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Journal of Hematology & Oncology

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The distinct biological implications of Asxl1 mutation and its roles in leukemogenesis revealed by a knock-in mouse model

  • Yueh-Chwen Hsu1,
  • Yu-Chiao Chiu2,
  • Chien-Chin Lin1, 3,
  • Yuan-Yeh Kuo4,
  • Hsin-An Hou5,
  • Yi-Shiuan Tzeng4,
  • Chein-Jun Kao3,
  • Po-Han Chuang3,
  • Mei-Hsuan Tseng5,
  • Tzu-Hung Hsiao2,
  • Wen-Chien Chou1, 3, 5Email author and
  • Hwei-Fang Tien5Email author
Journal of Hematology & Oncology201710:139

https://doi.org/10.1186/s13045-017-0508-x

Received: 8 May 2017

Accepted: 30 June 2017

Published: 11 July 2017

Abstract

Background

Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia-like disease in mice. However, actual effects of a “physiological” dose of mutant ASXL1 remain unexplored.

Methods

We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system.

Results

Heterozygotes (Asxl1 tm/+ ) marrow cells had higher in vitro proliferation capacities as shown by more colonies in cobblestone-area forming assays and by serial re-plating assays. On the other hand, donor hematopoietic cells from Asxl1 tm/+ mice declined faster in recipients during transplantation assays, suggesting compromised long-term in vivo repopulation abilities. There were no obvious blood diseases in mutant mice throughout their life-span, indicating Asxl1 mutation alone was not sufficient for leukemogenesis. However, this mutation facilitated engraftment of bone marrow cell overexpressing MN1. Analyses of global gene expression profiles of ASXL1-mutated versus wild-type human leukemia cells as well as heterozygote versus wild-type mouse marrow precursor cells, with or without MN1 overexpression, highlighted the association of in vivo Asxl1 mutation to the expression of hypoxia, multipotent progenitors, hematopoietic stem cells, KRAS, and MEK gene sets. ChIP-Seq analysis revealed global patterns of Asxl1 mutation-modulated H3K27 tri-methylation in hematopoietic precursors.

Conclusions

We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies.

Keywords

Asxl1MN1Hematopoietic stem cellEngraftment

Background

Additional sex combs-like 1 (ASXL1) is the human homolog of Drosophila additional sex combs (Asx) [1], frequently mutated in acute myeloid leukemia (AML) and other myeloid malignancies [24]. 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 [6, 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 [9].

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 [1014].

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) [17, 18].

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 [19, 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 MN1, mutant 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.

Methods

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.

Chromatin immunoprecipitation sequencing (ChIP-Seq)

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 ASXL1 tm/+ and wild-type bone marrow cells. ASXL1 tm/+ (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 [23, 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 [25, 26]. Sequencing reads and identified peaks were visualized with Integrative Genome Viewer (IGV) [27, 28].

Statistical analysis

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 1.

Results

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 ATTAIGGGGGPGGGG (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. 1a).
Fig. 1

Generation of the Asxl1 G643WfsX12 knock-in mice. a Schematic illustration of the generation strategies of the knock-in mouse model. b DNA sequences of Asxl1 G643WfsX12 heterozygous, homozygous and wild-type bone marrow cells. Asxl1 mutant had an additional guanine inserting into the 8-G cassette leading to Asxl1 G643WfsX12 mutation. Since cells with heterozygous Asxl1 mutant had both 8-G wild-type and 9-G mutant alleles, there were overlapping signals caused by 9-G mediated frame shift after the 8-G/9-G cassette

The additional guanine inserted into the 8-G cassette at exon 13 was confirmed by DNA sequencing (Fig. 1b). Both 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.

Asxl1 tm/+ 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 Asxl1 tm/+ 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 Asxl1 tm/+ 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, Asxl1 tm/+ 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 Asxl1 tm/+ 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 Asxl1.
Fig. 2

In vitro assays to compare the numbers and potencies of HSPCs between the Asxl1 G643WfsX12 heterozygous mice and wild type controls. a Cobblestone-area-forming cell assays on bone marrow cells from Asxl1 tm/+ mice showed more colonies than those from wild-type control mice (N = 3 each). b Colony-forming cell assays and serial plating assays showed no significant difference in the ability of forming colonies and differentiation into different lineages in the first plating between Asxl1 tm/+ bone marrow cells and wild controls. However, in the second plating Asxl1 tm/+ cells formed more colonies than wild-type controls. This trend lasted to the third plating but only two wild-type samples had sufficient cells for the third plating, so we were unable to perform t test in the third plating (first plating: wild-type N = 5, Asxl1 tm/+ N = 7; second plating: wild-type N = 5, Asxl1 tm/+ N = 6; third plating: wild-type N = 2, Asxl1 tm/+ N = 6). *p < 0.05

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-c-Kit+Sca-1+ (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 Asxl1 tm/+ 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 Asxl1 tm/+ 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.
Fig. 3

