IL-1β promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells

Myeloproliferative neoplasms (MPNs) are clonal disorders of the hematopoietic stem cell (HSC) in most cases caused by activating mutations in JAK2, CALR, or MPL that increase the proliferation of erythroid, megakaryocytic, and myeloid lineages.^1-7 MPNs can be subclassified as polycythemia vera, essential thrombocythemia, and primary myelofibrosis.^8,9 JAK2-V617F is the most frequent driver gene mutation in patients with MPN, but JAK2-V617F can also be detected as clonal hematopoiesis of indeterminate potential (CHIP) in healthy individuals, with a frequency much higher than the incidence of MPN.^10-13 Thus, the acquisition of JAK2-V617F is not the rate-limiting step for developing MPN, and there are factors that can suppress or promote the expansion of the JAK2 mutated clone and thereby control the transition from CHIP to MPN.

Inflammation is a hallmark of advanced MPN.¹⁴ Chronic immune stimulation in patients with a history of infectious or autoimmune diseases was associated with increased risk of myeloid malignancies including MPN.^15-17 Therefore, we hypothesized that inflammation might be one of the factors promoting the transition from JAK2-V617F–positive CHIP to MPN. Interleukin-1β (IL-1β), a pleiotropic cytokine with diverse innate and adaptive immune functions, is a master regulator of inflammatory state^18,19 and has been implicated in promoting several hematological malignancies.^20,21 Previous studies have shown that IL-1β is favoring progression to myelofibrosis at advanced stages of MPN.^22,23 

Here, we examined the role of IL-1β in the very early stages of MPN initiation by JAK2-V617F using competitive bone marrow (BM) transplantations at high dilutions (1:100), with very few (1-3) long-term HSCs (LT-HSCs). The initiation of MPN from a single mutant HSCs is fundamentally different from the progression of chronic phase MPN to myelofibrosis, which is mediated by profibrotic cytokines such as transforming growth factor β and is linked to augmented megakaryopoiesis and the emergence of myofibroblasts and monocyte-derived profibrotic fibrocytes.

We found that IL-1β secreted by JAK2-mutant hematopoietic cells promotes the early expansion of the JAK2-V617F clone and requires the presence of the IL-1 receptor type 1 (IL-1R1) on the responding BM stromal cells. Genetic ablation of IL-1β or IL-1R1 and treatment with a neutralizing IL-1β antibody reduces MPN disease initiation.

We used conditional JAK2-V617F transgenic mice,^24,SclCre^ER mice,^25,UBC-GFP mice,^26,IL-1β–knockout mice,²⁷ and IL-1R1–knockout mice.²⁸ SclCre^ER;V617F (VF) mice were characterized previously.²⁹ Cre^ER was activated by intraperitoneal injections of 2 mg tamoxifen (Sigma Aldrich) for 5 consecutive days. All mice were of pure C57BL/6N background and kept under specific pathogen-free conditions with free access to food and water in accordance with Swiss federal regulations. All animal experiments were approved by the Cantonal Veterinary Office of Basel-Stadt, Switzerland.

Blood samples and clinical data of patients with MPN were collected at the University Hospital Basel, Switzerland. Blood samples from healthy controls were obtained from the local blood donation center (Stiftung Blutspendezentrum SRK beider Basel). The study was approved by the local ethics committee (Ethik Kommission Beider Basel). Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki.

BM from transgenic mice was harvested 6 to 8 weeks after tamoxifen induction, mixed with wild-type BM competitor cells, and injected into the tail vein of lethally irradiated recipient mice (12 Gy). Blood was drawn from the tail vein every 4 to 6 weeks. Green fluorescent protein (GFP)-chimerism was assessed by flow cytometry, and complete blood counts were performed on Advia120 Hematology Analyzer with Multispecies software version 5.9.0-MS (Bayer). Aspirin (Sigma) was added to drinking water (150 μg/mL) and bottles were changed every 3 to 4 days. Mouse immunoglobulin G2a (IgG2a) anti-mouse IL-1β antibody (01BSUR; Novartis Pharma AG, Basel, Switzerland),^30-32 or mouse IgG2a isotype were injected intraperitoneally once per week (10 mg/kg every week, intraperitoneal).

