Loss of the ten-eleven translocation methylcytosine dioxygenase 2 (Tet2) gene, which is commonly mutated in hematological malignancies, dysregulates inflammatory pathways, including the interleukin-1 (IL-1) pathway [1,2,3]. Roles for IL-1 signaling have been reported in terminally differentiated hematopoietic cells and in non-cell autonomous contexts [3, 4]. However, our group demonstrated that inhibition of inflammatory pathways can suppress clonal hematopoiesis (CH), indicating potential direct roles for hematopoietic stem and progenitor cells (HSPCs) in inflammation . As TET2 mutations are often present in HSPCs and provide these cells with a competitive advantage, dysregulation of the IL-1 pathway in HSPCs may contribute to leukemogenesis and may catalyze the progression of preleukemic states to malignancy .
Mutations in the TET2 gene are detected in a variety of myeloid malignancies, including acute myeloid leukemia (AML) . Similarly, Tet2−/− transgenic mice and recipient mice transplanted with Tet2−/− bone marrow (BM) exhibit splenomegaly, monocytosis, extramedullary hematopoiesis, and expansion of the Lin-;Sca1+;c-Kit+ (LSK) population . Acute and chronic IL-1 exposure expands myeloid cells at the expense of lymphoid cells; however, chronic exposure ultimately depletes the ability of hematopoietic stem cells (HSCs) to self-renew . While previous studies have investigated the exogenous effects of IL-1α and IL-1β on hematopoiesis and on mature hematopoietic cells, IL-1R1, the primary of ten IL-1 receptors, binds to multiple proteins, including IL-1α, IL-1β, IL-1 receptor antagonist, IL-38, and its co-receptor IL-1 receptor accessory protein, underscoring that the full spectrum of the consequences of IL-1R1-dependent signaling in HSPCs is not yet known .
Based on these findings, we hypothesized that loss of the Il-1r1 gene would rescue the hematological abnormalities associated with Tet2 deficiency at the HSPC level. Both Il-1r1 and Tet2 are expressed in multiple hematopoietic cell types, including high expression in HSPCs (Supplementary Fig. 3A, B) . To determine whether loss of Il-1r1 can ameliorate malignancy, we generated Tet2−/−;Il-1r1−/− mice and analyzed peripheral blood (PB) counts in a large cohort. The frequencies of myeloid cells were elevated in Tet2−/− mice; however, these cell types were restored to wild-type (WT) levels in Tet2−/−;Il-1r1−/− mice (Fig. 1A). In addition to an increase in myeloid cells, lymphocyte frequency was reduced in Tet2−/− mice, demonstrating a myeloid shift at the expense of lymphocytes (Fig. 1A). Higher red cell distribution width (RDW-CV) was recently reported as a measure of pro-inflammatory states and correlated with an increased risk of AML in humans . Consistent with a pro-inflammatory state due to Tet2 loss, RDW-CV was increased in Tet2−/− mice and was relieved by inactivation of Il-1r1 (Fig. 1B). In representative mice from this larger cohort, Tet2−/− mice had larger spleen sizes and weights, which were corrected by loss of Il-1r1 (Fig. 1C and Supplementary Fig. 1A). These mice showed similar alleviations of elevated myeloid frequencies and suppressed lymphocyte frequencies, supporting a role for Il-1r1 at the stem-cell level (Supplementary Fig. 1B–D). To examine this possibility, the levels of HSPCs were measured by flow cytometry. Elevated levels of LSKs, long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitor (MPP) pools 2, 3/4, 3, and 4, and common myeloid progenitors (CMPs) were detected in Tet2−/− mice, and these increases were rescued in Tet2−/−;Il-1r1−/− mice, suggesting that loss of Il-1r1 rescues the expansion of HSPCs associated with Tet2 deficiency (Fig. 1D; Supplementary Fig. 1E–K). Comparable to other progenitor populations, Lin-;Sca1+ cells, which represent a subset of lymphoid progenitors that differ from common lymphoid progenitors (CLPs), were also increased in Tet2−/− mice (Fig. 1D) . However, Lin-;CD127+ progenitors within this Lin-;Sca1+ population were suppressed, indicating the presence of a block in lymphopoiesis at this stage (Fig. 1D). The increases in LSKs, LT-HSCs, ST-HSCs, and MPPs may represent a compensatory response to this blockage. Il-1r1 inactivation relieved inhibition of Lin-;CD127+ cells and normalized the levels of mature lymphoid cell types (Fig. 1D). These findings show that Il-1r1 loss can rescue Tet2-associated HSPC abnormalities. Together, they support roles for IL-1R-dependent signaling at the level of HSPCs, in the correction of myeloid disease, in the modulation of the pro-inflammatory state associated with Tet2 deficiency, and in the balance of myeloid and lymphoid cell types.
