The online version of this article (doi:10.1186/s13045-017-0508-x) contains supplementary material, which is available to authorized users.
Acute myeloid leukemia
Asxl1 G643WfsX12 heterozygous mice
Bone marrow cells
Cobblestone-area-forming cell assay
Chromatin immunoprecipitation sequencing
Common myeloid progenitors
Competitive repopulating unit assay
Gene Set Enrichment Analysis
Tri-methylation of Histone 3 at lysine 27
Hematopoietic stem cells
Hematopoietic stem cells and progenitors
Long-term culture-initiating cells
Wild-type control mice
Additional sex combs-like 1 (
ASXL1) is the human homolog of Drosophila
additional sex combs (
1], frequently mutated in acute myeloid leukemia (AML) and other myeloid malignancies [
4]. Germline heterozygous nonsense mutation of
ASXL1 results in Bohring-Opitz syndrome, a congenital disease with multi-system developmental abnormalities [
5]. ASXL1 binds a deubiquitinase BAP1 to form a critical complex for H2A K119 deubiquitination through the catalysis of polycomb repressive complex 1 [
7]. The deubiquitination activity is enhanced when BAP1 is complexed with truncated form of ASXL1 [
8]. BAP1 deletion produces phenotypes mimicking human chronic myelomonocytic leukemia in mice [
9]. Thus, it is likely that ASXL1-BAP1 axis is important to prevent leukemogenesis [
We previously analyzed the clinical implications of
ASXL1 mutation in a large cohort of patients and found that this mutation occurred in 10.8% (54/501) of de novo AML patients and predicted a shorter survival [
10]. Several studies also showed that
ASXL1 mutation was a poor prognostic factor in myeloid malignancies [
Since the discovery of
ASXL1 mutation in myeloid malignancies in 2009 [
15], many studies about its pathophysiology have been reported. However, controversies exist among these reports. For example, in vivo deletion of
Asxl1 was shown to result in subtle phenotypes including defects in the frequencies of myeloid and lymphoid cells in blood, marrow or other hematopoietic organs in mice but not myelodysplastic syndrome (MDS) or leukemia [
16]. However, in other studies, knockout of
Asxl1 led to systemic developmental defects including MDS-like presentation, with alteration of the self-renewal and repopulation capacities of the mutant hematopoietic stem/progenitor cells and global reduction of H3K27 tri-methylation (H3K27me3) [
The pathophysiological effect of
ASXL1 truncation mutations in human myeloid malignancies is another matter of debate. For example, it was suggested that
ASXL1 mutation was a loss-of-function mutation because of failure in detecting mutant protein in human leukemia cells [
14]. However, the findings that overexpression of truncating mutation in hematopoietic cells of mice displayed human MDS features with de-repression of
Hoxa9 in another study [
19] and detectability of truncating proteins in human cell lines bearing
ASXL1 truncating mutations argued for gain-of-function or dominant negative effects of
ASXL1 mutations [
20]. These controversies are likely due to different methods of genetic engineering of the animals or forced overexpression of the mutation. Overall, the pathophysiological alterations in human acute myeloid leukemia (AML) cells bearing
ASXL1 mutations have not been explored systematically.
To overcome these problems, we generated and analyzed a mouse model bearing human-like
Asxl1 mutation followed by extensive phenotypic and molecular characterizations on this mouse model. In our model, the
Asxl1 mutation was knocked in to the endogenous
Asxl1 allele, thus the mice have “physiological dose” of mutation, as we see in the patients. For translating to clinical situations, we also investigated the global expression profiles of our large AML cohort to delineate the pathophysiology related to
ASXL1 mutations. We found that bone marrow cells from
Asxl1 heterozygotes formed more colonies in cobblestone-area-forming assays and the ability to form colonies persisted longer in serial colony-forming cell assays. On the other hand, in vivo transplantation assays showed that donor bone marrow cells from
Asxl1 mutant mice declined faster in their recipients than those from the wild-type mice. While forced overexpression of mutant
Asxl1 in mouse bone marrow hematopoietic cells could lead to MDS-like disease [
19], our mice bearing a “physiological dose” of mutant
Asxl1 did not show obvious trend of developing blood diseases throughout their life span. However, with overexpression of
Asxl1 hematopoietic stem cells and progenitors (HSPCs) were more likely to engraft in recipient mice than wild-type HSPCs, suggesting that
Asxl1 mutation could lower the threshold of engraftment driven by
MN1 overexpression. Global expression profiling in mutant versus wild-type
Asxl1 mouse cells as well as in
ASXL1-mutated versus wild-type human AML cells, with or without concurrent
MN1 overexpression, disclosed pathophysiological pathways involved in
Asxl1 mutation. ChIP-Seq experiments showed global
Asxl1 mutation-modulated H3K27me3 patterns in HSPCs.
