JMML tumor cells disrupt normal hematopoietic stem cells by imposing inflammatory stress through overproduction of IL-1β - PubMed (original) (raw)
JMML tumor cells disrupt normal hematopoietic stem cells by imposing inflammatory stress through overproduction of IL-1β
Yuhan Yan et al. Blood Adv. 2022.
Abstract
Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.
© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.
Figures
Figure 1.
Normal HSCs are aberrantly activated by _Ptpn11_E76K/+ neoplastic cells, leading to accelerated differentiation and exhaustion. (A) Genomic DNA was extracted from LSK cells (Lin−Sca-1+c-Kit+), myeloid cells (Mac-1+Gr-1+), monocytes (CD115+Gr-1+), and B cells (B220+) isolated from the BM of _Ptpn11_E76K/+ _LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates. The abundance of the inhibitory neo cassette with a stop codon in the targeted Ptpn11 allele was determined by quantitative polymerase chain reaction (n = 5 mice per genotype). (B) BM cells harvested from 5- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates were assayed for the frequencies of common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte erythroid progenitors (MEPs), and common lymphoid progenitors (CLPs; n = 6 mice per genotype). (C) BM cells (2 × 104 cells) collected from 5- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates (n = 3 mice per genotype) were processed for colony-forming unit assays. (D-E) Frequencies of HSC-enriched LSK (Lin−Sca-1+c-Kit+) cells (D) and HSCs (Lin−Sca-1+c-Kit+CD150+CD48−Flk2−) (E) in the BM and spleens of 5- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates (n = 6 mice per genotype) were determined by multiparameter fluorescence-activated cell sorting (FACS) analyses. (F-H) BM cells freshly isolated from _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates were assayed by FACS analyses to determine apoptotic cells (n = 6 mice per genotype) (F), cell cycle distribution (n = 5 mice per genotype) (G), and levels of phosphorylated Erk (p-Erk), p-Akt, and p-NF-κB in HSCs (n = 4 mice per genotype) (H). (I-J) Bone sections prepared from 4- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates were processed for immunofluorescence staining with the indicated antibodies. Spatial relationship between HSCs (Lin−CD48−CD41−CD150+) and endothelial (CD31+CD144+) cells was examined (representative images from n = 5 mice per genotype are shown); the distance of these 2 types of cells was calculated (I). Spatial relationship between HSCs (Lin−CD48−CD41−CD150+) and mesenchymal stem progenitor cells (MSPCs; Nestin+) was examined (representative images from n = 6 mice per genotype are shown); HSCs within 8 μm of MSPCs were considered to be close to MSPCs (J). (K-N) BM cells harvested from 3-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice (CD45.2+RFP−) and WT RPF transgenic mice (CD45.2+RFP+) were mixed at the HSC ratio of 1:1. The mixed BM cells and BM cells isolated from WT RPF transgenic mice (CD45.2+RFP+) were transplanted IV into lethally irradiated WT BoyJ mice (CD45.1+; n = 8 and 6 mice for mixed BM cells and WT BM cells, respectively). Sixteen weeks after transplantation, recipient mice were euthanized. Spleen weights (normalized against body weights) were documented (K). Frequencies of Mac-1+Gr-1+ cells in different donor-derived subpopulations in the peripheral blood (PB) were examined at the indicated time points (L). The pool sizes of HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) (M) and the cell cycle distribution of HSCs (Lin−Sca-1+c-Kit+CD150+) in different donor-derived populations (N) were determined at 16 weeks after transplantation as above. *P = .05, **P = .01, ***P = .001. DAPI, 4′,6-diamidino-2-phenylindole; GM-CSF, granulocyte-macrophage colony-stimulating factor; ns, not significant.
Figure 1.