To test the hypothesis that IL-1β favors MPN disease initiation by promoting the early expansion of the JAK2-V617F clone, we used competitive BM transplantations at limiting dilutions that mimic the monoclonal origin of MPN.³³ As BM donors, we used triple-transgenic SclCre^ER;JAK2-V617F;UBC-GFP (VF;GFP) mice that allow tracking of the transplanted BM cells by detection of GFP. VF;GFP mice were crossed with IL-1β^−/− mice to obtain VF;IL-1β^−/−;GFP mice. Expression of the VF transgene was induced with tamoxifen. After 6 to 8 weeks, 2 × 10⁴ unfractionated BM cells containing only 1 to 3 JAK2-mutant LT-HSCs were mixed with a 100-fold excess of IL-1β^−/− BM competitor cells and transplanted into recipient mice (Figure 1A).³³ Within 36 weeks, ∼60% of these recipients developed MPN phenotype characterized by elevated hemoglobin and/or platelet counts and splenomegaly (Figure 1B). Mice with MPN phenotype also showed increased IL-1β levels in the plasma and BM, whereas mice without MPN phenotype displayed very low IL-1β levels (Figure 1C). Very similar results were obtained in experiments when WT BM competitor cells were used instead of IL-1β^−/− competitors (supplemental Figure 1), indicating that WT competitor cells did not contribute to the increased IL-1β production.

Transplantation of VF;IL-1β^−/−;GFP BM with IL-1β^−/− competitor cells at a 1:100 ratio produced considerably fewer recipients with MPN phenotype than mice that received VF;GFP BM, and only 1 mouse showed thrombocytosis (Figure 1B, lower panel). Recipients with MPN phenotype did not show increased IL-1β levels, suggesting that excess IL-1β was produced by the JAK2-V617F expressing cells (Figure 1C). Recipients transplanted with VF;GFP BM showed higher GFP chimerism (Figure 1D), higher percentage of engraftment (90% vs 66%) defined as GFP chimerism of >1% in peripheral blood Gr1⁺ granulocytes, and a higher percentage of MPN initiation (48% vs 12%) than mice transplanted with VF;IL-1β^−/−;GFP BM (Figure 1E). We have previously shown that VF and VF;IL-1β^−/− mice have comparable frequencies of HSCs in the BM.²² 

When only recipients with MPN phenotype were compared, blood counts and peripheral blood GFP chimerism were similar, except for slightly lower platelet count and GFP chimerism in platelets of VF;IL-1β^−/−;GFP recipients (Figure 1F). Furthermore, mice transplanted with VF;IL-1β^−/−;GFP BM that developed MPN phenotype showed lower GFP chimerism in hematopoietic stem and progenitor cells (HSPCs; Figure 1G), less reticulin fibrosis and osteosclerosis in the BM, and partial restoration of splenic architecture (Figure 1H). Inflammatory cytokines were largely unchanged, except for a trend toward lower IL-6 levels (Figure 1I). Loss of IL-1β in VF donor cells resulted in reduced levels of IL-1α in BM of mice that developed MPN (Figure 1J). Interestingly, loss of IL-1β in VF donor cells also increased the levels of IL-1R antagonist (IL-1Ra). As a result, the ratio of IL-1Ra to IL-1α increased in VF;IL-1β^−/− recipients with MPN phenotype, correlating with the less severe MPN phenotype and less myelofibrosis than recipients of VF BM.