Generation of Asxl1 mutation knock-in mice
The cognate mouse mutation is predicted to be c.1925dupG; p.G643WfsX12, encoding 654 amino acids mimicking the most common form of human mutant ASXL1 protein, compared to 1514 residues in wild-type Asxl1 protein. Potential chimeras were crossed with wild-type C57BL/6 mice to facilitate the confirmation of germ-line transmission, their offspring who harbored
Asxl1 mutation were backcrossed with C57BL/6 to generate inbred strains then maintained at C57BL/6 background. Heterozygous mice were mated with wild-type mice to get heterozygous mice and littermate control mice. Heterozygous mice were mated with each other to get homozygous mice. Mice between 2- to 6-month age were used for experiment except those were assigned to long-term observation cohort. All animals were housed in specific pathogen-free animal facility and all procedures were approved by IACUC of the National Taiwan University College of Medicine.
We used Lin
- bone marrow cells as a surrogate to identify genome-wide histone modification affected by
Asxl1 mutations. Chromatin lysate was harvested and sonicated with a sonicator (Bioruptor®Pico) to shear the DNA into a length ~200 bp, then it was hybridized with anti-H3K27me3 (Millipore, Germany). Immunoprecipitated DNA was sent to the National Center for Genome Medicine and sequenced by Illumina HiSeq 2000 sequencer with 100 × 2 bp paired-end sequencing.
ChIP-Seq data analysis
Sequencing reads were aligned to the mm10 mouse reference genome by Burrows-Wheeler Alignment tool (BWA; version 0.7.15). We used the Model-based Analysis of ChIP-Seq tool (MACS2) to detect peaks of reads between sample and input sequences in
ASXL1tm/+ and wild-type bone marrow cells.
ASXL1tm/+ (or wild-type) specific peaks were called by intersecting the identified peaks with BEDTools v2.17.0 [
21]. These condition-specific peaks were annotated by Peak Annotation and Visualization (PAVIS) and analyzed for the enrichments in gene regions [
22]. To realize the biological functions governed by the interaction of
Asxl1 mutation and H3K27me3, we analyzed peaks-associated genes by The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 with default settings [
24]. Furthermore, we performed motif analysis on sequences around the condition-specific peaks (±250 bps from peak center) by MEME-ChIP web tool included in the MEME Suite [
26]. Sequencing reads and identified peaks were visualized with Integrative Genome Viewer (IGV) [
In vitro and in vivo experiments were performed at least three times independently. Data were processed in Microsoft Excel or GraphPad Prism software. Student’s
t test, paired
t test, ANOVA or chi-square test were used to compare the differences in values between groups.
For the other experimental procedures, please see the Additional file
Generation of Asxl1 G643WfsX12 gene knock-in mice
In human AML, the most common mutation is c.1934dupG; p.G646WfsX12 (up to 66%) [
10]. The mouse and human ASXL1 proteins share 74% identity in amino acid sequence. The changed amino acid G646 is within a stretch of highly conserved region ATTAIGGGG
GPGGGG (designated as a bold and underlined G) [
10]. An additional guanine was inserted into this 8-G cassette located at mouse
Asxl1 exon 13 to mimic this frequent human
ASXL1 mutation (Fig.
1a). This insertion causes frame shift in the reading frame and introduces a premature stop codon so that the mutant
Asxl1 would be shorter than wild-type form and lack the c-terminal region which contains a plant homeodomain (PHD). Therefore, the cognate mouse mutation c.1925dupG; p.G643WfsX12 is expected to bear similar pathophysiological consequence as human’s. In our mouse model, mutant
Asxl1 expression was driven by the endogenous
Asxl1 promoter. Therefore, mutant
Asxl1 would be expressed identically as the endogenous
Asxl1, not restrictive to hematopoietic cells (Fig.
