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T. D’Altri et al.
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Figure 5. The ASXL1G643W variant is associated with increased resistance to chemotherapy. (A) Schematic outline of the chemotherapy treatment set-up. Briefly, sub- lethally irradiated recipient mice were transplanted with bone marrow (BM) cells harvested from leukemic mice. Twenty-one days after transplant, recipient mice were treated with standard induction chemotherapy for 5 days. Three days later, blood was harvested for analysis and leukemic mice were subsequently monitored for disease development. (B) Analysis of peripheral leukemic numbers in mice after chemotherapy/vehicle treatment. Each data point represents the fold change dif- ference between the vehicle-treated and chemotherapy-treated groups for a given clone (n=12 mice per clone). The two different genotypes, Asxl1+/+; CebpaΔ/p30 and Asxl1G643W/G643W CebpaΔ/p30, were represented by four and three clones, respectively. The responses of individual clones are indicated in the Online Supplementary Figure S4. A one-tailed Mann-Whitney U test was used to assess statistical significance. (C) Kaplan-Meyer survival curves of leukemic mice after chemotherapy/vehicle treatment. The data represent the aggregate of two different leukemic clones (n=6 recipients of each clone, clones 1-2 and 5-6) for each of the two different genotypes (Asxl1+/+; CebpaΔ/p30 and Asxl1G643W/G643W; CebpaΔ/p30).
measured by the decrease of leukemic cells in the periph- eral blood 3 days post-treatment cessation (Figure 5B). This translated into an increased latency for the treated ASXL1WT cohort whereas chemotherapy had no impact on survival of the ASXL1G643W mutant cohort (Figure 5C).
Taken together, these findings demonstrate that muta- tion of ASXL1 renders CEBPA mutated AML largely resist- ant to chemotherapy.
Discussion
Myeloid diseases such as AML are developing from pre- malignant clones which mostly harbor lesions in epigenet- ic regulators.3,4 One of these regulators is ASXL1 which is consequently frequently mutated in both MDS and AML as well as in premalignant settings such as CHIP. This mutational profile raises the question whether ASXL1 plays a functional role in full-blown AML or whether it merely provides a fertile ground in which AML can evolve.
There has been considerable confusion concerning the molecular mechanisms by which ASXL1 mutations sus- tain AML development or hematological deficiencies, specifically whether ASXL1 mutations act as dominant negatives. Some of this confusion is related to the inherent difficulties in detecting ASXL1 by western blotting (issues which we also experienced) thus making it nearly impos- sible to determine whether the mutated protein is present or not. However, overexpression of ASXL1 variants was associated with hematological malignancies raising the possibility that the mutated protein could act as a domi-
nant negative.6,14,15 Our analysis of heterozygous and homozygous Asxl1G643W mice in the context of normal hematopoiesis suggests that the Asxl1G643W variant has a dose-dependent impact in this context. Given that com- plete loss of Asxl1 leads to more pronounced hematopoi- etic phenotypes, the most restrained explanation from the in vivo work is that the ASXL1G643W variant is hypomorphic or, alternatively, that it exerts a combination of dominant negative and hypomorphic effects.10-12
Expression of the Asxl1G643W variant in the context of CEBPA mutant AML significantly accelerated AML devel- opment and was associated with marked resistance to induction chemotherapy. These findings are not only in perfect alignment with the co-occurrence of CEBPA and ASXL1 lesions in human AML, but also with the overall poor prognosis of ASXL1 mutated human AML. Hence the resistance towards chemotherapy is likely underlying the poor prognosis of AXL1 mutated AML.33-35 It would be interesting to test if other mouse models can recapitulate this behavior beyond CEBPA mutant AML.
Interestingly, gene expression analysis demonstrated that Asxl1G643W/G643W; CebpaD/p30 exhibited downregulation of signa- tures associated with immune activation perhaps reflecting that the Asxl1 lesion renders the developing leukemia less visible to the immune system. We also observed a reduc- tion in activation of the DNA damage pathways in ASXL1 mutated cells which could potentially reflect efficient clear- ing of ongoing genomic insults. Here our finding of the marked upregulation of Traip is of particular interest as the corresponding protein has recently been identified as a master regulator of DNA crosslink repair.32 Thus, our data strongly suggest that Asxl1 lesions have functional conse-
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haematologica | 2021; 106(4)