Finally, BM transplantations into IL-1β^−/− recipients using IL-1β^−/− competitor cells demonstrated that VF BM cells alone were the source for the increased IL-1β levels in the plasma and BM (supplemental Figure 2A, upper panel). Unexpectedly, IL-1β^−/− recipients transplanted with VF;IL-1β^−/− BM showed higher frequencies of engraftment and MPN disease initiation than expected and almost equal to IL-1β^−/− recipients transplanted with VF BM (supplemental Figure 2A, middle panel). GFP chimerism was reduced when all recipient mice were considered (supplemental Figure 2A, lower panel), but when only mice that developed MPN were compared, very few alterations were noted (supplemental Figure 2B). At 36 weeks, IL-1β^−/− recipients transplanted with VF;IL-1β^−/− BM showed a trend toward reduced reticulin fibrosis, reduced GFP chimerism in HSPCs, and partial restoration of splenic architecture (supplemental Figure 2C-D), and a trend toward reduction of IL-6 and KC/GRO levels (supplemental Figure 2E). The reason why engraftment and MPN disease initiation increased when IL-1β was absent in both donors and recipients is currently unknown. No increase in IL-1Ra or in the ratio between IL-1α and IL-1Ra was found in IL-1β^−/− recipients transplanted with VF;IL-1β^−/− BM (supplemental Figure 2F), suggesting that lack of upregulation of IL-1RA might contribute to the more efficient MPN disease initiation. Complete loss of IL-1β partially prevented this outcome, possibly because of diminished anti–IL-1 inflammatory responses in the BM.

Overall, our data show that loss of IL-1β in hematopoietic cells reduced MPN initiation, suggesting that IL-1β produced by the JAK2-mutant cells is promoting early expansion of the JAK2-V617F clone and conversion to MPN.

To examine the effects of IL-1β loss on mutant HSC function, we performed secondary transplantations into WT recipients (Figure 2). As donors, we selected 3 recipients of VF;GFP BM and 3 recipients of VF;IL-1β^−/−;GFP BM that displayed MPN phenotype (supplemental Figure 3). BM cells were pooled and used for noncompetitive (Figure 2A-C) and competitive secondary transplantations (Figure 2D-F). Secondary recipients of VF;IL-1β^−/−;GFP BM showed significantly reduced blood counts and lower GFP chimerism in the peripheral blood, BM, and spleen than recipients of VF;GFP BM (Figure 2B-C,E-F; supplemental Figure 4A-B). By comparing the frequencies of engraftment (defined as >1% in Gr1⁺ granulocytes at 18 weeks) between the competitive and noncompetitive secondary transplantations, and using the "extreme limiting dilution analysis" algorithm,³⁴ we estimated that the numbers of functional HSCs were approximately fivefold reduced in donor mice that initially received VF;IL-1β^−/− BM compared with in donors initially transplanted with VF BM (Figure 2G). Overall, the HSC pool in primary recipient mice transplanted with 1 to 3 HSCs from VF donors expanded to a total of ∼300 functional HSCs after 36 weeks, whereas primary recipients transplanted with 1 to 3 HSCs from VF;IL-1β^−/− donors had only ∼50 functional HSCs. These results indicate that JAK2-V617F mutant HSCs require IL-1β for optimal expansion of the HSC population with long-term repopulation capacity.

Next, we examined the role of IL-1R1 in MPN disease initiation. VF;GFP mice were crossed with IL-1R1^−/− mice to obtain VF;IL-1R1^−/−;GFP mice that were used as donors for competitive BM transplantations into WT recipients (Figure 3A-E), or IL-1R1^−/− recipients (Figure 3F-J). GFP-chimerism in peripheral blood was higher in mice transplanted with VF;GFP BM than in recipients of VF;IL-1R1^−/−;GFP BM, when all mice that showed engraftment were considered (Figure 3D). Transplantations into WT recipients resulted in high frequency of engraftment, but fewer recipients transplanted with VF;IL-1R1^−/−;GFP BM developed MPN phenotype (27%) than recipients of VF;GFP BM (55%) (Figure 3E). GFP chimerism was similar when only mice with MPN phenotype were compared (supplemental Figure 5A), but loss of IL-1R1 from JAK2-mutant cells resulted in reduced GFP chimerism in HSPCs in the BM and spleen (supplemental Figure 5B). Recipients of VF;IL-1R1^−/−;GFP BM displayed low IL-1β levels in the plasma and BM, and no differences were noted between mice with or without MPN phenotype (Figure 3C). Nevertheless, IL-1β levels were higher in WT recipients transplanted with VF;IL-1R1^−/− BM than in those transplanted with VF;IL-1β^−/− BM (supplemental Figure 5C). Although VF;IL-1β^−/− cells are unable to produce IL-1β, cells from VF;IL-1R1^−/− mice can produce IL-1β, although at lower levels, because of loss of the positive IL-1β-feedback mediated by the IL-1R1.^18,19,35,36 Loss of IL-1R1 in JAK2-mutant cells also reduced reticulin fibrosis and osteosclerosis in the BM and partially corrected the splenic architecture in mice that developed MPN phenotype (supplemental Figure 5D). Cytokine levels did not change, except for decreased IL-1β in plasma (supplemental Figure 5E).