The additional guanine inserted into the 8-G cassette at exon 13 was confirmed by DNA sequencing (Fig.
Asxl1 G643WfsX12 heterozygotes and homozygotes were fertile. The pups’ genotypes fit Mendelian ratio through gestation period till birth (Additional file
1: Figure S1). Homozygous new-born mice suffered from high rate of post-natal death, with only 7% of viability after weaning. Autopsy of the dead new born mice did not show any obvious organ abnormalities (data not shown). Lack of nursing was probably the main reason of these post-natal lethal events, but the exact causes remains to be elucidated. Due to the difficulty in gathering sufficient mice for observation, we hence focused our long-term observation on heterozygous and wild-type mice.
Asxl1tm/+ hematopoietic cells had higher short-term in vitro proliferation capacities
We initially performed several in vitro assays to evaluate the population frequency and differentiation potencies of HSPCs in the bone marrow of
Asxl1tm/+ and wild-type mice. We first used cobblestone-area forming cell (CAFC) assays to evaluate the frequencies of hematopoietic precursor cells in bone marrows. The cobblestone areas were counted one week after seeding and we found that
Asxl1tm/+ cells formed more cobblestone areas than
Asxl1-wild cells (
N = 3 each,
p = 0.028) (Fig.
2a). Colony-forming cell (CFC) assay were also performed to test the effects of
Asxl1 mutant on cell differentiation. Serial plating was performed every 7 days to estimate population frequencies of hematopoietic precursors in the initial plating. In initial plating,
Asxl1tm/+ and wild-type bone marrow cells formed similar numbers of each type of colonies. However, total colony number as well as granulocyte colonies (CFU-G) formed by
Asxl1tm/+ cells were more than wild-type cells in second plating and this trend last to the third plating (Fig.
2b, Additional file
1: Figure S2A to S2C). These results indicate that mutated
Asxl1 confers stronger short-term in vitro proliferation capabilities to hematopoietic precursors than the wild-type
Bone marrow cells of Asxl1 G643WfsX12 heterozygotes showed compromised long-term in vivo repopulation and self-renewal capabilities
To evaluate the in vivo influence of
Asxl1 mutation on hematopoiesis in a long-term basis, we employed bone marrow transplantation assays. Five hundred Lin
+ (LSK) bone marrow cells sorted from
Asxl1 mutant and wild-type mice, respectively, together with 200,000 helper cells, were transplanted into wild-type recipient mice for competitive repopulation unit assays. The peripheral blood of the transplanted mice was sampled monthly and evaluated for reconstitution efficiency in a 4-month period. We found that recipient mice transplanted with LSK bone marrow cells from
Asxl1tm/+ donors had less donor-derived cells in peripheral blood and marrow when compared to those receiving wild-type LSK bone marrow cells (Fig.
3a). Interestingly, B cells in the peripheral blood of
Asxl1tm/+ mice were particularly reduced (Fig.
3c). These data suggest that
Asxl1 mutant LSK cells have reduced in vivo long-term repopulation capacities compared with wild-type LSK cells.
Next, we performed serial bone marrow transplantation assays to rigorously test the potency of in vivo self-renewal ability of the
Asxl1tm/+ HSPCs. In this setting, whole bone marrow cells were serially transplanted into recipients without helper cells. To evaluate the reconstitution efficiency, peripheral blood of the recipient mice transplanted with either
Asxl1tm/+ bone marrow cells or wild-type bone marrow cells were sampled 2 months after every round of transplantation. We found that the frequencies of total cells and T cells, but not B or myeloid cells, in the recipient mice’s peripheral blood derived from
Asxl1tm/+ mice declined faster compared with those derived from wild-type controls (Fig.
4). The results suggest that
Asxl1 mutation renders a compromised long-term in vivo self-renewal capability in a variety of lineages compared to wild-type cells in vivo.
