ASXL1 mutations accelerate bone marrow fibrosis via EGR1-TNFA axis-mediated neoplastic fibrocyte generation in myeloproliferative neoplasms

Abstract

Apart from the central role of the activated JAK/STAT signaling pathway, ASXL1 mutations are the most recurrent additional mutations in myeloproliferative neoplasms and occur much more commonly in myelofibrosis than in essential thrombocythemia and polycythemia vera. However, the mechanism of the association with ASXL1 mutations and bone marrow fibrosis remains unknown. Here, integrating our own data from patients with myeloproliferative neoplasms and a hematopoietic-specific Asxl1 deletion/Jak2V617F mouse model, we show that ASXL1 mutations are associated with advanced myeloproliferative neoplasm phenotypes and onset of myelofibrosis. ASXL1 mutations induce skewed monocyte/macrophage and neoplastic monocyte-derived fibrocyte differentiation, consequently they enhance inflammation and bone marrow fibrosis. Consistently, the loss of ASXL1 and JAK2V617F mutations in hematopoietic stem and progenitor cells leads to enhanced activation of polycomb group target genes, such as EGR1. The upregulation of EGR1, in turn, accounts for increased hematopoietic stem and progenitor cell commitment to the monocyte/macrophage lineage. Moreover, EGR1 induces the activation of TNFA and thereby further drives the differentiation of monocytes to fibrocytes. Accordingly, combined treatment with a TNFR antagonist and ruxolitinib significantly reduces fibrocyte production in vitro. Altogether, these findings demonstrate that ASXL1 mutations accelerate fibrocyte production and inflammation in myeloproliferative neoplasms via the EGR1-TNFA axis, explaining the cellular and molecular basis for bone marrow fibrosis and the proof-ofconcept for anti-fibrosis treatment.

Introduction

Myeloproliferative neoplasms (MPN) are malignant clonal diseases originating from hematopoietic stem cells, characterized by the proliferation of one or more myeloid lineages and an increasing risk of transformation to acute myeloid leukemia.¹ Primary myelofibrosis (PMF) is the subtype with the worst prognosis.² Moreover, approximately 15% of patients with essential thrombocythemia (ET) or polycythemia vera (PV) develop post-ET/PV MF over time, which is similar to PMF in treatment and outcome.¹ Somatic mutations in Janus kinase 2 (JAK2), calreticulin (CALR), or myeloproliferative leukemia protein (MPL) are regarded as driver mutations that activate the JAK/STAT signaling pathway and are essential for the MPN phenotype.^3-5 JAK2^V617F is the most common driver mutation and is present in more than 95% of cases of PV and more than 50% of ET and MF (including PMF and post-ET/PV MF) patients.²

Although inappropriate JAK/STAT pathway activation exists in most MF patients, the JAK1/JAK2 inhibitor ruxolitinib has a limited effect on reversing fibrosis grades in MF patients.⁶ Meanwhile, there are reports of several animal models with Jak2^V617F which induce PV or ET-like phenotypes while MF is rare.^{7- 9} Besides driver mutations, additional mutations are common in MF patients,^10,11 and the mouse models with concomitant Jak2^V617F and Ezh2, Tet2 or Dnmt3a loss showed accelerated MF as well. However, the mechanisms are not fully delineated.^12-14

ASXL1 mutations are the most recurrent nondriver mutations in MF and are much more common in PV and ET patients.^10,11 As one of the mammalian homologs of the Drosophila Asx,¹⁵ polycomb group (PcG) genes, ASXL1 acts as an essential cofactor for the nuclear deubiquitinase BRCA1-Associated Protein 1 (BAP1)^16,17 and as a critical mediator of Polycomb Repressive Complex 2 (PRC2),¹⁸ participating in the epigenetic control of gene expression. Frameshift and nonsense mutations are the major types of ASXL1 mutation, resulting in C-terminal truncation and usually loss of ASXL1 expression.¹⁸ Wild-type ASXL1 plays an essential role in normal hematopoiesis. Asxl1 knockout mice show impaired hematologic progenitor differentiation and development of myelodysplasia and myelodysplastic syndrome/MPN.¹⁹ In Jak2^V617F mice, heterozygous knockout of Asxl1 in germline accelerates MF progression,²⁰ while how ASXL1 loss results in transcription deregulation and aberrant lineage differentiation in MPN remains poorly understood.

In this study, we analyzed the clinical characteristics of ASXL1 mutations in MF patients and generated hematopoietic-specific Asxl1 knockout and Jak2^V617F knockin mouse models. Our data showed that ASXL1 mutations could promote monocyte/macrophage-mediated inflammation and neoplastic monocyte-derived fibrocyte-induced bone marrow (BM) fibrosis by activating the EGR1-TNFA axis in both ASXL1-mutated MF patients and Asxl1 knockout/Jak2^V617F mice, offering novel potential therapeutic strategies for anti-fibrosis treatment.

Methods

Patients and animals

Three hundred and two consecutive MF patients were investigated in this study. Diagnoses were classified according to 2016 World Health Organization (WHO) MPN definitions.²¹ All patients gave written informed consent compliant with the Declaration of Helsinki. Studies involving medical records and human tissues were approved by the Ethics Committee of the blood disease hospital, Chinese Academy of Medical Sciences & Peking Union Medical College. For mouse model studies, cre-inducible Jak2^{LSL V617F/+} mice, Asxl1^flox/flox mice and hematopoietic-specific Vav1-Cre transgenic mice were used. All mice were on a C57BL/6 background. Details are contained in the Online Supplementary Methods. The experimental protocols were approved by the Institutional Animal Care and Use Committee of State Key Laboratory of Experimental Hematology.

In vitro monocyte/macrophage differentiation assay

Murine BM was isolated and enriched for c-kit using CD117 MicroBeads (Miltenyi) and separated using an AutoMACS Pro separator (Miltenyi). BM c-kit⁺ cells were plated in methylcellulose medium (Methocult M3234, Stem Cell Technologies) supplemented with mouse interleukin (IL)3 (Peprotech, 10 ng/mL) for 1×10⁴ cells per well. After 8 days, colonies were counted, then isolated, pooled, and resuspended in phosphate-buffered saline, and stained with F4/80 antibodies (Biolegend, 123115) for flow cytometric analysis on a FACS Canto II flow cytometer (BD Biosciences). Additionally, Wright-Giemsa-stained cytospin smears were prepared for morphological analysis.