Competitive repopulation assays. The peripheral blood a and bone marrow b of mice receiving Asxl1 tm/+ cells showed lower chimerism than those receiving wild-type cells in a 4-month observation period. c At 4 months after transplantation, donor-derived B cells in the peripheral blood of recipient mice receiving Asxl1 tm/+ cells were particularly reduced compared with those receiving wild-type donor cells

Next, we performed serial bone marrow transplantation assays to rigorously test the potency of in vivo self-renewal ability of the Asxl1 tm/+ 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 Asxl1 tm/+ 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 Asxl1 tm/+ 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.
Fig. 4

Serial bone marrow transplantation assays. At 2 months after secondary transplantation, the chimerism of total blood cells (a) B cells (b) T cells (c) and myeloid cells (d) in the peripheral blood of recipients receiving Asxl1 tm/+ cells declined faster than wild-type donors. *p < 0.05

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 Asxl1 tm/+ and wild-type littermates were analyzed and compared by FACS analysis. We noted that bone marrow LSK cells, long-term (Lin-Sca-1+c-Kit+CD150+CD48-) and short-term hematopoietic stem cells (Lin-Sca-1+c-Kit+CD150+CD48+), multipotent progenitors (Lin-Sca-1+c-Kit+CD150-CD48+), common myeloid progenitors (CMP, Lin-Sca-1-c-Kit+CD34+FcγRlo), granulocyte-monocytic progenitors (GMP, Lin-Sca-1-c-Kit+CD34+FcγRhi), and megakaryocyte-erythroid progenitors (MEP, Lin-Sca-1-c-Kit+CD34-FcγRlo) were all not different between the Asxl1 tm/+ 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 Asxl1 tm/+ bone marrow cells as shown above.

Asxl1 G643WfsX12 alone was not sufficient for development of blood malignancies in mice

A cohort of Asxl1 tm/+ 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 Asxl1 tm/+ 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) Asxl1 tm/+ mice had higher WBC (p = 0.0091) and RBC counts (p = 0.03893) (Fig. 5b). There were more T cells in bone marrows of Asxl1 tm/+ 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-c-Kit+Sca-1- (LK) bone marrow cells, there was no significant difference in the proportion of CMP, GMP, and MEP between Asxl tm/+ 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.
Fig. 5

The body weights and hemograms of old Asxl1 mutant mice (18 months old). a Body weights of Asxl1 heterozygous mice were significantly lower than wild-type mice. b Old male Asxl1 tm/+ mice tended to have higher RBC and WBC counts in peripheral blood. c There were higher percentages of T cells in old Asxl1 tm/+ mice while both mice had similar frequencies of B cells and myeloid cells in the bone marrow. d There was no significant difference in CMP, GMP, and MEP between Asxl1 tm/+ and wild-type control mice. *p < 0.05

Asxl1 G643WfsX12 lowered the engraftment threshold of MN1-overexpressing cells

Since 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) [2931], 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 MN1 in Asxl1 tm/+ or wild-type Lin- bone marrow cells by retroviral transduction. Proliferation of wild-type and Asxl1 tm/+ cells were not different as examined by BrdU incorporation assays (Fig. 6c). When MN1 was transduced into Asxl1 tm/+ 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 Asxl1 tm/+ 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 MN1-transduced Lin- 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 MN1 overexpressed 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 MN1-driven engraftment.
Fig. 6

Interaction between mutant Asxl1 and MN1 overexpression. a According to our patient data, ASXL1 mutation was associated with higher MN1 expression. b Among patients with higher MN1 expression, those with ASXL1 mutation had shorter overall survival than those without this mutation (median 4.5 months vs. not reached). c Asxl1 tm/+ had no impact on proliferation of bone marrow cells. d Asxl1 tm/+ and wild-type bone marrow cells overexpressed MN1 did not show significant difference in proliferation. e Three independent LTC-IC assays showed that under MN1 overexpression background, Asxl1 tm/+ bone marrow cells had higher frequency of long-term culture initiation cells than wild-type controls

Table 1

The number of mice successfully reconstituted in transplantation assay of MN1 overexpressed cells with WT or mutant Asxl1

Cell dose

WT + MN1

Asxl1 tm/+ +MN1

Number tested

Number reconstituted

Number tested

Number reconstituted

5000

6

6

8

8

1000

12

7

11

9

500

6

1

7

7

p = 0.036 by chi square test

Abbreviations: WT Asxl1 wild type, MN1 MN1 overexpression

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 Asxl1 tm/+ 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 MN1.
Fig. 7

Gene expression and gene set analyses of the interplay between ASXL1 mutations and MN1 overexpression. a Proportions of significantly differentially expressed genes and b enriched gene sets between samples with mutant and wild-type ASXL1 in mice and AML patients. Significant differential expression was defined by Student’s t test p < 0.05; gene set enrichment analysis was conducted by GSEA, with a threshold on GSEA p value at 0.0005. cf GSEA enrichment plots of selected oncogenic gene signatures. For each microarray dataset, all genes were sorted by the significance of differential expression between samples carrying mutant and wild-type ASXL1 (denoted by left and right arrows). Gene sets were tested for overrepresentation at either side of the ranked list, of which the overrepresentation was measured by a running enrichment score (ES; green curves). Positive and negative ES represent enrichments in ASXL1-mutated and wild-type samples, respectively. Significance of an ES was assessed by random permutation of the gene list. c, d All of the five gene signatures showed MN1-transduction specific enrichments in mice. e, f Concordant enrichments were seen in AML patients, with no dependency on the expression of MN1. These oncogenic functions may partially account for the change in the threshold of MN1-driven engraftment in the presence of Asxl1 mutation