The HSPC components of Asxl1 G643WfsX12 heterozygous mice were largely similar to those of the wild-type littermates
The amount of HSPCs in the bone marrow of
Asxl1tm/+ and wild-type littermates were analyzed and compared by FACS analysis. We noted that bone marrow LSK cells, long-term (Lin
-) and short-term hematopoietic stem cells (Lin
+), multipotent progenitors (Lin
+), common myeloid progenitors (CMP, Lin
lo), granulocyte-monocytic progenitors (GMP, Lin
hi), and megakaryocyte-erythroid progenitors (MEP, Lin
lo) were all not different between the
Asxl1tm/+ heterozygotes and the wild-type mice (Additional file
1: Figure S2D). These data suggest that
Asxl1 mutation does not affect the amount of hematopoietic cell components
in vivo by surface marker analysis, although both in vitro and in vivo experiments indicate presence of its biological activities in
Asxl1tm/+ bone marrow cells as shown above.
Asxl1 G643WfsX12 alone was not sufficient for development of blood malignancies in mice
A cohort of
Asxl1tm/+ and wild-type control mice were collected to observe the influence of
Asxl1 mutation on overall health status in an 18- to 24-month period (
N = 33 for
Asxl1tm/+ mice and
N = 38 for wild-type controls). Heterozygous mice were significantly lighter than wild-type mice (Fig.
5a). While there were no significant differences in hemograms in the peripheral blood or marrow hematopoietic components in younger mice, there were subtle hematopoietic phenotypes when the mice were old at 18 months. Old male (18-month age)
Asxl1tm/+ mice had higher WBC (
p = 0.0091) and RBC counts (
p = 0.03893) (Fig.
5b). There were more T cells in bone marrows of
Asxl1tm/+ mice (
p = 0.049) than wild-type controls, while both mice had similar frequency of HSPCs (Fig.
5c, Additional file
1: Figure S3). Within the Lin
- (LK) bone marrow cells, there was no significant difference in the proportion of CMP, GMP, and MEP between
Asxltm/+ and wild-type mice (Fig.
5d). The autopsy also did not show any difference in the incidence of splenomegaly between the two groups. The only six viable
Asxl1 homozygous mice did not exhibit obvious abnormalities in hemogram or in autopsy findings at age of 18 months (Additional file
1: Figure S4). During the life span of the mice,
Asxl1 G643WfsX12 showed no tendency to induce any kind of blood malignancies; hence, we concluded that
Asxl1 G643WfsX12 alone was not sufficient for leukemogenesis in vivo.
Asxl1 G643WfsX12 lowered the engraftment threshold of MN1-overexpressing cells
Asxl1 mutation alone did not produce obvious blood diseases in mice, we sought to determine if this mutation functions as a facilitator for leukemogenesis. In our patients with array data (
N = 349, among whom the mutation status of
ASXL1 was known in 343) [
31], we noted that those bearing
ASXL1 mutation tended to have higher
MN1 expression (
P = 0.056, Fig.
6a). Moreover, among the 225 patients who received standard chemotherapy, those with higher
MN1 expression (≥ median) as well as
ASXL1 mutation had shorter overall survival compared to those with higher
MN1 expression but without
ASXL1 mutation (Fig.
6b), suggesting a possible cooperative effect between these two genetic aberrancies in AML patients.
MN1 over-expression is a sufficient driving event for mouse leukemogenesis [
32]. Therefore, we were interested to know whether
Asxl1 mutation facilitates the engraftment of
MN1 over-expression in mice. To this end, we overexpressed
Asxl1tm/+ or wild-type Lin
- bone marrow cells by retroviral transduction. Proliferation of wild-type and
Asxl1tm/+ cells were not different as examined by BrdU incorporation assays (Fig.
MN1 was transduced into
Asxl1tm/+ and wild-type bone marrow cells, respectively, we still could not observe significant difference in proliferation rate between these two types of cells (Fig.
6d). However, long-term culture-initiation cell (LTC-IC) assay showed that when
MN1 was overexpressed in
Asxl1tm/+ bone marrow cells, there was higher percentage of long-term colony forming cells compared to
MN1 overexpressed wild-type bone marrow cells (Fig.