Transplantations into recipients deficient for IL-1R1^−/− also resulted in high frequency of engraftment (Figure 3F-J), but MPN disease initiation in IL-1R1^−/− deficient recipients transplanted with VF;GFP BM was reduced to 29% (Figure 3J), compared with 55% in WT recipients (Figure 3E). In mice that developed MPN phenotype, blood counts did not change, but GFP chimerism in the peripheral blood was slightly reduced with complete loss of IL-1R1 (supplemental Figure 6A), whereas GFP chimerism remained unchanged in HSPCs (supplemental Figure 6B). Compared with WT recipients, IL-1R1^−/− recipients transplanted with VF;GFP showed reduced grade of reticulin fibrosis in the BM, normalization of splenic architecture, (supplemental Figure 6C), and reduced levels of inflammatory cytokines (supplemental Figure 6D). However, loss of IL-1R1 in both donor and recipient did not affect reticulin fibrosis or the levels of inflammatory cytokines (supplemental Figure 6C-D).

Taken together, these results show that increased expression of IL-1β by the JAK2-mutant hematopoietic cells and the presence of IL-1R1 on both mutant hematopoietic cells and WT nonhematopoietic cells are required for efficient MPN disease initiation and progression.

Because reducing proinflammatory signaling by IL-1β knockout in VF donor cells or deletion of IL-1R1 in the recipient cells both reduced MPN disease initiation in competitive transplantations at high dilution, we examined whether the same effect can be observed with pharmacologic inhibition of inflammation. We first tested the effects of aspirin, which was added to the drinking water on day 1 after transplantation (supplemental Figure 7). We observed a slight reduction in the frequencies of engraftment (96% in the controls vs 70% in the aspirin-treated mice), but there were no differences in the blood counts or frequencies of MPN disease initiation (supplemental Figure 7).

Next, we examined the effects of a neutralizing anti–IL-1β antibody in mice transplanted with BM from a VF;GFP donor (Figure 4A). Treatment with anti–IL-1β antibody or isotype started on day 1 after transplantation and was continued for 18 weeks. Anti–IL-1β antibody reduced the percentage of mice with engraftment, defined as >1% GFP at 18 weeks and fewer mice developed MPN phenotype than mice treated with isotype control (Figure 4B). GFP chimerism was already higher at 6 weeks and further increased in VF mice that developed MPN phenotype under isotype treatment than in VF mice that engrafted but failed to develop MPN (Figure 4C). In contrast, no differences in GFP chimerism were observed after 6 weeks under anti–IL-1β antibody treatment, but GFP chimerism decreased between week 6 and 18 under anti–IL-1β antibody treatment in VF mice that engrafted but failed to develop MPN (Figure 4D). HSPCs from mice with MPN phenotype displayed high GFP chimerism that did not change by anti–IL-1β antibody treatment, whereas HSPCs from mice with engraftment >1% but without MPN phenotype, resembling a state of CHIP, showed low GFP chimerism and, in some cases, a decrease in chimerism was noted upon anti–IL-1β treatment (Figure 4E). Control mice transplanted with BM from WT:GFP donor showed no differences in phenotype or percentages of engraftment between the anti–IL-1β antibody and isotype treatment groups (supplemental Figure 8). Interestingly, anti–IL-1β antibody treatment did not decrease the GFP chimerism in HSPCs from the BM or spleen compared with isotype control (supplemental Figure 8D).