In vitro fibrocyte differentiation assay and quantification

Murine BM nucleated cells or patients’ BM mononuclear cells were resuspended in conditions that promote the differentiation of monocytes to fibrocytes.^22,23 Cells were cultured in 24-well tissue culture plates with 5×10⁵ cells/500 mL. After 5 days, immunofluorescence staining was performed to identify fibrocytes. Details of the protocols are provided in the Online Supplementary Methods.

Immunohistochemical and image quantification of patients’ samples

Bone marrow biopsy sections were dewaxed, rehydrated and retrieved. Sections were blocked in 10% donkey/10% goat serum or 10% donkey serum and then incubated with primary antibodies overnight followed by secondary staining. Next, AutoFluo Quencher (Applygen) was applied to quench autofluorescence. Finally, glass coverslips were mounted onto the slides using Mounting Medium with DAPI (Abcam). Images were captured by confocal microscopy (PerkinElmer UltraVIEW VoX system) and quantified using Fiji-ImageJ software. Details of the protocols are contained in the Online Supplementary Methods.

Gene-expression profiling and bioinformatics analysis

Murine BM c-kit⁺ cells were enriched for bulk RNA sequencing, assay for transposase-accessible chromatin (ATAC) with sequencing and chromatin immunoprecipitation (ChIP) sequencing. Detailed protocols are contained in the Online Supplementary Methods.

A more detailed description of the methods is published in the Online Supplementary Appendix.

Results

ASXL1 mutations are associated with severe disease phenotypes in patients with myelofibrosis

To determine the clinical impact of ASXL1 mutations on MF patients, we analyzed data from 302 MF patients in our single center; 250 (82.8%) patients displayed driver mutations, including 174 (57.6%) JAK2^V167F, 63 (20.9%) CALR, and 13 (4.3%) MPL mutations (Online Supplementary Figure S1A). 98 (32.5%) patients harbored ASXL1 mutations. Figure 1A shows the landscape of localizations and types of ASXL1 mutation. Frameshift mutations were the most common mutation type (N=50, 51.0%) followed by nonsense (N=46, 46.9%) and missense mutations (N=2, 2.0%).

Figure 1B-D and Online Supplementary Table S1 summarize the clinical and laboratory characteristics of MF patients according to ASXL1 mutations. In this cohort, ASXL1 mutations were correlated with lower hemoglobin levels, higher monocyte counts, increasing CD34⁺ cells in peripheral blood (PB), larger spleen sizes, and higher MF grades, consistent with prior studies.¹⁰ Similar results were also found in the driver mutation positive (driver^MT) cohort (N=250) (Figure 1E-G, Online Supplementary Table S2). We next analyzed the co-mutations in MF patients with or without ASXL1 mutations. Considering the total cohort, compared with ASXL1 wildtype (ASXL1^WT) patients, ASXL1-mutated (ASXL1^MT) patients more commonly had CALR, KRAS, and ZRSR2 mutations (Online Supplementary Figure S1B), while considering the driver^MT cohort, CALR and NRAS mutations were more frequent in the ASXL1^MT patients than in the ASXL1^WT patients (Online Supplementary Figure S1C). Altogether, these data suggest that ASXL1 mutations are associated with severe disease phenotypes in MF patients.

Asxl1 deletion is associated with enhanced extramedullary hematopoiesis in the spleen and onset of bone marrow fibrosis in Asxl1-/-Jak2V617F/+ mice

To further address the consequences of ASXL1 mutations on MPN in vivo, we utilized Vav1-Cre mice, Asxl1^flox/flox and Jak2^V617F/+ knockin alleles to achieve hematopoietic cellspecific Jak2^V617F/+/Asxl1^flox/flox (Asxl1^-/-Jak2^VF), Jak2^V617F/+ (Jak2^VF), and Asxl1^flox/flox (Asxl1^-/-) mice. In control with wildtype (WT) mice, both Asxl1^-/-Jak2^VF and Jak2^VF mice developed erythrocytosis and died of thrombosis at an early stage of the disease (Figure 2A, Online Supplementary Figure S2A, B). Compared with age-matched Jak2^VF mice, Asxl1^-/-Jak2^VF mice showed lower white blood cell, lymphocyte and platelet counts, and higher monocyte counts in PB (Figure 2A). Furthermore, the percentages of c-kit⁺ cells in PB were significantly increased in Asxl1^-/-Jak2^VF mice compared to other genotypes (Figure 2B).

Consistent with PB findings, Asxl1^-/-Jak2^VF mice showed comparable erythropoiesis in BM and spleens (Online Supplementary Figure S2C), and a decreased proportion of B lymphocytes (B220⁺) in BM compared with Jak2^VF mice (Online Supplementary Figure S2D). Analysis of the hematopoietic stem and progenitor cell (HSPC) compartment revealed comparable percentages of Lin^-Scal1⁺c-kit⁺ (LSK) cells, granulocyte/macrophage progenitors, and megakaryocyte/erythroid progenitors in the BM of Asxl1^-/-Jak2^VF and Jak2^VF mice (Figure 2C, Online Supplementary Figure S2E), whereas in the comparison with Jak2^VF mice, the proportions of granulocyte/macrophage progenitors and megakaryocyte/erythroid progenitors were significantly higher in spleens of Asxl1^-/-Jak2^VF (Figure 2D, Online Supplementary Figure S2E), in line with the increased spleen weights of Asxl1^-/-Jak2^VF mice (Figure 2E). We next performed methylcellulose colony-forming assays to verify the effect of Asxl1 deletion on HSPC functions in Asxl1^-/-Jak2^VF mice. Colonyforming capacities of nucleated cells were enhanced in spleen cells from Asxl1^-/-Jak2^VF mice compared with those of other genotypes, whereas there was no difference in BM colony-forming capacity between mice of different genotypes (Online Supplementary Figure S3A, B), indicating more activated extramedullary hematopoiesis in the spleens of Asxl1^-/-Jak2^VF mice.

