In this issue of Haematologica, Shi et al. report on a critical role of ASXL1 mutations in driving bone marrow fibrosis via a EGR1-TNFA axis in both murine models and patients with primary myelofibrosis.1
Additional sex combs like 1 (ASXL1) mutations are among the most common molecular biological abnormalities in patients with primary myelofibrosis, but the effect of these mutations on prognosis remains controversial. Recent studies demonstrated that ASXL1 mutations alone are not detrimental but confer a worse prognosis when associated with a mutation in TP53 or high-risk genes.2 In line with these findings, it was demonstrated that ASXL1 mutations are early driver events in primary myelofibrosis but might be acquired later in the disease course of secondary myelofibrosis.3 This raises the question of the effect of ASXL1 mutations on hematopoietic stem and progenitor cells. In their study, Shi et al. sought to shed light on the mechanism of aberrant lineage differentiation and transcription deregulation related to ASXL1 mutations in myeloproliferative neoplasms (MPN), using patients’ biopsies and the hematopoietic-specific Vav-Cre-driven murine model named Asxl1-/- Jak2VF.
In their article, Shi and colleagues1 once again confirm that ASXL1 mutations, regardless of the “MPN driver” mutation, are associated with a more severe disease phenotype (e.g., larger spleens, higher fibrosis grades, lower hemoglobin) and higher monocyte frequency but do not specifically differentiate between primary myelofibrosis and secondary myelofibrosis or additional mutations. The hematopoietic-specific Jak2VF murine model with deletion of Asxl1 represents a model for early acquisition of ASXL1 mutations comparable to ASXL1 being an early event in primary myelofibrosis.3 In line with their own and earlier clinical data, Shi et al. demonstrate that loss of Asxl1 triggers earlier onset of fibrosis and a generally more severe phenotype and also induces a differentiation bias towards the monocyte/macrophage lineage. Monocytosis in patients with primary myelofibrosis was previously associated with inferior survival4 and could be explained by a more severe inflammatory state. As ASXL1 mutations were associated with monocytosis in patients and the murine model, the authors explored the hypothesis of monocyte-derived fibrocytes contributing to more severe fibrosis. Fibrocytes are still only very broadly defined as spindle-shaped cells expressing markers of both hematopoietic cells (CD34, CD43, CD45, CD68, LSP-1, and major histocompatibility complex class II) and stromal cells (collagen I, collagen III, and fibronectin) and have been associated with primary myelofibrosis.5 Shi et al. show an association of an increased frequency of fibrocytes in patients carrying an ASXL1 mutation when compared to controls but functional evidence of active extracellular matrix production of these cells contributing to fibrosis still remains to be demonstrated. Surprisingly, the authors did not find a significant difference in Gli1+ and LepR+ staining in their relatively small cohort of patients (n=4 ASXL1mut vs. n=8 ASXL1WT) which were previously reported to expand as fibrosis-driving cells in response to a MPN clone.6,7 This might be due to the fact that both are known to be expressed at low levels and are difficult to detect by immunofluorescence without signal amplification. Another critical point is the preparation of tissue, specifically fixation and decalcification, which have significant impact on bone marrow staining. Recent work by van Egeren and colleagues8 just described a population of CD34– bone marrow monocytes using single-cell RNA sequencing and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells. It would now be interesting to explore if there is also an association with ASXL1 co-mutations.
Shi et al. sought to dissect transcriptional differences upon co-mutation/loss of Asxl1 in their murine model. Using bulk RNA sequencing of the heterogeneous population of cKit+ hematopoietic stem and progenitor cells, the authors show that inflammation-related pathways such as Nfkb, TNFα and IL-17, are upregulated in Asxl1-/- Jak2VF bone marrow ckit+ cells and confirmed higher serum levels of TNFβ in ASXL1 mutant patients and Asxl1-/- Jak2VF mice. Given the strong association they observed between the double mutants/co-mutations, it would have been of particular interest to determine the effect of the co-mutation on CD14+ monocytes, for example, and not only progenitor cells. Interestingly, Shi and colleagues observed and validated the upregulation of Egr1 in LSK, GMP and monocytes of Asxl1-/- Jak2VF mice. This is an interesting link to fibrosis as Egr1 expression was described in solid organ fibrosis to be induced by fibrogenic (pro-inflammatory) stimuli and to regulate the expression of extracellular matrix components, matrix remodeling enzymes and fibrogenic cytokines such as TGF-β, leading to myofibroblast differentiation. Shi et al. further leveraged RNA sequencing, assay for transposase-accessible chromatin (ATAC) sequencing and chromatin immunoprecipitation sequencing to investigate the transcriptional and epigenetic alterations in Asxl1-/- Jak2VF double mutants and highlight increased chromatin accessibility associated with increased levels of histone marks on enhancers, also specifically on the Egr1 locus.
This is a strong point towards a role of EGR1 in more advanced fibrosis.
Recent pivotal studies have transformed our understanding of mutation acquisition in MPN9,10 and the timing of acquisition of an ASXL1 mutation in MPN patients seems to be crucial for the phenotype. This raises the question of what role the timing of ASXL1 mutations in MPN has on disease and fibrosis initiation and progression, and if similar pathways and genes are activated. The “ASXL1-erating” effect on fibrosis kinetics in MPN was clearly demonstrated and it will be interesting to see in the future the functional effect of an EGR1/TNFα axis which could potentially act as a point of therapeutic intervention.
- Received August 17, 2022
- Accepted August 19, 2022
No conflicts of interest to disclose.
HG and RKS wrote and edited the manuscript.
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- Williams N, Lee J, Mitchell E. Life histories of myeloproliferative neoplasms inferred from phylogenies. Nature. 2022; 602(7895):162-168. https://doi.org/10.1038/s41586-021-04312-6PubMedGoogle Scholar
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