We confirmed that in our transplantations with VF;GFP BM at 1:100, mice that developed MPN phenotype displayed strongly elevated IL-1β levels in the BM and plasma compared with in mice transplanted with WT;GFP BM, whereas VF;GFP transplanted mice without MPN phenotype had marginally elevated IL-1β levels only in the BM (Figure 5A). Anti–IL-1β antibody treatment massively reduced IL-1β levels in all treated mice. Primitive BM mesenchymal stromal cells (MSCs), sympathetic nerve fibers, and Schwann cells were reduced at 18 weeks in the BM from isotype treated VF;GFP mice compared with WT;GFP mice. Interestingly, this decrease was also observed in VF;GFP transplanted mice without MPN phenotype that display low GFP chimerism and resemble a state of CHIP (Figure 5C-E). Thus, the damage to the niche cells does not appear to require strongly elevated IL-1β levels that are found in mice that developed MPN phenotype. Anti–IL-1β antibody largely normalized the frequencies of primitive MSCs and the densities of sympathetic nerve fibers and Schwann cells in all treated mice (Figure 5C-E).

To analyze the distribution of IL-1β protein in BM sections, we used in situ BM 3D fluorescence imaging with proximity ligation assay (PLA) that can visualize single IL-1β molecules.^37-39 WT mice showed low densities of IL-1β PLA signals, whereas VF mice that developed MPN phenotype displayed the highest densities of PLA signals (Figure 6; supplemental Figure 9A). IL-1β PLA signals were enriched in close proximity with CD11b⁺ myeloid cells in mice of all genotypes, and these gradients were reduced in VF mice that were treated with anti–IL-1β antibody.

In VF mice, the IL-1β PLA signals were also enriched in the vicinity of CD41⁺ megakaryocytes and these signals were strongly reduced in mice treated with anti–IL-1β antibody (Figure 6). These results show that JAK2-V617F upregulates IL-1β expression not only in myeloid cells but also in megakaryocytes and suggest that the treatment with anti–IL-1β antibody interrupts the feedback activation of IL-1β. Consistent with the IL-1β PLA data, expression of IL-1β messenger RNA was also increased in sorted monocytic and megakaryocytic cells from the BM of VF mice compared with that from WT mice (supplemental Figure 9B-C).

To investigate the cell autonomous mechanisms of IL-1β in JAK2-mutant HSCs and MPN disease initiation, we performed messenger RNA sequencing of JAK2-mutant LT-HSCs (Lin⁻Sca1⁺cKit⁺CD48^–CD150⁺). BM cells from VF, VF;IL-1β^−/−, VF;IL-1R1^−/−, or WT donors that have not been induced with tamoxifen before euthanasia were transplanted into lethally irradiated recipient mice (Figure 7A). These recipient mice received tamoxifen injections starting 16 weeks after transplantation and were euthanized 2 or 4 weeks after tamoxifen induction when they already displayed mild MPN phenotype (Figure 7B).

LT-HSCs from mice transplanted with BM from VF;IL-1R1^−/− mice displayed significant increase in the expression of JAK2-V617F compared with recipients transplanted with BM from VF mice, whereas no differences were observed between VF and VF;IL-1β^−/− mice (Figure 7C). To identify significantly enriched biological pathways in the RNA sequencing data sets, we used the WebGestalt online tool,⁴⁰ and used the weighted set cover method of WebGestalt to identify the least redundant and most representative gene sets from the data set (Figure 7D; supplemental Figure 10).

At 2 weeks after tamoxifen induction, we observed no significant enrichment (false discovery rate < 0.05) of any biological pathways (supplemental Figure 10). At 4 weeks after tamoxifen, we identified several significantly enriched Gene Ontology pathways (false discovery rate < 0.05) between VF vs WT, VF vs VF;IL-1β^−/−, and VF vs VF;IL-1R1^−/−genotypes (Figure 7D). Interestingly, in all 3 comparisons, LT-HSCs from VF mice displayed significantly higher response to interferon signaling than LT-HSCs from WT, VF;IL-1β^−/−, or VF;IL-1R1^−/− mice. Furthermore, heat map analysis showed increased expression of several genes including IL-1β, Tnf, Cxcl4, Cxcl3, Cxcl10, Ccl3, and Ccl5 from the top 3 Gene Ontology pathways (response to interferon-β, response to interferon-γ, and cytokine-mediated signaling pathway) that are commonly upregulated in VF LT-HSCs compared with LT-HSCs from the other 3 genotypes (Figure 7E). Similarly, the loss of IL-1β or IL-1R1 in JAK2-V617F-expressing LT-HSCs resulted in a reduction In the expression of the same genes (Figure 7E). Overall, our results suggest that loss of IL-1β or IL-1R1 in LT-HSCs expressing JAK2-V617F reverse the inflammatory signature to make them in this respect more WT-like.