BM histology analysis revealed typical features of MPN with trilineage hyperplasia, especially increased megakaryocytes and atypia in both Jak2^VF and Asxl1^-/-Jak2^VF mice (Figure 3). Interestingly, reticulin and collagen fiber infiltration was present in Asxl1^-/-Jak2^VF mice at 16 weeks of age, but not in other genotypes (Figure 3). Additionally, Asxl1^-/-Jak2^VF and Jak2^VF spleen specimens exhibited effacement of normal splenic architecture and extramedullary hematopoiesis was more obvious in Asxl1^-/-Jak2^VF mice relative to Jak2^VF mice (Figure 3, Online Supplementary Figure S3C).

To further confirm that these findings are cell-autonomous, we transplanted BM nucleated cells from Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice into lethally irradiated recipients (Online Supplementary Figure S4A). The survival of recipients of cells from Asxl1^-/-Jak2^VF mice was worse than that of mice transplanted with cells of other genotypes (Online Supplementary Figure S4B). At 24 weeks after transplantation, Asxl1^-/-Jak2^VF –cell recipients gradually developed monocytosis and thrombocytopenia compared with Jak2^VF–cell recipients (Online Supplementary Figure S4C). Meanwhile, the proportions of c-kit⁺ cells in PB were significantly higher in Asxl1^-/-Jak2^VF recipients (Online Supplementary Figure S4D). Additionally, HSPC compartment analysis showed increased myeloid progenitors (Lin^-Sca1^-c-kit⁺) in spleens, not in BM of recipients of Asxl1^-/-Jak2^VF cells in comparison with Jak2^VF-cell recipients (Online Supplementary Figure S4E), despite comparable spleen weights in these two groups (Online Supplementary Figure S4F). Histological analysis revealed increased reticulin and collagen fibers in BM and enhanced extramedullary hematopoiesis in spleens of recipients of Asxl1^-/-Jak2^VF cells compared with Jak2^VF–cell recipients at 38-40 weeks after transplantation (Online Supplementary Figure S4G).

Taken together, our findings in Asxl1^-/-Jak2^VF mice are consistent with clinical findings in ASXL1^MT MF patients, indicating that ASXL1 mutations are associated with MPN disease progression.

Skewed inflammatory monocyte/macrophage differentiation in ASXL1MT myelofibrosis patients and Asxl1-/-Jak2VF mice

The overproduction of inflammatory cytokines is a hallmark feature in MPN especially in MF.²⁴ We thus compared the circulating cytokine levels in PV and MF patients and observed that MF patients had a more severe inflammatory environment than PV patients (Online Supplementary Figure S5A, Online Supplementary Table S3). Notably, ASXL1^MT MF patients showed higher levels of tumor necrosis factor (TNF)-α and IL-10 than ASXL1^WT MF patients (Online Supplementary Figure S5A, Online Supplementary Table S3). Moreover, a set of inflammatory cytokines and chemokines, including TNF-α, CCL2 and CCL5 were elevated in Asxl1^-/-Jak2^VF mice compared with Jak2^VF mice (Online Supplementary Figure S5B).

Figure 1.ASXL1 mutations are associated with severe disease phenotypes in myelofibrosis patients. (A) Landscape of localizations and mutational types of 98 ASXL1 mutations in 302 patients with myelofibrosis (MF). (B-D) Spleen sizes (B) (N=87 for ASXL1^MT patients and n=196 for ASXL1^WT patients), proportions of CD34⁺ cells in peripheral blood (PB) (C) (N=38 for ASXL1^MT patients and N=79 for ASXL1^WT patients), and MF grades (D) (N=98 for ASXL1^MT patients and N=204 for ASXL1^WTpatients) of MF patients with different ASXL1 mutational status. (E-G) Spleen sizes (E) (N=75 for Driver^MTASXL1^MT patients and N=157 for Driver^MTASXL1^WT patients), proportions of CD34⁺ cells in PB (F) (N=34 for Driver^MTASXL1^MT patients and N=68 for Driver^MTASXL1^WT patients) and MF grades (G) (N=85 for Driver^MTASXL1^MT patients and N=165 for Driver^MTASXL1^WT patients) of driver^MT MF patients with different ASXL1 mutational status. ASXN: additional sex combs N-terminus domain; ASXH: additional sex combs homology domain; PHD: plant homeodomain. LCM: left costal margin. In (B), (C), (E) and (F), the results are presented as the median ± interquartile range. A Mann-Whitney U test was performed between the medians of two groups. In (D) and (G), the results are presented as percentages. A Mann-Whitney U test was performed between ordinal variables. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Several cell populations, such as monocytes, granulo-cytes and megakaryocytes, are responsible for overproduction of cytokines in MF.²⁵ Remarkably, we observed that both ASXL1^MT MF patients and Asxl1^-/-Jak2^VF mice had elevated monocyte counts in PB (Figure 2A, Online Supplementary Tables S1 and 2), which are the major cell origin of cytokines in MF.^26,27 Subsequent subtype assays of PB monocytes (CD115⁺CD11b⁺) in mouse models revealed elevated Ly6C⁺ monocytes (inflammatory monocytes),²⁸ but not Ly6C^- monocytes in Asxl1^-/-Jak2^VF mice (Online Supplementary Figure S5C). In addition, monocyte-derived dendritic cells, which accumulate during inflammatory conditions,²⁹ were increased in the PB of Asxl1^-/-Jak2^VF mice (Figure 4A, Online Supplementary Figure S5D). Consistent with PB findings, Asxl1^-/-Jak2^VF mice showed higher proportions of monocytes (CD11b⁺CD115⁺) in BM and spleens compared with Jak2^VF mice (Figure 4B, C, Online Supplementary Figure S6A), while no difference was found in granulocytes (CD11b⁺CD115^-) (Online Supplementary Figure 6B).