6e), implying that
Asxl1 mutation promoted stem cell activities of marrow cells in
MN1 overexpression background. To test this hypothesis, we transplanted several different doses of
- bone marrow cells, together with 200,000 helper cells, into lethally irradiated recipients. Bone marrow cells of the recipient mice were harvested between 4 to 5 weeks after transplantation to evaluate the reconstitution efficiency. More than 1%
MN1 over-expressing cells in the marrow cells of recipient mice was defined to be successfully reconstituted. At 5000-cell dose, 100% of recipient mice transplanted with either
Asxl1 mutant or wild-type bone marrow cells were successfully reconstituted. However, while most recipient mice transplanted with low-dose
MN1-transduced cells could be reconstituted in the presence of
Asxl1 mutation (9 out of 11 at 1000 test cells and 7 out of 7 at 500 test cells were successfully reconstituted), significantly lower proportion of recipient mice transplanted with the same doses of
MN1-transduced cells without
Asxl1 mutation were successfully reconstituted (7 out of 12 at 1000 test cells and 1 out of 6 at 500 test cells,
p = 0.036 by Chi-square test) (Table
1 and Additional file
1: Figure S5). Our results suggest that
Asxl1 G643WfsX12 can lower the threshold of
The number of mice successfully reconstituted in transplantation assay of
MN1 overexpressed cells with WT or mutant
Microarray analyses showed the cooperative effects of Asxl1 mutation and MN1 overexpression
Our mouse model provided an ideal platform to investigate the impacts of
Asxl1 mutation per se on global gene expression patterns, since the genetic backgrounds of our mice were far less complicated than those in human leukemia cells. In addition, we were interested in the mechanisms underlying the supportive role of
Asxl1 mutation in the engraftment of
MN1 overexpressing bone marrow cells. To these ends, we collected Lin
- marrow cells from
Asxl1tm/+ and wild-type control mice with or without
MN1 transduction (wild-type,
Asxl1 mutation, wild-type +
MN1 overexpression, and
Asxl1 mutation +
MN1 overexpression) for microarray analyses to explore differential gene expression patterns as well as molecular functions conferred by
Asxl1 mutation per se and/or its interplay with
MN1 overexpression. Of note, only 4.6% genes were significantly differentially expressed between
Asxl1-mutated and wild-type cells (Fig.
7a; left bar); the number of perturbed functional gene sets was also very modest (3.2%, comparing
Asxl1 mutation vs. wild-type cells, Fig.
7b; left bar). However, the differences became obvious when comparing
Asxl1-mutated cells overexpressing
MN1 vs. wild-type cells overexpressing
MN1 cells: up to 12.2% differentially expressed genes among all genes (Fig.
7a, second bar from the left), higher than that achieved by randomly shuffling the microarray dataset for 100 times (empirical
p < 0.01), with correspondingly large scale of perturbed gene sets, up to 23.9% (Fig.
7b, second left bar). These data were consistent with our findings that
Asxl1 alone did not render obvious blood diseases in the mice but it might play a cooperative role with
To compare our mouse model with human disease, we profiled gene expression of leukemia cells from a total of 343 AML patients and compared the expression patterns between samples with (
N = 50) and without (
N = 293)
ASXL1 mutation. For 172 AML patients with higher
MN1 expression (above the median level), we also compared the expression patterns between those with (
N = 29) and without (
N = 143)
ASXL1 mutation. The disturbance of global gene expression profiles and gene sets related to
Asxl1 mutation were quite comparable and obvious in total cohort (Fig.
7a, right two bars) and in the subgroup of patients with higher
MN1 expression (Fig.
7b, right two bars).
Gene set enrichment analysis revealed oncogenic functions perturbed by the interaction between Asxl1 mutation and MN1 overexpression
A deeper look into the lists of significantly differential gene sets derived by GSEA revealed a handful of crucial oncogenic functions perturbed by the interaction between
Asxl1 mutation and
MN1 overexpression. Hypoxia-related genes were implied to be relevant factors of leukemogenesis [
34]. In our data, while the expression of genes of a hypoxia signature did not show an overall change in mice with
Asxl1 mutation vs. wild-type littermates (GSEA
p = 0.272; Fig.
7c), they were significantly co-upregulated in
Asxl1tm/+, compared to wild-type mice, in the presence of
p = 0.007; Fig.
7d). Similar enrichments were seen in signatures representing multipotent progenitors and hematopoietic stem cells, as well as genes related to oncogenic
MEK, in an
MN1-dependent manner (all
p values <0.0005 in
. wild-type mice transduced with
7d; compared with
p > 0.05 in
Asxl1 mutation vs. wild-type mice without
MN1 overexpression, except for genes related to
7c, p value 0.012). Such positive enrichment toward
Asxl1 mutation was corroborated in AML patients, regardless of the abundance of
p values <0.0005; Fig.