Normal HSCs are aberrantly activated by _Ptpn11_E76K/+ neoplastic cells, leading to accelerated differentiation and exhaustion. (A) Genomic DNA was extracted from LSK cells (Lin−Sca-1+c-Kit+), myeloid cells (Mac-1+Gr-1+), monocytes (CD115+Gr-1+), and B cells (B220+) isolated from the BM of _Ptpn11_E76K/+ _LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates. The abundance of the inhibitory neo cassette with a stop codon in the targeted Ptpn11 allele was determined by quantitative polymerase chain reaction (n = 5 mice per genotype). (B) BM cells harvested from 5- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates were assayed for the frequencies of common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte erythroid progenitors (MEPs), and common lymphoid progenitors (CLPs; n = 6 mice per genotype). (C) BM cells (2 × 104 cells) collected from 5- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates (n = 3 mice per genotype) were processed for colony-forming unit assays. (D-E) Frequencies of HSC-enriched LSK (Lin−Sca-1+c-Kit+) cells (D) and HSCs (Lin−Sca-1+c-Kit+CD150+CD48−Flk2−) (E) in the BM and spleens of 5- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates (n = 6 mice per genotype) were determined by multiparameter fluorescence-activated cell sorting (FACS) analyses. (F-H) BM cells freshly isolated from _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates were assayed by FACS analyses to determine apoptotic cells (n = 6 mice per genotype) (F), cell cycle distribution (n = 5 mice per genotype) (G), and levels of phosphorylated Erk (p-Erk), p-Akt, and p-NF-κB in HSCs (n = 4 mice per genotype) (H). (I-J) Bone sections prepared from 4- to 6-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ littermates were processed for immunofluorescence staining with the indicated antibodies. Spatial relationship between HSCs (Lin−CD48−CD41−CD150+) and endothelial (CD31+CD144+) cells was examined (representative images from n = 5 mice per genotype are shown); the distance of these 2 types of cells was calculated (I). Spatial relationship between HSCs (Lin−CD48−CD41−CD150+) and mesenchymal stem progenitor cells (MSPCs; Nestin+) was examined (representative images from n = 6 mice per genotype are shown); HSCs within 8 μm of MSPCs were considered to be close to MSPCs (J). (K-N) BM cells harvested from 3-month-old _Ptpn11_E76K/+_LysM_-Cre+ mice (CD45.2+RFP−) and WT RPF transgenic mice (CD45.2+RFP+) were mixed at the HSC ratio of 1:1. The mixed BM cells and BM cells isolated from WT RPF transgenic mice (CD45.2+RFP+) were transplanted IV into lethally irradiated WT BoyJ mice (CD45.1+; n = 8 and 6 mice for mixed BM cells and WT BM cells, respectively). Sixteen weeks after transplantation, recipient mice were euthanized. Spleen weights (normalized against body weights) were documented (K). Frequencies of Mac-1+Gr-1+ cells in different donor-derived subpopulations in the peripheral blood (PB) were examined at the indicated time points (L). The pool sizes of HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) (M) and the cell cycle distribution of HSCs (Lin−Sca-1+c-Kit+CD150+) in different donor-derived populations (N) were determined at 16 weeks after transplantation as above. *P = .05, **P = .01, ***P = .001. DAPI, 4′,6-diamidino-2-phenylindole; GM-CSF, granulocyte-macrophage colony-stimulating factor; ns, not significant.
Figure 2.