Figure 2.Asxl1 deletion is associated with enhanced extramedullary hematopoiesis in Asxl1^-/-Jak2^VF mice. (A) Hemoglobin, white blood cell, neutrophil, lymphocyte, monocyte, and platelet counts in peripheral blood (PB) were assessed at 12 weeks of age in Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice (N=13–15 per group). (B) Representative flow cytometric plots (upper) and the proportions (lower) of c-kit⁺ cells in PB of Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=8–9 per group). (C, D) The proportions of LSK cells (Lin^-Sca-1⁺c-kit⁺), granulocyte/macrophage progenitors (Lin^-Sca-1^-c-kit⁺CD34⁺FcγRII/III^high), common myeloid progenitors (Lin^-Sca-1^-c-kit⁺CD34⁺FcγRII/III^low) and megakaryocyte-erythroid progenitors (Lin^-Sca-1^-c-kit⁺CD34^-FcγRII/III^-) in bone marrow (C) and spleens (D) from Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=8-9 per group). (E) Representative images (upper) and the weights (lower) of spleens from Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=11-15 per group). In (A–E), the results are presented as mean ± standard error of the mean. A two-tailed unpaired Student t test was performed between means of two groups. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Hb: hemoglobin; WBC: white blood cells; NEUT: neutrophils; LYM: lymphocytes; MONO: monocytes; PLT: platelets; GMP: granulocyte/macrophage progenitors; CMP: common myeloid progenitors; MEP: megakyocyte-erythroid progenitors; SSC: side scatter; BM: bone marrow.

Monocyte-derived macrophages are also critical in chronic inflammation. They are highly heterogeneous cells that can rapidly polarize to M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages in response to microenvironmental signals.³⁰ We next analyzed macrophage populations and M1/M2 polarization in mouse models. Gr1^-CD115^intF4/80⁺SSC^low cells were defined as macrophages in flow cytometry analysis and further classified as M1 (CD80⁺CD206^-) and M2 (CD80^-CD206⁺) subtypes.³¹ The proportions of macrophages, predominantly M1 macro-phages, were markedly increased in BM and spleens of Asxl1^-/-Jak2^VF mice compared with the proportions in Jak2^VF mice (Figure 4D, E; Online Supplementary Figure S6C). Similarly, using immunostaining, we observed that ASXL1^MT MF patients had higher numbers of CD45⁺CD68⁺ cells, which are composed of monocytes and macro-phages, in BM specimens than those in ASXL1^WT MF patients (Online Supplementary Figure S7, Online Supplementary Table S4).

To confirm the origins of macrophages in mouse models, we performed genotyping identification in sorted Asxl1^-/-Jak2^VF BM macrophages and detected both the Jak2^V617F mutation and the Asxl1 deletion (Online Supplementary Figure S6D, E). We next did a noncompetitive bone marrow transplantation assay (Asxl1^-/-Jak2^VF BM nucleated cells [CD45.2] to lethally irradiated recipients [CD45.1]) and measured the percentages of donor (CD45.2) and recipient (CD45.1) cells in macrophages. Nearly 90% of the total and M1 macrophages were positive for CD45.2 in BM and spleens of Asxl1^-/-Jak2^VF recipients (Online Supplementary Figure S6F). These data suggest that the increased macrophages are neoplastic macrophages derived from monocytes rather than primary tissue-resident macrophages.

Figure 3.Morphology of enhanced extramedullary hematopoiesis and myelofibrosis in Asxl1^-/-Jak2^VF mice. Representative images of hematoxylin and eosin (H&E), Reticulin and Masson trichrome staining in femur and representative images of H&E staining in spleen biopsy specimens from Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 16 weeks of age. Asxl1^-/-Jak2^VF and Jak2^VF mice showed increased megakaryocytes and atypia in bone marrow and spleen specimens (arrow). Original magnification 40×, scale bar, 50 mm. BM: bone marrow.

On the basis of the above findings, we questioned whether Asxl1 deletion would lead to the differentiation bias of Asxl1^-/-Jak2^VF HSPC toward the monocyte/macrophage lineage. To examine this, we isolated Asxl1^-/-Jak2^VF and Jak2^VF BM c-kit⁺ cells and seeded them in methylcellulose supplemented with mouse IL-3 (10 ng/mL) in vitro. On day 8, no difference was found in the numbers of colonies between these two groups (Online Supplementary Figure S8A), while flow cytometric and morphological analysis of cells obtained from colonies showed higher proportions of macrophages (F4/80⁺) in Asxl1^-/-Jak2^VF mice than in Jak2^VF mice (Online Supplementary Figure S8B, C), indicating a skewed monocyte/macrophage differentiation of Asxl1^-/-Jak2^VF HSPC.

Figure 4.ASXL1^MT myelofibrosis patients and Asxl1^-/-Jak2^VF mice have increased inflammatory monocytes/macrophages. (A) The proportions of monocyte-derived dendritic cells (CD11c^intCD11b^highMHC II⁺Ly6C⁺) in peripheral blood of Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=4-6 per group). (B, C) The proportions of monocytes (CD11b⁺CD115⁺) in bone marrow (B) and spleens (C) of Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=6-7 per group). (D) Representative flow cytometric plots (left) and the proportions of total macrophages (Gr-1^-CD115^intF4/80⁺SSC^low) (middle) and M1(CD80⁺CD206^-)/M2 (CD80^-CD206⁺) subtypes (right) in bone marrow of Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=7-9 per group). (E) Representative flow cytometric plots (left) and the proportions of total macrophages (Gr-1^-CD115^intF4/80⁺SSC^low) (middle) and M1(CD80⁺CD206^-)/M2 (CD80^-CD206⁺) subtypes (right) in spleens of Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age (N=7-9 per group). In (A–E), the results are presented as mean ± standard error of mean. A two-tailed unpaired Student t test was performed between means of two groups. ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. PB: peripheral blood; moDC: monocyte-derived dendritic cells; BM: bone marrow; SSC: side scatter; FSC: forward scatter.

Altogether, these data indicate that, in the context of a constitutively activated JAK/STAT pathway, ASXL1 mutations induce an inflammatory monocyte/macrophage differentiation bias and enhance inflammation in ASXL1^MT MF.

