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Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment

Abstract

Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway1, are found in 50% of patients with Noonan syndrome2. These patients have an increased risk of developing leukaemia3, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity4,5,6,7. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1β and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.

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Figure 1: Ptpn11E76K/+ mutation in Nestin+ MSPCs aberrantly activates neighbouring wild-type HSCs, inducing MPN in Ptpn11E76K/+Nestin-Cre+ mice.
Figure 2: MPN that developed in Ptpn11E76K/+Vav1-Cre+ mice is less severe and less progressive than MPN developed in Ptpn11E76K/+Mx1-Cre+ mice.
Figure 3: IL-1β and CCL3 are overproduced by Ptpn11E76K/+ MPN cells and MSPCs, respectively.
Figure 4: Ptpn11E76K/+ MSPCs recruit monocytes to the vicinity by excessive production of CCL3, leading to the hyperactivation and displacement of neighbouring HSCs from the niche.

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Acknowledgements

This work was supported by The National Institutes of Health grants HL130995 and DK092722 (to C.K.Q.).

Author information

Affiliations

Authors

Contributions

L.D. generated microenvironmental cell-type-specific knock-in mice, analysed MPN development and progression, performed HSC imaging and MSPC signalling analyses, and tested the therapeutic effects of CCL3 receptor antagonists. W.-M.Y. trained L.D. and H.Z. in techniques, performed cytokine array analyses, and analysed patient specimens. H.Z. generated MSPC-specific knock-in mice, characterized MPN development and progression, and analysed HSC phenotypes. S.T.B. and M.P. identified and collected patient specimens. M.L.L. provided patient specimens and thoroughly discussed the work. M.Z., G.H., H.E.B., and D.T.S. provided critical advice on experimental designs and interpretation of the data, and edited the manuscript. C.-K.Q. designed the experiments and directed the entire project. L.D. and C.K.Q. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Cheng-Kui Qu.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks I. Ghobrial, B. Neel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Ptpn11E76K/+ mutation in MSPCs induces MPN by aberrant activation of neighbouring wild-type HSCs in Ptpn11E76K/+Nestin-Cre+ mice.

a, Peripheral blood collected from 7–12-month-old Ptpn11E76K/+Nestin-Cre+ mice with MPN and Ptpn11+/+Nestin-Cre+ littermates were analysed for percentages of neutrophils and lymphocytes (n = 15 mice per group). b, BM cells (2 × 104 cells) freshly collected from Ptpn11E76K/+Nestin-Cre+ mice with MPN and Ptpn11+/+Nestin-Cre+ littermates (n = 4 mice per group) were assayed for haematopoietic colony-forming units in 0.9% methylcellulose IMDM medium containing 30% FBS, glutamine (10−4 M), β-mercaptoethanol (3.3 × 10−5 M), and IL-3 (1.0 ng ml−1) or GM-CSF (1.0 ng ml–1). After 7 days of culture at 37 °C in a humidified 5% CO2 incubator, haematopoietic cell colonies (primarily CFU-GM) derived from myeloid progenitors were counted under an inverted microscope. c, Femurs, spleens, livers and lungs were processed for histopathological examination (haematoxylin and eosin staining) (n = 4 mice per group). Representative pictures are shown. dh, BM cells and splenocytes were collected from Ptpn11E76K/+Nestin-Cre+ mice with MPN and Ptpn11+/+Nestin-Cre+ littermates. CD115+Gr-1+ monocytes in the BM (n = 4 mice per group) (d), frequencies of LSKs (Lin-Sca-1+c-Kit+) in the BM (n = 8 mice per group) (e), absolute number of HSCs in two femurs and two tibias (n = 10 mice per group) (f), frequencies of LSKs in the spleen (n = 8 mice per group) (g), and apoptotic cells in the HSC population in the BM (n = 6 mice per group) (h) were assayed by multiparameter FACS analyses. i, BM cells collected from wild-type BoyJ mice were transplanted into 6-month-old Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice. Recipients were monitored for MPN development for 6–8 months. Percentages of donor cell (CD45.1+)-derived Mac-1+ myeloid cells in the peripheral blood (n = 5 mice per group) and BM (n = 8 mice per group) of recipients were determined. j, Frequencies of HSCs in the BM from Ptpn11E76K/+Nestin-Cre+ mice that had not yet manifested MPN and Ptpn11+/+Nestin-Cre+ littermates (n = 4 mice per group) were assayed as above. Data shown in a, b, dj are mean ± s.d. of all mice examined; *P < 0.05; **P < 0.01; ***P < 0.001. Source Data for this figure are available online.

Source data

Extended Data Figure 2 Ptpn11E76K/+ mutation in the BM stroma enhances MPN development from mutant HSCs with the same mutation in Ptpn11E76K/+Mx1-Cre+ mice.

a, Tissues collected from Ptpn11+/+Mx1-Cre+, Ptpn11E76K/+Mx1-Cre+ (8 weeks after pI–pC administration), and Ptpn11E76K/+Vav1-Cre+ mice at 16-week old (n = 3 mice per group) were processed for histopathological examination (haematoxylin and eosin staining). Representative pictures are shown. bd, Ptpn11E76K/+Vav1-Cre+ mice (4 weeks old) (n = 3 mice per group) were administered pI–pC or PBS, as described in Methods. Spleen weights (b), Mac-1+Gr-1+ myeloid cells in the BM, spleen, and liver (c), the cycling status of HSCs (d) were analysed 16 weeks after pI–pC administration. e, Timed pregnant Ptpn11E76K/+Mx1-Cre female mice (13.5 days post coitum) that were mated with Ptpn11+/+Mx1-Cre+ male mice were administered pI–pC as above. Ptpn11E76K/+Mx1-Cre+ pups delivered by these female mice were identified. The efficiencies of neo deletion from targeted Ptpn11 alleles in haematopoietic cells and MSPCs of these mice were approximately 95%. Mac-1+Gr-1+ cells in the peripheral blood of Ptpn11+/+Mx1-Cre+ (n = 3 mice), Ptpn11E76K/+Mx1-Cre+ (n = 3 mice), and Ptpn11E76K/+Vav1-Cre+ (n = 7 mice) mice at the same age were monitored at the indicated time points. Data shown in be are mean ± s.d. of all mice examined; **P < 0.01; ***P < 0.001; N.S., not significant. Source data are available online.

