PCR duplicate reads were marked with sam- blaster.30 Resulting BAM files were further processed according to GTAK best practices including INDEL re-alignment and base qual- ity recalibration (https://software.broadinstitute.org/gatk/best-prac- tices/bp_3step.php?case=GermShortWGS). Somatic variants were detected using Varscan2 somatic command.31 Raw variants were processed with processSomatic command with the P-value set to 0.05 to obtain high confident variants. Variants were further processed using fpFilter per script to remove potential false posi- tives (https://github.com/ckandoth/variant-filter). Resulting variants were annotated using Variant Effect Predictor and filtered against germline variants in both dbSNP and ExAC, while keeping delete- rious and clinically significant variants.32 For samples without germline controls, variants were called using MuTect using a panel of normal derived from in-house cohort of AML remission sam- ples, as described previously.29,33 Oncoplots were drawn using maftools Bioconductor package.34 All the somatic mutations for whole exome sequencing and about 90% mutations reported for targeted exome sequencing were validated using PCR amplifica- tion and Sanger sequencing.
Mice
Mice with Asxl2 gene-trap allele [referred to as knockout (KO) allele in this study] have been described previously.24 Asxl2 het- erozygous KO mice were maintained on C57BL/6J (B6) and 129Sv (129) genetic backgrounds. Asxl2 homozygous null mice [and cor- responding wild-type (WT) controls] used in all experiments were B6 x 129 F1 obtained by crossing Asxl2 heterozygous B6 and 129 mice. Mouse colonies were housed and maintained at the animal facility of Comparative Medicine Centre, National University of Singapore (NUS). All mice experiments were approved by Institutional Animal Care and Use Committee, NUS, Singapore.
Flow cytometry
Cells were incubated with fluorochrome-conjugated antibodies for 30 min on ice, washed and resuspended in SYTOX Blue Dead Cell Stain (ThermoFisher Scientific) before acquisition on FACS LSR II flow cytometer (BD Biosciences). Sorting of cells was per- formed on FACSAria cell sorter (BD Biosciences) and data were analyzed using FACSDIVA software (BD Biosciences). See Online Supplementary Table S2 for the list of antibodies used for flow cyto- metric staining.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 7 software.
Accession numbers
The accession numbers for the sequencing data reported in this study are SRP122878 (exome sequencing) and GSE106798 (RNA sequencing).
Results
Mutational profile of RUNX1-RUNX1T1 AML
We performed whole-exome sequencing of 10 paired samples of newly-diagnosed and relapsed AML with RUNX1-RUNX1T1 rearrangement from an Asian cohort,
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