To investigate whether Il-1r1 deficiency rescues Tet2-associated hematological malignancies in a cell autonomous manner, we performed a competitive transplantation of BM containing HSPCs from C57 (CD45.2), Boy/J (CD45.1), Tet2−/− (CD45.2), Il-1r1−/− (CD45.2), and Tet2−/−;Il-1r1−/− (CD45.2) donor mice into CD45.1- and CD45.2-expressing F1 recipient mice and evaluated the effects of Il-1r1 loss on engraftment and on Tet2−/− HSPCs and mature hematopoietic cell types (Supplementary Fig. 2A). Inactivation of Il-1r1 reduced the increased engraftment of CD45.2-expressing cells and the high numbers and frequencies of white blood cells (WBC) and myeloid cells detected in mice transplanted with Tet2−/− BM (Fig. 2A; Supplementary Figs. 2B–G and 4A, B). Similar corrections of myeloid cells were observed in PB smears and Gr-1+ myeloid cells (Fig. 2A, B). As in the transgenic mice, loss of Il-1r1 corrected spleen sizes and weights (Fig. 2C; Supplementary Fig. 2H). Consistent with relief of lymphocyte suppression, an increased lymphocyte frequency and elevated percentages of CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and plasmacytoid dendritic cells (pDCs), all cells of lymphoid origin, were detected in mice transplanted with Tet2−/−;Il-1r1−/− BM, demonstrating that loss of Il-1r1 at the HSPC level can restore the levels of multiple lymphoid cell types (Supplementary Fig. 4C–N). Similar to the transgenic mice, increased levels of LSK, MPP2, and MPP3/4 cells associated with Tet2 deficiency were rescued in mice transplanted with Tet2−/−;Il-1r1−/− BM, corroborating a role for Il-1r1 in the regulation of cell populations that contain leukemia-initiating cells (Supplementary Fig. 3C–E). In addition, in mice transplanted with Tet2−/− BM, Lin-;c-Kit+ cells were reduced, while Lin-;Sca1+ cells were significantly elevated (Supplementary Fig. 3F, G). These changes were reversed in mice transplanted with Tet2−/−;Il-1r1−/− BM, further supporting profound shifts in myeloid and lymphoid populations (Supplementary Fig. 3F, G). Collectively, these findings suggest that Il-1r1 loss abrogates hematological malignancy and corrects disruption of the myeloid-lymphoid balance via cell autonomous mechanisms in HSPCs.