7e and f). In aggregate, the logistic relationship between
Asxl1 mutation and
MN1 overexpression is summarized as (1)
Asxl1 mutation promoted engraftment of bone marrow cells in
MN1 overexpression background (Fig.
6e and Table
1); (2) AML patients with both
ASXL1 mutation and high
MN1 expression had inferior survival when compared with
ASXL1-wild-type and high
MN1 expression (Fig.
6b); (3) In the background of
Asxl1 mutation in mice and in human AML patients was associated with upregulation of signatures of hematopoietic stem/progenitor cells and related to hypoxia, KRAS, and MEK pathways.
ChIP-Seq analysis revealed Asxl1 mutation-modulated binding profiles of H3K27me3
Several studies have linked functions of
Asxl1 mutation to H3K27me3, an inactive mark associated with transcriptional repression [
19]. In order to investigate their interactions in our mouse model, we performed histone extraction followed by western hybridization to evaluate the global H3K27me3 levels in bone marrow cells. There was no significant difference in global H3K27me3 levels between
Asxl1tm/+ bone marrow cells and wild-type bone marrow cells (Additional file
1: Figure S6). Then, we analyzed if there was different global H3K27me3 pattern between
Asxl1tm/+ and wild-type Lin
- bone marrow cells via ChIP-Seq analysis. Comparing sequencing reads of ChIP products and input controls, we identified ~70 k H3K27me3-binding peaks in each of the
Asxl1tm/+ and wild-type samples. Of note, considerable proportions of them were
Asxl1tm/+ cells-specific (25,695; 37.0%) or wild-type (26,850; 37.7%) cells-specific. These peaks are highly concordant with gene loci in the mouse genome (Fig.
Mn1 harbored three H3K27me3-binding sites, of which one was
8a, left lower panel). We then analyzed the distribution of the condition-specific peaks in gene regions. Significant enrichment of peaks was found in upstream (within 5 k bps; 7.2 and 6.8% of
Asxl1tm/+- and wild-type-specific peaks, respectively; both
p < 0.001) and downstream regions of gene bodies (within 1 k bps; 1.4%,
p = 0.031; and 1.3%,
p = 0.023, respectively) compared to randomly distributed peaks across the genome (Fig.
8b). Other genomic categories, such as 5’ and 3’ untranslated regions (UTRs) and exons, were not enriched (all
p values >0.05), suggesting the preference of
Asxl1 mutation-associated H3K27me3 occupancy in gene regulatory regions.
To further investigate
Asxl1 mutation-modulated targets of H3K27me3, we analyzed enriched motifs on the peaks by the MEME-ChIP web tool, which performs motif discovery, enrichment, and visualization from DNA sequences of interest. Interestingly, while
Asxl1tm/+ and wild-type specific peaks do not overlap with each other, they carried very similar motifs: RGRAA, TVTGTR, and TTTAWW (all
E values <0.001; Fig.
8c), indicating that the modulation of
Asxl1 mutation in H3K27me3 occupancy is independent of the binding motifs.
In order to investigate the effects of such selective targeting on gene expression and biological functions, we linked the peaks with gene expression microarrays. Lists of H3K27me peaks-associated genes with concordant significant downregulation are provided in Additional file
1: Table S1. Notably, the down-regulated genes in
Asxl1 wild-type cells were significantly associated with concordant
Asxl1 wild-type- specific H3K27m3 peaks (15.69% of the
Asxl1 wild-type-specific downregulated genes harbored concordant
Asxl1 wild-type-specific H3K27m3 peaks, compared to 11.69% of non-downregulated genes; Fisher’s exact test
p = 0.001; Fig.
8d, upper panel). However,
Asxl1tm/+ cells-specific peaks were not significantly associated with down-regulated genes in
Asxl1tm/+ cells (Fisher’s exact test
p = 0.52; Fig.
8d, lower panel), implying that in
Asxl1 mutated cells, the association between H3K27m3 and gene downregulation is disrupted when compared with
Asxl1 wild-type condition. Taken together, our ChIP-Seq data demonstrated distinct
Asxl1 mutation-modulated binding profiles of H3K27me3.