ASXL1 mutations result in increased monocyte-derived fibrocyte differentiation in ASXL1MT myelofibrosis patients and Asxl1-/-Jak2VF mice

Mesenchymal stromal cell (MSC)-derived myofibroblasts were previously considered as the major collagen-producing cells in MPN.^32-34 We performed Gli1, Leptin Receptor (Lep-tinR), and a-SMA immunostaining in MF patients and chose blood vessel as a positive control (Online Supplementary Figure S9A). However, no difference was found in MSC-derived myofibroblasts (Gli1⁺ and/or LeptinR⁺ and a-SMA⁺) as well as Gli1⁺ cells and LeptinR⁺ cells between ASXL1^WT and ASXL1^MT MF patients (Online Supplementary Figures S9B-D and S10, Online Supplementary Table S4). Recently, several studies identified neoplastic monocyte-derived fibrocytes as a separate contributor to BM fibrosis.^22,23 Considering the increased monocytes in both ASXL1^MT MF patients and Asxl1^-/-Jak2^VF mice, we sought to determine whether monocyte-derived fibrocytes play a critical role in BM fibrosis formation. Monocyte-derived fibrocytes are positive for both hematopoietic markers and collagen markers.^22,23 Accordingly, we performed CD45 and ProCollagenI (ProCol-I) immunostaining of BM specimens from MF patients and observed increased fibrocytes (CD45⁺ProCol-I⁺) in ASXL1^MT MF patients compared with ASXL1^WT MF patients (Figure 5A, B; Online Supplementary Table S4).

We next isolated BM nucleated cells from Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice and cultured them in conditions that promote the differentiation of monocytes to fibrocytes.^22,23 On day 5, the numbers of long spindle-shaped CD45⁺CollagenI (Col-I)⁺ fibrocytes derived from Asxl1^-/-Jak2^VF BM nucleated cells were higher than those derived from cells of other genotypes (Figure 5C, D). Genotyping detected the Jak2^V617F mutation and Asxl1 deletion in cultured fibrocytes, confirming that the fibrocytes were originated from malignant clones (Online Supplementary Figure S11A–B). We also measured the proportions of fibrocytes (CD45⁺Col-I⁺, CD11b⁺Col-I⁺ or CD68⁺Col-I⁺) using flow cytometry and observed that Asxl1^-/-Jak2^VF mice exhibited markedly increased fibrocytes in both BM and spleens compared with other genotypes (Figure 5E, F; Online Supplementary Figure S11C–D).

Overall, these results establish that increased neoplastic monocyte-derived fibrocytes may be associated with acceleration of BM fibrosis in ASXL1^MT MF patients and Asxl1^-/-Jak2^VF mice.

Asxl1 deletion results in derepression of polycomb group target genes in Asxl1-/-Jak2VF mice

ASXL1 deletion impairs hematopoiesis and accelerates myeloid malignancies via aberrant histone modifications and dysregulated transcription.³⁵ We thus performed bulk RNA sequencing, ATAC sequencing and ChIP sequencing on Asxl1^-/-Jak2^VF and Jak2^VF BM c-kit⁺ cells to elucidate the transcriptional and associated epigenetic alterations after Asxl1 deletion. The expression profiles of Asxl1^-/-Jak2^VF BM c-kit⁺ cells showed distinct clusters from Jak2^VF cells in principal component analysis (Figure 6A). As shown by the heatmap, 2,352 genes were significantly upregulated and 1,504 genes significantly downregulated in Asxl1^-/-Jak2^VF BM c-kit⁺ cells compared with Jak2^VF cells (fold change >2, P<0.05) (Figure 6B). Interestingly, gene set enrichment analysis showed that the upregulated genes in Asxl1^-/-Jak2^VF were significantly associated with bona fide PcG target genes, as identified by the overlap between H3K27me3 and H2AK119ub1 ChIP-sequencing experiments on Asxl1^-/-Jak2^VF and Jak2^VF BM c-kit⁺ cells (Figure 6C, D).

This is consistent with the genetic categorization of ASXL1 as a PcG gene.³⁶ Integrated analysis of RNA-sequencing and ATAC-sequencing data showed that there was a significant increase of chromatin accessibility associated with upregulated genes (Figure 6E) and these sites with gained accessibility were enriched with increased levels of H3K4me1 and H3K27ac, histone marks of active enhancers in Asxl1^-/-Jak2^VF BM c-kit⁺ cells (Figure 6E). Figure 6F shows the changes of representative PcG target genes Jun and Egr1. Taken together, these results demonstrate that Asxl1 deletion results in the derepression of PcG target genes by activating their enhancers in Asxl1^-/-Jak2^VF BM c-kit⁺ cells.

Activated EGR1-TNFA axis enhances monocyte/macrophage and fibrocyte differentiation in ASXL1MT myelofibrosis patients and Asxl1-/-Jak2VF mice

To explore the critical driving genes for disease pheno-types, we next performed bulk RNA sequencing on BM c-kit⁺ cells of all genotypes and finally focused on 45 genes that were upregulated in Asxl1^-/-Jak2^VF BM c-kit⁺ cells compared with those in the other three genotypes (Figure 7A). Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed the enrichment of several inflammation-related pathways including TNF, IL-17 and NF-KB pathways (Figure 7B). Notably, in line with the activated TNF pathway, TNF-a levels were elevated in serum of ASXL1^MT MF patients and Asxl1^-/-Jak2^VF mice (Online Supplementary Figure S5A, B). Several inflammation-related genes, such as Egr1, Fos, Cxcl2 and Ccl4, were also up-regulated in Asxl1^-/-Jak2 BM c-kit⁺ cells (Figure 7C), which was validated by real-time quantitative polymerase chain reaction analysis (Figure 7D). Among Tnfa- and inflammation-related genes, the PcG target gene Egr1, was of special interest to us and validated by western blot in BM c-kit⁺ cells (Online Supplementary Figure S12). Upregulated Egr1 can stimulate HSPC along the monocyte/macrophage lineage.³⁷ We further measured its expression in LSK cells, granulocyte/macrophage progenitors and monocytes, and detected comparable upregulation in different cell populations in Asxl1^-/-Jak2^VF and Jak2^VF mice (Online Supplementary Figure S13), which was reminiscent of monocyte/macrophage bias in Asxl1^-/-Jak2^VF mice and ASXL1^MT MF patients. We confirmed the upregulated EGR1 expression in BM mononuclear cells of ASXL1^MT MF patients compared with PV and ASXL1^WT MF patients (Figure 7E, Online Supplementary Table S5). Increased chromatin accessibility and enhancer activation were consistently observed at the Egr1 locus in Asxl1^-/-Jak2^VF BM c-kit⁺ cells (Figure 6F).