To investigate whether inactivation of IL-1 signaling in BM cells alleviates systemic inflammation associated with Tet2 deficiency, serum cytokine levels were measured. Consistent with previous studies, loss of Tet2 led to increases in multiple cytokines and chemokines, including tumor necrosis factor α (TNFα) and the interferon-γ (IFN-γ)-inducible genes IFN-γ-induced protein 10 (IP-10/CXCL10) and monokine induced by IFN-γ (MIG/CXCL9) (Supplementary Fig. 5A–C) [1, 2]. These cytokines and chemokines were restored to WT levels in mice transplanted with Tet2−/−;Il-1r1−/− BM (Supplementary Fig. 5A–C). TNFα promotes the expansion of Tet2−/− cells in vitro, indicating a non-cell autonomous role . However, we demonstrated that TNFα levels were also elevated in mice transplanted with Tet2−/− BM and that this increase was rescued by Il-1r1 loss in a cell autonomous manner. TNFα and IFNγ can control the levels of Lin-;Sca1+ cells and Sca1 expression and can promote myeloid expansion and regeneration, providing opportunities for antagonistic regulation of lymphoid and myeloid populations [13,14,15]. Together, these results support roles for IL-1 signaling in HSPCs in modulating the myeloid-lymphoid balance and in determining the pro-inflammatory status of Tet2−/− mature hematopoietic cells. Based on these findings, we propose a mechanism by which loss of Tet2 leads to a pro-inflammatory state that is characterized by high levels of TNFα and IFN-γ and that causes a myeloid bias at the expense of lymphoid cells (Supplementary Fig. 2I). This shift was evidenced by the elevation of CMPs and the suppression of Lin-;CD127+ lymphoid progenitors in Tet2-deficient contexts. The loss of IL-1R1-dependent signaling rescued these disruptions in normal hematopoiesis, abrogating myeloid disease and bolstering its potential as a therapeutic target (Supplementary Fig. 2J).
To determine the clinical relevance of IL-1R1 expression in patients with myeloid malignancies, we examined two publically-available datasets for IL-1R1 expression levels and correlated these levels with survival. Both pediatric and adult AML patients with higher levels of IL-1R1 expression exhibited decreased survival, suggesting a role for IL-1R1 in AML pathogenesis (Supplementary Fig. 6A, B). To evaluate whether the effects of IL-1R1 on survival are specific to distinct AML subtypes, survival was analyzed in the context of high and low IL-1R1 expression in ten adult AML subtypes. High expression of IL-1R1 conferred reduced survival in subtypes containing mutations in the CBFB-MYH11, NPM1, or p53C genes (Supplementary Fig. 7A–K). IL-1 signaling has been implicated in the expansion of CD34+ human AML cells, further supporting its clinical relevance . These results underscore the potential therapeutic implications of IL-1R-dependent signaling in myeloid malignancies and suggest that patient stratification may be needed.
In summary, we have shown that loss of Il-1r1 in Tet2−/− HSPCs rescued several abnormalities associated with Tet2 deficiency, including the elevation of LSK cells, the pro-inflammatory state, and the myeloid-lymphoid imbalance. Furthermore, high expression of IL-1R1 had a clinically significant impact on AML survival. Collectively, these findings underscore a potential therapeutic role for IL-1 signaling in the myeloid aspects of hematological malignancies and preleukemic conditions at the stem-cell level.
The IL-1R1 expression data are publically available. Other data generated in this study are available from the corresponding author on reasonable request.
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The authors would like to thank the members of the Indiana University Melvin and Bren Simon Cancer Center Flow Cytometry Resource Facility for their outstanding technical support. The Indiana University Melvin and Bren Simon Comprehensive Cancer Center Flow Cytometry Resource Facility (FCRF) is funded, in part, by NCI grant P30 CA082709, NIDDK grant U54 DK106846, and by NIH instrumentation grant 1S10D012270. The authors would also like to acknowledge the In Vivo Therapeutics Core for support in irradiation of mice and Dr. Mingjiang Xu for supplying Tet2−/− mice. We would also like to thank Ms. Tracy Winkle for administrative support.
Grant Support: This work was supported by NIH grants R01CA173852, R01CA134777, R01HL146137, and R01HL140961, and Riley Children’s Foundation (to RK). SB was supported by a T32 DK007519 “Regulation of Hematopoietic Cell Production” to Dr. Hal Broxmeyer, a Cancer Biology Training Program Fellowship from the Melvin and Bren Simon Cancer Center, a Ruth L. Kirschstein National Research Service Individual Fellowship (F30), and a Walter A. and Laura W. Deutsch Research Endowment from the Herman B. Wells Center for Pediatric Research.
The authors declare no competing interests.
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Burns, S.S., Kumar, R., Pasupuleti, S.K. et al. Il-1r1 drives leukemogenesis induced by Tet2 loss. Leukemia 36, 2531–2534 (2022). https://doi.org/10.1038/s41375-022-01665-3