For the first time, we have demonstrated the pathophysiological functions of a “physiological” dose of
Asxl1 mutations in vivo and in vitro. In contrast to the previous studies with enforced overexpression of mutant ASXL1 protein in a background of two wild-type alleles of endogenous
19], our model facilitated investigation of a more clinically relevant
In our study, we noted while
Asxl1 mutation promoted engraftment of
MN1-overexpressing cells and showed increased colony formation and cobblestone area formation, the LSK cells bearing
Asxl1 mutation had inferior repopulation capacities when compared with wild-type cells in vivo. This counterintuitive observation could be explained by two possibilities: (1) in our in vivo transplantation assays (Figs.
4), we assessed the activities of HSCs. But
MN1 overexpression targeted committed progenitor cells, not HSCs [
35]. This may explain the discrepancies between these experimental results; (2) in serial transplantation, the marrow stem cells were taken and expanded in a previously irradiated microenvironment, not normal hematopoietic niche. Spangrude et al. have shown a vastly inferior repopulation capacity of LSK cells repeatedly exposed to such perturbed microenvironment [
36]. Such radiation perturbation on microenvironment was absent in colony formation and cobblestone area formation assays, and less severe in
MN1 overexpressing cell transplantation assays. We could not rule out the possibility that
Asxl1 mutant cells were particularly susceptible to this factor, thus showing decreased repopulation capacity in serial transplantation assays, while similar phenomenon was not shown in the other assays without serial irradiation. Moreover, Kamminga et al. showed that although a gradual decrease of the percentage of LSK cells was observed when LSK cells were used as donor cells in serial transplantation, only minor decrease was observed for the clonogenic CAFC activity of the purified cells [
37]. These results suggested that in vivo repopulation ability of LSK cells might be affected by residue host cells or competitor cells. They also highlighted the limitation of current animal assays to detect the “real” in vivo hematopoietic stem cell activities.
In our model,
Asxl1 G643WfsX12 mutation did not lead to leukemia or other blood malignancies in a 18-24-month observation period, indicating that a physiological dose of
Asxl1 G643WfsX12 was not sufficient for leukemogenesis. Nevertheless, the mutation could enhance engraftment of
MN1 overexpressing cells, suggesting that
Asxl1 mutation could function as a cooperative hit of
MN1 overexpression to promote the engraftment of bone marrow cells. This is consistent with the clinical observation that
ASXL1 mutant burden often increases in disease progression and mutations in
41], as well as other genes encoding epigenetic modifiers, were often acquired early in the disease and were almost never found in isolation [
Gene expression microarray and GSEA showed limited difference between
Asxl1tm/+ and wild-type control bone marrow cells under steady state, consistent with our observation that
Asxl1tm/+ mice did not develop obvious blood diseases. In
MN1 overexpression background, the expression patterns and physiological pathways between
Asxl1 mutation and wild-type became distinctive (Fig.
7), implying the promoting effects of
Asxl1 mutation on
MN1 overexpression-induced engraftment of bone marrow cells. The high number of differentially expressed genes and perturbed biological pathways in human
ASXL1-mutated versus wild-type AML cells demonstrated a far more complicated milieu in human AML cells compared with mice HSPCs with
Asxl1 mutation per se (Fig.
From our microarray studies, we found that
Asxl1 mutation alone in mice had little effects on both gene expression profiles and biological pathways, while the perturbation became obvious in the presence of
MN1 overexpression. How
MN1 overexpression augments the genomic effects of
Asxl1 mutation is not completely defined in our study, but we found that
Asxl1 mutation plus
MN1 overexpression, but not
Asxl1 mutation alone, was associated with enrichment of signatures representing multipotent progenitors and hematopoietic stem cells, as well as genes related to oncogenic
Hypoxia-related genes are considered critical for the survival of leukemia initiation cells [
43]. The enrichment in hematopoietic stem cell and multipotent progenitor gene sets further implies the supporting function of
ASXL1 mutation in blood malignancies.