Figure 5.Increased monocyte-derived fibrocytes in both ASXL1^MT myelofibrosis patients and Asxl1^-/-Jak2^VF mice. (A) Representative immunofluorescence imaging of fibrocytes (ProCol-I⁺CD45⁺) in bone marrow (BM) specimens from patients with myelofibrosis (MF). Original magnification 60×; scale bar, 10 mm. (B) The number of fibrocytes in BM specimens of ASXL1^WT and ASXL1^MT MF patients (N=8 for ASXL1^WT patients and N=8 for ASXL1^MT patients, median= 20.5 cells/10 high power field [HPF] for ASXL1^WT patients and 74.0 cells/10 HPF for ASXL1^MT patients). (C) Representative immunofluorescence images of fibrocytes (ColI⁺CD45⁺) from BM nucleated cells of 14-week-old Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice cultured in conditions that promote differentiation to fibrocytes. Left: original magnification 20×; scale bar, 40 mm; right: original magnification 60×; scale bar, 10 mm. (D) The numbers of fibrocytes derived from Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT BM nucleated cells cultured for 5 days (N=3-4 per group). (E, F) The proportions of fibrocytes in BM (E) and spleens (F) of Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT mice at 14-16 weeks of age determined using flow cytometry (N=5-6 per group). In (B), the results are presented as median ± interquartile range. A Mann-Whitney U test was performed between medians of two groups. In (D-F), the results are presented as mean ± standard error of mean. A two-tailed unpaired Student t test was performed between means of two groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 6.Asxl1 deletion results in derepression of polycomb group target genes in Asxl1^-/-Jak2^VF bone marrow c-kit⁺ cells. (A) Principal component analysis plot showing the gene-expression profile of bone marrow (BM) c-kit⁺ cells from Asxl1^-/-Jak2^VF and Jak2^VF mice. Each dot represents an independent biological sample. (B) Heatmap showing significantly upregulated and downregulated genes in Asxl1^-/-Jak2^VF BM c-kit⁺ cells compared with Jak2^VF BM c-kit⁺ cells (fold change >2, P<0.05). (C) Gene set enrichment analysis (GSEA) showed that polycomb group (PcG) target genes were significantly depressed in Asxl1^-/-Jak2^VF BM c-kit⁺ cells when compared to Jak2^VF BM c-kit⁺ cells. PcG target genes were defined by the 4,700 regions co-occupied by H3K27me3 and H2AK119ub1 from ChIP-sequencing data of Asxl1^-/-Jak2^VF and Jak2^VF BM c-kit⁺ cells. (D) Representative enriched PcG target genes in Asxl1^-/-Jak2^VF BM c-kit⁺ cells in GSEA. (E) Metaplots and heatmaps of ATAC sequencing, and H3K4me1 and H3K27ac ChIP sequencing at upregulated genes in Asxl1^-/-Jak2^VF BM c-kit⁺ cells and Jak2^VF BM c-kit⁺ cells. (F) Snapshot of the genomic view for H3K4me1 and H3K27ac ChIP sequencing, ATAC sequencing and RNA sequencing at the representative PcG target gene Jun and Egr1 loci. ATAC: assay for transposase-accessible chromatin; Chip: chromatin immunoprecipitation.

Figure 7.Activated EGR1 enhances monocyte/macrophage differentiation in Asxl1^-/-Jak2^VF mice. (A-C) Venn diagram (A), KEGG pathway enrichment analysis (B), and heatmap (C) of upregulated genes in Asxl1^-/-Jak2^VF compared with Jak2^VF, Asxl1^-/- and WT BM c-kit⁺ cells (fold change >2, P<0.05). (D) Relative expressions of Fos, Ccl4, Egr1 and Cxcl2 mRNA were measured in Asxl1^-/-Jak2^VF, Jak2^VF, Asxl1^-/- and WT bone marrow (BM) c-kit⁺ cells by real-time quantitative polymerase chain reaction (RT-qPCR) and normalized with one sample of Jak2^VF mice (N=3-4 per group). (E) Relative expressions of EGR1 mRNA in BM mononuclear cells from polycythemia vera (PV), ASXL1^MT and ASXL1^WT myelofibrosis patients measured by RT-qPCR and normalized with one sample of PV patients (N=13 per group). (F) Representative flow cytometric plots (left) and the proportions (middle) of F4/80⁺ cells and photomicrographs of Wright-Giemsa-stained cytospin smears (right) obtained from colonies generated by Asxl1^-/-Jak2^VF BM c-kit⁺ cells transduced with either empty vector or Egr1 short hairpin RNA (N=3 independent experiments). In (D-F), the results are presented as mean ± standard error of mean. A two-tailed unpaired Student t test was performed between means of two groups. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. MF: myelofibrosis; SSC: side scatter; EV: empty vector; shRNA: short hairpin RNA.

We then assessed the causal effect of Egr1 on mono-cyte/macrophage differentiation. After being transduced with lentivirus-expressing control (empty vector) or specific short hairpin RNA (shRNA) against Egr1, Asxl1^-/-Jak2^VF BM c-kit⁺ cells were sorted for expression of green fluorescence protein (GFP) and seeded in methylcellulose supplemented with mouse IL-3 (10 ng/mL) in vitro. On day 8, we observed that Egr1 knockdown significantly reduced the percentage of macrophages (F4/80⁺) derived from Asxl1^-/-Jak2^VF BM c-kit⁺ cells (Figure 7F).