Because IL-1β serum levels were increased in patients with MPN and correlated with the JAK2-V617F VAF,²² we examined whether genetic polymorphisms located in the IL1B gene correlated with higher IL-1β serum levels in patients with MPN (supplemental Figure 11). Several functionally relevant polymorphisms in the IL1B gene have been reported.^41,42 We selected 2 IL1B gene polymorphisms that were associated with increased production of IL-1β in nonhematological cancers,^43-45 to compare their frequencies in patients with MPN and normal controls (supplemental Figure 11A). We found that the frequencies of the homozygous AA genotype of rs16944 (G511>A) located in the promoter region of IL1B and of the homozygous GG genotype of rs1143627 (A31>G) located closer to the IL1B coding regions were almost threefold higher in patients with JAK2-V617F–positive MPN than in normal controls. Furthermore, patients with MPN with AA and GG genotypes displayed higher IL-1β serum levels than patients with MPN with GG/GA or AA/AG genotypes (supplemental Figure 11B). Although validation in a larger cohort of patients with MPN is needed, our data support the model that presence of IL-1β favors the expansion of the JAK2-mutant clone.

In this study, we investigated whether inflammation mediated by IL-1β promotes the early expansion of the JAK2-V617F mutant clone and can thereby favor the conversion from a CHIP-like state to MPN. This process is fundamentally different from the progression from chronic phase MPN to myelofibrosis, which we studied previously.²² Competitive BM transplantations at high dilutions allowed us to examine oligoclonal or monoclonal MPN disease initiation starting from 1 to 3 LT-HSCs per transplanted mouse.³³ Genetic ablation of IL-1β from JAK2-mutant donor HSCs significantly reduced the frequency of engraftment and lowered the frequency of conversion from CHIP to MPN among mice that engrafted (Figure 1), and these HSCs were also less functional in secondary transplantations (Figure 2), consistent with a role of IL-1β for optimal HSC expansion and long-term repopulation capacity. Although the complete genetic loss of IL-1β in donor and recipients eliminated the differences in engraftment and MPN disease initiation compared to VF (supplemental Figure 2), treatment with anti–IL-1β antibody decreased engraftment and MPN disease initiation (Figure 4). Our data establish IL-1β as an important trigger for the conversion from a CHIP-like state to MPN in carriers of JAK2-V617F.

The IL-1R1–knockout data are in-line with the IL-1β–knockout results. Because the IL-1R1 is required for the positive autocrine feedback loop that augments IL-1β production,^18,19,35,36 loss of IL-1R1 in VF;IL-1R1^−/− hematopoietic cells prevented the massive overproduction of IL-1β observed in VF hematopoietic cells. IL-1β produced by the VF;IL-1R1^−/− hematopoietic cells was still adequate to assure a high percentage of engraftment, but the levels were not sufficient to reach the same high frequency of conversion from CHIP to MPN observed with VF hematopoietic cells (Figure 3A). Loss of IL-1R1 in the recipients did not reduce the frequency of engraftment but lowered the conversion to MPN (Figure 3B). Thus, IL-1β promotes engraftment in a cell autonomous fashion, also in secondary transplantations, and is not entirely dependent on the presence of the IL-1R1, whereas the conversion to MPN required the presence of the IL-1R1 in the BM niche. Consistent with the genetic deletion of IL-1β, treatment with anti–IL-1β antibody reduced both the frequency of engraftment and the conversion to MPN (Figure 4B).

We have previously shown that in the VF mouse model, IL-1β mediates loss of sympathetic nerve fibers and the Schwann cells that surround them, resulting in a reduction of nestin-positive BM MSCs,²¹ and these changes were also associated with progression to myelofibrosis.²² The reduction of BM MSCs was also observed in isotype-treated mice transplanted with VF;GFP BM that did not develop MPN phenotype and displayed low GFP chimerism resembling a state of CHIP (Figure 5C-E). These results suggest that reducing the frequency of MSCs alone is not sufficient to assure conversion to MPN. Conversely, preserving high frequencies of MSCs by anti–IL-1β antibody treatment did reduce both the rate of engraftment and conversion to MPN. The mechanism of why the presence of MSCs interferes with the expansion of the JAK2-V617F clone is currently unknown and will be the subject of future studies.