KRAS is considered relevant in leukemia formation [
46]; the enrichment in
KRAS gene set confers the possibility that mutant ASXL1 act as a cofactor in disease development. MAPK/ERK pathway is crucial for hematopoiesis and aberrant MAPK/ERK pathway is associated with cancer formation [
47]. RAS signaling are also considered to be involved in AML transformation at both genetic and epigenetic levels [
48]. Bone marrow cells of our mouse model were supposed to have
Asxl1 mutation alone, but in
ASXL1-mutated human AML cells, we expected there were additional genetic perturbations. One of the advantages of our mouse model was that it enabled us to interrogate the functions of
Asxl1 mutation per se, in a “simpler” genetic background. This was probably why we saw different biological effects of
MN1 overexpression between BM cells in our mouse model and human AML cells with more complex genetic background.
Asxl1 has been considered to be associated with the regulation of H3K27me3, we performed ChIP-Seq to investigate the alteration of global H3K27me3 pattern in
Asxl1tm/+ bone marrow cells. Considerable numbers of H3K27me3 peaks specific to
Asxl1tm/+ and to wild-type bone marrow cells were noted and preponderantly located within 5 k upstream and 1 k downstream of gene bodies. These results indicate that
Asxl1 mutation can modulate the global pattern of histone methylation in a non-random manner, preferentially immediate to the gene bodies. In addition, in
Asxl1 mutated cells, the correlation between H3K27m3 and gene down-regulation appears attenuated when compared with
Asxl1 wild-type context, suggesting functional implications of Asxl1 functions in H3K27me3 modulation. Taken together, our systematic analyses unveiled crucial oncogenic functions perturbed by the interplay between
Asxl1 mutation and
MN1 overexpression that may partially account for the cooperative role of
Asxl1 mutations in
MN1-associated leukemia in human and mouse settings and the functional impacts of
ASXL1 mutation in human AML.
Taken together, for the first time, our study reveals the in vitro and in vivo effects of a “physiological” dose of
Asxl1 mutation. Although mutant
Asxl1 does not act as a sufficient driver in blood malignancies, it facilitates engraftment of cells overexpressing
MN1. Our study also enlightens the effects on global H3K27m3 profiles by
Asxl1 mutation and several potential biological pathways underlying mutant
We thank the technical services provided by the Transgenic Mouse Model Core Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology, Taiwan, the Gene Knockout Mouse Core Laboratory of National Taiwan University Center of Genomic Medicine, and the National Center for Genome Medicine.
MN1 expression construct was a kind gift from Dr. R. Keith Humphries. We would like to acknowledge the service provided by the Flow Cytometric Analyzing and Sorting Core Facilities at National Taiwan University Hospital and First Core Laboratory of National Taiwan University College of Medicine. We thank the Taiwan Mouse Clinic (MOST 105-2325-B-001-010) which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the Ministry of Science and Technology of Taiwan for technical support in complete blood count and tissue section experiment. We thank the Drs. Hsing-Chen Tsai, Dr. Tai-Chung Huang, and Yen-Wei Chen for the technical support in ChIP-Seq sample preparation and analysis. We also thank the National Center for Genome Medicine for the technical and bioinformatics service in ChIP-Seq analysis. We appreciated the staff of the Eighth Core Lab, Department of Medical Research, National Taiwan University Hospital for technical support during the study.
The study was supported by a National Taiwan University Hospital − National Taiwan University joint research grant (UN103-051), Ministry of Science and Technology of Taiwan (MOST102-2325-B-002-028, 103-2314-B-002-130-MY3, 103-2314-B-002-131MY3 and 104-2923-B-002-001), Far Eastern Hospital and NTUH joint grant 105-FTN24, and Ministry of Health and Welfare of Taiwan (MOHW106-TDU-B-211-144005).
Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author upon reasonable request.
YCH wrote the paper, performed the experiments, and analyzed data. YCC wrote the paper and analyzed the data. WCC and HFT planned, designed and coordinated the research, and wrote the manuscript. CCL, YYK, HAH, YST, CJK, PHC, MHT, and THH provided important materials and help in the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The collection of patients’ leukemia cells for microarray studies was approved by the Institutional Review Board of the National Taiwan University Hospital. Animals used in this study were housed in a specific pathogen-free animal facility and all procedures were approved by IACUC of National Taiwan University College of Medicine (IACUC approval number: 20120346).
Consent for publication
The authors declare that they have no competing interests.
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