JMML cells produce excessive IL-1β that plays a key role in driving HSC attrition. (A) Purified WT HSCs (Lin−Sca-1+c-Kit+CD150+CD48−Flk2−) were cocultured with BM cells isolated from _Ptpn11_E76K/+_LysM_-Cre+ and Ptpn11+/+_LysM_-Cre+ mice in 2-chamber transwell systems in StemSpan medium supplemented with thrombopoietin (TPO; 50 ng/mL), Flt3 ligand (50 ng/mL), and stem cell factor (SCF; 50 ng/mL) for 8 days. Percentages of Mac-1+Gr-1+ cells derived from HSCs were determined. Experiments were performed 3 times, and similar results were obtained in each. Results shown are the mean ± standard deviation (SD) of triplicates from 1 experiment. (B-C) BM plasma collected from 16-week-old _Ptpn11_E76K/+_LysM_-Cre+ mice and Ptpn11+/+_LysM_-Cre+ control mice (n = 3 mice per genotype) were processed for chemokine-cytokine array analyses. Representative results from 1 pair of the mice are shown (B). Levels of IL-1β in the BM plasma were quantified by enzyme-linked immunosorbent assay (n = 5 mice per genotype) (C). (D) WT HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) were cultured in StemSpan medium supplemented with TPO (100 ng/mL), Flt3 ligand (50 ng/mL), and SCF (100 ng/mL) in the presence or absence of IL-1β (10 ng/mL) and/or IL-1rα (10 ng/mL). Frequencies of Mac-1+Gr-1+ cells were determined 7 days later. (E-I) Ptpn11+/+_LysM_-Cre+_IL_-1R+/+, Ptpn11+/+_LysM_-Cre+_IL_-_1R_−/−, _Ptpn11_E76K/+_LysM_-Cre+_IL_-1R+/+, and _Ptpn11_E76K/+_LysM_-Cre+_IL_-_1R_−/− mice were generated and euthanized at the age of 4 months. Spleen weights (normalized against body weights) were documented (n = 8-9 mice per genotype) (E). Percentages of Mac-1+Gr-1+ cells in the peripheral blood, spleen, and BM were determined (n = 4-7 mice per genotype) (F). Frequencies of LSK cells and HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) in the BM (n = 7-10 mice per genotype) (G) and in the spleen (n = 6-10 mice per genotype) (H) and the cell cycle distribution in HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) in the BM (n = 4 mice per genotype) (I) were determined by multiparameter FACS analyses. (J) Purified HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) from WT and _IL_-_1R_−/− mice were cocultured with Mac-1+ cells isolated from 4-month-old Ptpn11+/+_LysM_-Cre+ and _Ptpn11_E76K/+_LysM_-Cre+ mice in StemSpan medium supplemented with TPO (100 ng/mL), Flt3 ligand (50 ng/mL), and SCF (100 ng/mL) in a 2-chamber transwell system for 8 days. Frequencies of Mac-1+Gr-1+ cells differentiated from HSCs in the upper chamber were assayed by FACS analyses. Experiments were performed 3 times, and similar results were obtained in each. Results shown are mean ± SD of triplicates from 1 experiment. (K) Cells from patients with JMML carrying PTPN11 mutations and healthy BM cells were cultured in StemSpan medium (serum free) supplemented with human SCF (hSCF; 50 ng/mL), hTPO (50 ng/mL), and hFlt3 ligand (50 ng/mL) for 3 days (experiment 1 [Exp. 1] and Exp 2). Culture medium was collected and analyzed for IL-1β by FACS with IL-1β antibody–conjugated beads. (L) CD34+ cord blood cells were isolated and cocultured with cells from patients with JMML or control cells in transwell systems in StemSpan medium (serum free) supplemented with hSCF (50 ng/mL), hTPO (50 ng/mL), and hFlt3 ligand (50 ng/mL). Five days (Exp. 1) or 12 days (Exp. 2) later, percentages of CD14+ cells and CD11c+ cells differentiated from CD34+ cells were determined by FACS analyses. *P = .05, **P = .01, ***P = .001. IFN-γ, interferon-γ; ns, not significant; TNF-α, tumor necrosis factor α.
References
- Birnbaum RA, O’Marcaigh A, Wardak Z, et al. Nf1 and Gmcsf interact in myeloid leukemogenesis. Mol Cell. 2000;5(1):189-195. -PubMed
- Emanuel PD, Bates LJ, Castleberry RP, Gualtieri RJ, Zuckerman KS. Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood. 1991;77(5):925-929. -PubMed
- Yu WM, Daino H, Chen J, Bunting KD, Qu CK. Effects of a leukemia-associated gain-of-function mutation of SHP-2 phosphatase on interleukin-3 signaling. J Biol Chem. 2006;281(9):5426-5434. -PubMed
- Chang TY, Dvorak CC, Loh ML. Bedside to bench in juvenile myelomonocytic leukemia: insights into leukemogenesis from a rare pediatric leukemia. Blood. 2014;124(16):2487-2497. -PubMed
- Emanuel PD. Juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia. Leukemia. 2008;22(7):1335-1342. -PubMed