Recent studies have reported associations between inflammatory cytokines and the expansion of CHIP clones carrying mutations other than JAK2-V617F. Tumor necrosis factor α promoted the expansion of hematopoietic cells with mutations in Dnmt3a or Asxl1,^46,47 and IL-1α and IL-1β mediated expansion of hematopoietic clones carrying Tet2 mutations.⁴⁸ For example, inhibiting inflammation, by aspirin, could decrease the probability of MPN disease initiation from JAK2-mutant HSCs. Although aspirin monotherapy slightly reduced the frequency of engraftment of VF BM, it did not decrease the frequency of MPN initiation (supplemental Figure 7). However, aspirin in combination with anti-PD1 showed therapeutic benefit in a Braf-V600E melanoma model.⁴⁹ JAK2-V617F clones induce strong IL-1–mediated responses, as seen by the relatively high levels of IL-1β and IL-6 in plasma as compared with other cytokines such as interferon-γ or KC/GRO (Figure 1E). Genetic deletion of IL-1β or IL-1R1 in LT-HSCs expressing JAK2-V617F made them more WT-like in respect to VF-induced responsiveness to interferon and cytokine signaling (Figure 7D-E). The anti–IL-1β antibody canakinumab is currently being tested in a clinical trial for clonal cytopenias of unknown significance.⁵⁰ 

Our results provide evidence that IL-1β production by JAK2-mutant hematopoietic cells generates an inflammatory environment in the BM that promotes JAK2-V617F clonal expansion and conversion to MPN. Inhibition of IL-1β by anti–IL-1β antibody may be beneficial in individuals with JAK2-V617F CHIP who are at increased risk of progression to MPN.

The authors thank Marc Donath for helpful discussions and members of our laboratory for critical reading of the manuscript. The authors thank Christian Beisel, Elodie Burcklen, and Philippe Demougin of the Genomics core facility for their support. They also thank the Bioinformatics core facility, Animal core facility, and Flow cytometry core facility of the Department of Biomedicine for excellent technical support. Bioinformatic calculations were performed at sciCORE (http://scicore.unibas.ch/) scientific computing center at the University of Basel.

This work was supported by grants from the Swiss National Science Foundation (310030_185297 /1, and 310030_185297 /2) and Swiss Cancer Research (KFS-4462-02-2018); the Stiftung für Hämatologische Forschung (R.C.S.); core support grants from Medical Research Council (MRC) to the Cambridge Stem Cell Institute, National Health Service Blood and Transplant (United Kingdom); European Union’s Horizon 2020 research (ERC-2014-CoG-648765); MRC-AMED grant MR/V005421/1; and a Programme Foundation Award (C61367/A26670) from Cancer Research UK (S.M.-F.).

Contribution: S.R. designed and performed the research, analyzed data, and wrote the manuscript; Y. Zhang and E.G. performed research and analyzed data; N.H., Q.K., C.B.S., M.U., H.H.-S., and Y. Zhu performed research; D.L.P. and J.R. analyzed sequencing data; M.S.B. analyzed clinical data; S.D. performed and analyzed histopathology of mouse tissues; C.J.F., T.S., and S.M.-F. designed research and analyzed data; R.C.S. designed the research, analyzed the data, and wrote the manuscript.

Conflict-of-interest disclosure: R.C.S. is a scientific adviser for, scientific advisory board member of, and has equity in Ajax Therapeutics; and has consulted for, and/or received honoraria from, Novartis, Bristol Myers Squibb/Celgene, AOP, GlaxoSmithKline, Baxalta, and Pfizer. N.H. owns stocks in Cantargia. C.J.F. is a full-time employee of Novartis Pharma AG. The anti–IL-1β antibody studies were carried out in the laboratory of R.C.S. with the antibody provided by Novartis. The remaining authors declare no competing financial interests.

Correspondence: Radek C. Skoda, Department of Biomedicine, University of Basel and University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland;.