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result in a dominant-negative version of the protein.6 In line with these findings, several reports have indicated that truncated ASXL1 enhances BAP1 complex activity, thereby promoting depletion of the H2AK119Ub mark and aberrant myeloid differentiation.14,15 More refined modeling of ASXL1 function in hematological malignan- cies has been performed by knockin of patient-specific ASXL1 lesions into the murine Asxl1 locus.16,17 These knockin mice generally exhibit more subtle phenotypes compared to the complete deletion of the gene. Although heterozygous knockin mice do no develop MDS or AML, the knockin alleles collaborate with other leukemic drivers such as MN1 and RUNX1 or accelerate AML development in an insertional mutagenesis setting.16,17
CEBPA is a key myeloid transcription factor which is mutated in approximately 10% of AML patients and bial- lelic CEBPA mutant AML constitutes a specific AML sub- type.18-20 These patients either harbor biallelic N-terminal lesions or, more frequently, combine these lesions with a C-terminal mutation. Whereas the N-terminal lesions pro- mote the expression of the N-terminally truncated p30 iso- form, C-terminal mutations result in variants that are unable to dimerize and are consequently inactive. Hence, the genetic lesions in biallelic CEBPA mutant AML con- verge at the expression of the N-terminally truncated p30 isoform in the form of CEBPA-p30 homodimers.20 In stark contrast to full-length CEBPA, CEBPA-p30 is not able to repress E2F-mediated cell cycle progression21 and recent work has also identified specific downstream targets of this oncogenic CEBPA variant.22 Importantly, mice in which CEBPA-p30 expression is driven from the endoge- nous Cebpa locus develop AML within the first year of their lives.21 Interestingly, mutations in ASXL1 are frequent in biallelic CEBPA mutant AML, but how these two sets of lesions interact functionally is currently unknown.23,24
In the present work, we generated a novel Asxl1 knockin line by introducing the most common disease- associated mutation (p.G643WfsX12) into the murine Asxl1 allele.2,25 In order to assess the importance of Asxl1 lesions in the context of biallelic CEBPA mutated AML, we combined lesions in these two proteins and found that the ASXL1 mutation accelerated the development of CEBPA-p30 driven AML. Gene expression analysis yield- ed potential drivers of the accelerated phenotype. Interestingly, ASXL1 mutated AML were largely refracto- ry to chemotherapy, thereby paralleling the findings from the human setting.
Methods
Generation of the Asxl1G643W knockin mice
The Asxl1G643W knockin line was generated using the double
nicking CRISPR-Cas9 system in embryonic stem cells (ESC), fol- lowed by blastocyst injection. Two pspCas9n-2A-Puro constructs containing the two Asxl1 target sequences were electroporated into C57BL/6N ESCs together with a 141-mer single stranded (ss) DNA correction template containing the desired mutation. ESC clones were screened for the presence of the mutation and correct- ly targeted clones were injected into mouse blastocysts. Please refer to the Online Supplementary Appendix for additional details.
F1 offspring were backcrossed into C57BL/6 and maintained on that background. Animals were housed in a specific-pathogen-free facility and all procedures were approved by the Danish Animal Ethical Committee.
In vivo acute myeloid leukemia development
Bone marrow (BM) cells were retrieved and frozen in fetal calf serum (FCS) with 10% DMSO. For leukemic experiments, stored BM was thawed and 2 million viable cells were trans- planted into lethally irradiated (900 Gy) recipients by tail vein injection. Three weeks later, recipients were subjected to three intraperitoneal injections with poly-IC (0.3 mg in 200 mL PBS, GE Healthcare) separated by 48 hours. Recipient mice were monitored for leukemic development and euthanized when moribund. Please refer to the Online Supplementary Appendix for
additional details.
For the chemotherapy experiments, we transplanted cohorts
of sublethally irradiated recipients with frozen secondary AML. Three weeks after transplant half the mice in each cohort were treated for 3 days with cytarabine 50 mg/kg and doxorubicine 1 mg/kg and for 2 days with cytarabine 50 mg/kg. The remaining mice received PBS as vehicle treatment. 25-30 mL of blood were harvested for analysis three days after the last injection. Leukemic cell numbers were determined by combining cell counting with CD45.1 (recipient)/ CD45.2 (donor) flow cytom- etry. For the survival study, the mice were observed for signs of disease and euthanized when moribund.
Statistics
Unpaired t-test was used to compare values in the different groups. Log-rank (Mantel-Cox) test was used to compare sur- vival distributions. For the chemotherapy data in Figure 5B a one-tailed Mann-Whitney U test was used.
RNA sequencing
Donor derived AML blasts (CD45.2, Ter119-, B220-, CD3-, Mac1low, Gr1low, c-Kit+) were sorted from frozen BM samples into RLT lysis buffer (Qiagen) and RNA was extracted using the RNA Microkit (Qiagen). 100 ng RNA was used for the library generation, using TruSeq-V2 kit (Illumina). The libraries were analyzed by Qbit (ThermoFisher) and Bioanalyzer (Agilent) and pooled in equimolar amounts. Multiplexed samples were sequenced on a NextSeq 500 (Illumina) yielding approximately 35-45 million reads per sample. Please refer to the Online Supplementary Appendix for additional details.
Results
Generation of the Asxl1G643W knockin mouse line
In order to model the role of ASXL1 lesions in hematopoietic malignancies in the best possible manner, we decided to generate a mouse line expressing the most common ASXL1 mutation (G643WfsX12, from hereon G643W) found in MDS patients.2,25 Specifically, we used a double nicking CRISPR-Cas9 system in combination with a 141 bp ssDNA donor strand to introduce the c.1934dupG mutation into the endogenous Asxl1 locus. This approach results in the insertion of a G within a stretch of eigth G located in the last exon of Asxl1 which in turn generates a frameshift and an in-frame stop codon
haematologica | 2021; 106(4)
Asxl1 lesions collaborate with CEBPA-p30 in AML
Flow cytometry analysis and cell sorting
For blood analysis, 50 mL blood was collected from the facial vein. Erythrocytes were depleted with BD PharmLyse. For BM analysis, cells were collected by crushing tibia, femur and ilium and filtered. Blood or BM nucleated cells were washed in PBS with 3% FCS and stained for 15 min at 4°C. Please refer to the Online Supplementary Appendix for additional details (antibodies and marker combinations).
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