Apart from participating in hematopoietic differentiation, EGR1 also acts as a master transcription factor to activate TNFA expression. Luciferase activity assay and electrophoretic mobility shift assay have detected the EGR1 binding site on the TNFA promoter in human monocytic cells.³⁸ We next measured the expression of Tnfa mRNA in Asxl1^-/-Jak2^VF BM c-kit⁺ cells transduced with empty vector or shRNA against Egr1. As shown in Figure 8A, Tnfa expression failed to be upregulated after knockdown of Egr1, and TNF-a production was reduced after knockdown of Egr1 in BM c-kit⁺ cells as well (Online Supplementary Figure S14). In MPN, TNF-a facilitates the expansion of JAK2^V617F-positive clones,³⁹ and its activation was also recently identified as an early event in fibrosis-driving MSC.⁴⁰ We thus speculated that TNF-a might promote the differentiation of fibrocytes in Asxl1^-/-Jak2^VF mice. In vitro experiments showed that the numbers of cultured fibrocytes derived from BM nucleated cells treated with murine TNF-a (2 ng/mL) were significantly higher than those of cells treated with dimethyl sulfoxide, and the addition of the TNF-a receptor (TNFR) antagonist R-7050 (1 mM) eliminated this effect (Figure 8B). Moreover, R-7050 (1 mM) alone could also decrease the production of fibrocytes (Figure 8B). We also examined the effect of TNF-a on fibrocyte differentiation in the other three genotypes and observed that TNF-a promoted fibrocyte differentiation regardless of Asxl1 and Jak2 mutational status (Online Supplementary Figure S15A-C). To further validate the effect of Egr1 on fibrocyte production in Asxl1^-/-Jak2^VF mice, we also transfected Asxl1^-/-Jak2^VF BM nucleated cells with Egr1 shRNA or an empty vector and performed an in vitro fibrocyte differentiation assay. As shown in Online Supplementary Figure S16, the number of fibrocytes significantly reduced after Egr1 knockdown in Asxl1^-/-Jak2^VF mice, confirming the effect of Egr1 on fibrocyte production.

Figure 8.TNF-a promotes monocyte-derived fibrocyte differentiation in Asxl1^-/-Jak2^VF mice. (A) Relative expressions of Egr1 and Tnfa mRNA in Asxl1^-/-Jak2^VF bone marrow (BM) c-kit⁺ cells infected with either empty vector or Egr1 shRNA (3 independent experiments). (B) Representative immunofluorescence imaging (left) and numbers (right) of cultured fibrocytes derived from Asxl1^-/-Jak2^VF BM nucleated cells treated with dimethyl sulfoxide, mouse TNF-a (2 ng/mL), the TNF-a receptor antagonist, R-7050 (1 mM), and mouse TNF-a (2 ng/mL) combined with R-7050 (1 mM) (N=3 per group). Original magnification 20× for images; scale bar, 40 mm. The results are presented as mean ± standard error of mean. A two-tailed unpaired Student t test was performed between means of two groups. *P<0.05, ***P<0.001, ****P<0.0001. EV: empty vector; DMSO: dimethyl sulfoxide; TNF-a: tumor necrosis factor-alpha. shRNA: short hairpin RNA.

Previous studies found no significant effects of ruxolitinib on fibrocyte differentiation in samples from patients with PMF.²³ We thus wondered whether combined inhibition of the JAK/STAT pathway and the TNFR antagonist would suppress fibrocyte differentiation. Excitingly, combining ruxolitinib (100 nM) with R-7050 (1 mM) enhanced the inhibitory effects on fibrocytes compared to the effects of ruxolitinib monotherapy (Online Supplementary Figure S17A), and the efficacy was confirmed in BM mononuclear cells from MF patients (Online Supplementary Figure S17B). Notably, ruxolitinib (100 nM) alone significantly reduced the number of cultured fibrocytes derived from Asxl1^-/-Jak2^VF BM nucleated cells while it did not reduce fibrocyte differentiation of BM mononuclear cells from MF patients (Online Supplementary Figure S17A, B), which was consistent with a previous study of PMF patients’ samples.²³

Collectively, our data indicate that an activated EGR1-TNFA axis is involved in monocyte-derived fibrocyte differentiation in ASXL1^MT MF and shed light on an attractive combination therapy for anti-fibrosis treatment.

Discussion

Mutated ASXL1 is associated with severe MF-related features in MF patients. Whether ASXL1 mutations are gainof-function or loss-of-function remains a question in myeloid malignancies. Several studies have shown that gain-of-function of truncated ASXL1 mutations contributes to myeloid malignancies,^41,42 while neither full-length nor truncated ASXL1 protein was found in ASXL1-mutated human myeloid leukemia cell lines and clinical samples.¹⁸ In this study, we generated a different kind of mouse model for Asxl1 knockout and Jak2^V617F MPN, using hematopoietic cell-specific expression as opposed to a prior germline study.²⁰ Our phenotype findings are consistent with previous results in germline Asxl1^+/- and Jak2^V617F mouse models, suggesting the crucial role of ASXL1 mutations in MPN progression. Moreover, we further explored the putative mechanism of ASXL1 mutations in MPN progression.

An activated JAK/STAT pathway enhances inflammatory cytokine production and participates in malignant clonal expansion, BM fibrosis and osteosclerosis in MPN.⁴³ Monocytes are the principal source of inflammatory cytokines in MF patients.²⁷ Both ASXL1^MT MF patients and Asxl1^-/-Jak2^VF mice exhibit expansion of monocytes, especially inflammatory-related Ly6C⁺ monocytes. Ly6C⁺ monocytes further differentiate into M1 macrophages or monocyte-derived dendritic cells in response to inflammatory stimuli and these differentiated cells, in turn, secrete cytokines,²⁸ creating a positive feedback loop, which results in a vicious cycle of inflammatory cytokine production. Hence, these data suggest that skewed monocyte and macro-phage differentiation results in enhanced inflammation in ASXL1^MT MF.