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 [33, 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 Asxl1 tm/+ , compared to wild-type mice, in the presence of MN1-overexpression (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 KRAS and MEK, in an MN1-dependent manner (all p values <0.0005 in Asxl1 tm/+ vs. wild-type mice transduced with MN1, Fig. 7d; compared with p > 0.05 in Asxl1 mutation vs. wild-type mice without MN1 overexpression, except for genes related to MEK, Fig. 7c, p value 0.012). Such positive enrichment toward Asxl1 mutation was corroborated in AML patients, regardless of the abundance of MN1 (all 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 MN1 overexpression, 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 Asxl1 tm/+ 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 Asxl1 tm/+ 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 Asxl1 tm/+ and wild-type samples. Of note, considerable proportions of them were Asxl1 tm/+ 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. 8a). Mn1 harbored three H3K27me3-binding sites, of which one was Asxl1 tm/+ -specific (Fig. 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 Asxl1 tm/+ - 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.
Fig. 8

ChIP-Seq analysis of H3K27me3 in Asxl1 tm/+ and wild-type bone marrow cells. a Integrative Genome Viewer plots of Asxl1 tm/+ and wild-type specific peaks in the mouse genome. Bottom panels, enlarged plots of two examples. b Distributions of the Asxl1 tm/+ modulated peaks in gene regions. Upstream and downstream lengths were set at 5 k and 1 k bps., respectively. The specific H3K27 trimethylation peaks are significantly enriched in upstream and downstream regions. *, binomial test p < 0.05; ***, p < 0.001. c Top enriched motifs on the Asxl1 tm/+ modulated peaks reported by the MEME-ChIP web tool. d Proportions of significant downregulated genes in Asxl1 wild-type (upper panel) and Asxl1 tm/+ (lower) cells harboring condition-specific H3K27me3 peaks. 15.69% of the Asxl1 wild-type-specific downregulated genes harbored wild-type-specific H3K27 trimethylation peaks, while only 11.69% of other genes in Asxl1 wild-type cells harbored wild-type-specific H3K27 trimethylation peaks. **, Fisher’s exact test p < 0.01; N.S., non-significant p

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 Asxl1 tm/+ 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, Asxl1 tm/+ cells-specific peaks were not significantly associated with down-regulated genes in Asxl1 tm/+ 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.

Discussion

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 Asxl1 [18, 19], our model facilitated investigation of a more clinically relevant Asxl1 mutation.

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. 3 and 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 ASXL1 [3841], as well as other genes encoding epigenetic modifiers, were often acquired early in the disease and were almost never found in isolation [42].

Gene expression microarray and GSEA showed limited difference between Asxl1 tm/+ and wild-type control bone marrow cells under steady state, consistent with our observation that Asxl1 tm/+ 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. 7a, b).

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 MEK.

Hypoxia-related genes are considered critical for the survival of leukemia initiation cells [34, 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 [4446]; 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.

Since 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 Asxl1 tm/+ bone marrow cells. Considerable numbers of H3K27me3 peaks specific to Asxl1 tm/+ 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.

Conclusions

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 ASXL1.

Abbreviations

AML: 

Acute myeloid leukemia

Asxl1 tm/+

Asxl1 G643WfsX12 heterozygous mice

BMC: 

Bone marrow cells

CAFC: 

Cobblestone-area-forming cell assay

CFC: 

Colony-forming cell

ChIP-Seq: 

Chromatin immunoprecipitation sequencing

CMP: 

Common myeloid progenitors

CRU: 

Competitive repopulating unit assay

GMP: 

Granulocyte-monocytic progenitors

GSEA: 

Gene Set Enrichment Analysis

H3K27me3: 

Tri-methylation of Histone 3 at lysine 27

HPC: 

Hematopoietic progenitors

HSC: 

Hematopoietic stem cells

HSPC: 

Hematopoietic stem cells and progenitors

LK: 

lin-c-Kit+Sca-1- cells

LSK: 

lin-c-Kit+Sca-1+ cells

LSK-

lin-c-Kit-Sca-1+ cells

LTC-IC: 

Long-term culture-initiating cells

MDS: 

Myelodysplastic syndrome

MEP: 

Megakaryocyte-erythroid progenitors

WT: 

Wild-type control mice

Declarations

Acknowledgements

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.

Funding

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.

Authors’ contributions

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

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Authors’ Affiliations

(1)
Graduate Institute of Clinical Medicine, National Taiwan University
(2)
Department of Medical Research, Taichung Veterans General Hospital
(3)
Department of Laboratory Medicine, National Taiwan University Hospital
(4)
Graduate Institute of Oncology, College of Medicine, National Taiwan University
(5)
Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital

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Copyright

© The Author(s). 2017

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