MF was thought to be a reactive phenomenon caused by the interaction between malignant hematopoiesis and the BM microenvironment, mediated by profibrotic cytokines.^34,44 Some studies found that Gli1⁺ and LeptinR⁺ WT MSC were functionally reprogrammed and differentiated into myofibroblasts and contributed to MF.^32,33,40 In our cohort, no difference was found in MSC-derived myofibroblasts between ASXL1^MT and ASXL1^WT MF patients, suggesting that other fibrosis-driving cells may be the major contributors to the acceleration of fibrosis in ASXL1^MT MF. Fibrocytes are derived from monocytes and initially identified in tissue fibrosis diseases such as end-stage liver or kidney diseases.^45,46 Neoplastic fibrocytes were first found in PMF patients by Verstovsek et al.²³ and recently reported to be present in Jak2^V617F mouse models as well.²² Deletion or inhibition of neoplastic fibrocytes can ameliorate MPN phenotypes in MPN mouse models, suggesting their crucial role in fibrosis formation.^22,23 Using mouse models and patients’ samples, our results suggest that ASXL1 mutations accelerate BM fibrosis by reprograming the fibrosis-driving potential of hematopoietic cells to fibrocytes, and further confirm that neoplastic fibrocytes are the major contributors to BM fibrosis. The deregulated cells identified upon Asxl1 deletion and the derepression of PcG target genes support the concept that ASXL1 acts as a PcG gene. Mechanistically, we demonstrated that Asxl1 deletion results in increased chromatin accessibility and enhancer activation at derepressed genes. Nevertheless, it still remains elusive why ASXL1 biochemically antagonizes PRC1 catalytic activity while genetically acting as a transcription repressor. Two recent studies in embryonic stem cell models showed that Bap1 loss results in pervasive accumulation of H2AK119ub1 and PRC titration away from its target promoters.⁴⁷, ^{4 8} Future studies will be required to test these mechanisms in the Asxl1^-/- mouse model.

Notably, Asxl1 deletion activates the enhancer at the PcG target gene Egr1 locus and consequently upregulates Tnfa in Asxl1^-/-Jak2^VF BM c-kit⁺ cells. Activated Egr1 increases Asxl1^-/-Jak2^VF HSPC commitment to monocyte/macrophage lineage and stimulates TNF-a secretion. Interestingly, we detected elevated TNF-a levels uniquely in ASXL1^MT MF patients, indicating a relationship between this cytokine and disease phenotype caused by ASXL1 mutations. TNF-α is an essential cytokine in MPN and its absence attenuates disease phenotypes in Jak2^V617F mice through limiting the expansion of clones.^26,39 Our study indicates that TNF-α most likely enhances fibrosis by promoting differentiation of monocytes to fibrocytes, and this effect is not malignant-specific. Thus, an Egr1-mediated monocyte/macrophage differentiation bias and TNF-α secretion synergistically resulted in increased fibrocyte production and accelerated BM fibrosis in ASXL1^MT MF. Previous research and our data have confirmed that ruxolitinib has little effect on fibrocyte differentiation in MF patients’ samples in vitro.²³ We therefore combined ruxolitinib with a TNFR antagonist and found remarkably reduced fibrocyte differentiation in vitro. Future in vivo experiments with genetic models and patient-derived xenograft models are necessary to confirm the efficacy and safety of the combination further.

In conclusion, our study illustrates the crucial role of ASXL1 mutation in MPN phenotypes and the onset of BM fibrosis. ASXL1 mutations activate the EGR1-TNFA axis in MPN, leading to monocyte/macrophage-mediated inflammation and neoplastic fibrocyte-induced BM fibrosis. Ruxolitinib together with a TNFR antagonist may mitigate fibrocyte production, providing an attractive theoretical approach to anti-fibrosis treatment.

Footnotes

• Received November 7, 2021
• Accepted June 28, 2022

Correspondence

* ZXS, JQL and YYZ. contributed equally as co-first authors.

^#ZJX, XDW, BL and GH contributed equally as co-senior authors

Z. Xiao zjxiao@ihcams.ac.cn

X. Wu wuxudong@tmu.edu.cn

B. Li libing@ihcams.ac.cn

G. Huang gang.huang@cchmc.org

Disclosures

No conflicts of interest to disclose.

Contributions

ZJX, XDW, BL, GH and ZXS conceived the idea of this study; ZXS, JQL,YYZ, BL, XDW, HG and ZJX designed the research; ZXS, JQL, YYZ, LY, YNC, PHZ, WJZ, YRY and HJH performed research; JYW, XY, TJQ, ZFX, LJP and SQQ collected clinical data; ZXS, JQL, YYZ and YNC analyzed data; ZXS, JQL, YYZ, ZXL and YNC performed statistical and bioinformatic analyses; ZXS, YYZ, BL, XDW, GH and ZJX wrote the manuscript; and all authors reviewed and approved the manuscript. The authorship order among co-first authors was assigned according to working hours and contributions.

Data-sharing statement

The murine RNA-sequencing, ATAC-sequencing and ChIP-sequencing data reported in this paper are available at the NCBI’s Gene Expression Omnibus (GEO) under accession number: GSE181291.

Funding

This study was supported in part by National Natural Science funds (N. 81530008, 81870104 and 82170139 to ZJX, 82070134 and 81600098 to BL, 81770129 to GH, and 81772676 and 31970579 to XDW), the National Key Research and Development Program (N. 2017YFA0504102 to XDW), Tianjin Natural Science funds (N. 18JCZDJC34900 to ZJX, 18JCJQJC48200 to XDW, and 19JCQNJC09400 to BL), the PUMC Youth Fund and Fundamental Research Funds for Central Universities (N. 3332019093 to JQL), CAMS Initiative Fund for Medical Sciences (N. 2016-I2M-1-001 and 2020-I2M-C&T-A-020 to ZJX, and 2020-I2M-C&T-B-090 to ZFX), and the Haihe Laboratory of Cell Ecosystem Innovation Fund (N. HH22KYZX0033 to ZJX).

Acknowledgments

The authors would like to thank Bin Li (State Key Laboratory of Experimental Hematology) and Ningpu Ban (Pathology Center, Blood Diseases Hospital, CAMS) for assistance in preparation of murine pathology sections. We thank the State Key Laboratory of Experimental Hematology Flow Cytometry Center, Experimental Animal Center and Image Center for assistance with the experiments.