Source data

Extended Data Figure 3 Donor-cell-derived MPN is developed in Ptpn11E76K/+Mx1-Cre+ broad knock-in mice and Ptpn11D61G/+ global knock-in mice, but not Ptpn11E76K/+Vav1-Cre+ haematopoietic cell-specific knock-in mice transplanted with wild-type BM cells.

BM cells (2 × 106) freshly collected from wild-type BoyJ mice (CD45.1+) were transplanted into lethally irradiated (1,100 cGy) Ptpn11E76K/+Mx1-Cre+, Ptpn11+/+Mx1-Cre+ (8 weeks after pI–pC administration), and Ptpn11E76K/+Vav1-Cre+ mice at 16-week old (CD45.2+). Spleen weights (n = 5 mice per group) (a), percentages of donor cell (CD45.1+) reconstitution (n = 8 mice per group) and percentages of donor cell-derived myeloid (Mac-1+Gr-1+) cells (n = 8 mice per group) in the peripheral blood of the recipients (b) were determined at the indicated time points following the transplantation. c, BM cells (1 × 106) freshly collected from wild-type BoyJ mice (CD45.1+) were transplanted into lethally irradiated 3–4-month old Ptpn11D61G/+ and Ptpn11+/+ (CD45.2+) mice (n = 14 and 17 mice, respectively). Recipients were monitored for MPN development for 8 months. Percentages of donor cell (CD45.1+)-derived Mac-1+ myeloid cells in the peripheral blood of recipients were determined. Representative results are shown. Data shown in a, b are mean ± s.d. of all mice examined; **P < 0.01; ***P < 0.001. Source Data for this figure are available online.

Source data

Extended Data Figure 4 Ptpn11E76K/+ mutation in MSPCs and osteoprogenitors, but not differentiated osteoblasts or endothelial cells, in the BM microenvironment induces MPN.

Cell-type-specific Ptpn11E76K knock-in mice as indicated were generated and monitored for MPN development. a, The ages of the microenvironmental cell-type-specific Ptpn11E76K/+ knock-in mice when they were euthanized for MPN diagnosis. b, Peripheral blood haematology was determined using the HemaTrue veterinary hematology analyzer. Mac-1+Gr-1+/− myeloid cells, B220+ B lymphoid, and CD3+ T-lymphoid cells in the BM were analysed by FACS. Karyotypes of MPN cells were examined by standard karyotyping analyses. HSCs in the peripheral blood were determined by multiparameter FACS. SSC/CD45 profiles were also determined by FACS. CD115+Gr-1+ monocytes were highlighted in red. Cytokine sensitivity of BM myeloid progenitors was determined by CFU assays with a range of GM-CSF concentrations. Transplantability of MPN cells was determined by transplantation of BM cells into lethally-irradiated BoyJ mice. Recipient mice were monitored for 6 months. All methods are described in Methods and/or related figure legends. c, BM-derived MSPCs were generated from the indicated mouse lines. The abundance of the neo cassette in genomic DNA was determined by qPCR (n = 3 mice per group). Data shown in c are mean ± s.d. of all mice examined. Statistical significance (***P < 0.001) was determined between the indicated cell-type-specific Ptpn11E76K/+ knock-in mice and Ptpn11E76K-neo/+Nestin-Cre control mice. Source Data for this figure are available online.

Source data

Extended Data Figure 5 Ptpn11E76K/+ MSPCs do not directly activate HSCs.

BM-derived MSPCs were enriched from Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice, as described in Methods. MSPCs at the 2nd or 3rd passages were plated in regular 24-well plates (a) or lower chambers of transwells (b). Forty-eight hours later when the cells were confluent, HSCs (75–200) (LinSca-1+c-Kit+CD150+CD48Flk2) sorted from Ptpn11E76K/+Mx1-Cre+ and Ptpn11+/+Mx1-Cre+ mice (8 weeks after pI–pC administration) were seeded in the same wells (a) or in upper chambers with the 0.4 μm pore size (b). The cells were co-cultured in StemSpan medium supplemented with cytokines TPO (50 ng ml−1), Flt3 ligand (50 ng ml−1), SCF (50 ng ml−1), IL-3 (20 ng ml−1), and IL-6 (20 ng ml−1). Frequencies of myeloid (Mac-1+Gr-1+) cells that differentiated from HSCs were assayed by FACS analyses after 7–10 days of co-culture. Experiments were performed three times and similar results were obtained in each (see Supplementary Information). Results shown are mean ± s.d. of triplicates from one experiment; N.S., not significant. Source data are available online.

Source data

Extended Data Figure 6 Cytokines/chemokines are aberrantly produced by Ptpn11E76K/+ MSPCs.

a, Spleen tissues freshly dissected from Ptpn11E76K/+Mx1-Cre+ and Ptpn11+/+Mx1-Cre+ mice (n = 3 mice per group) 12 weeks after pI–pC administration were gently smashed in PBS (0.1 g tissue per 1.0 ml). Supernatant collected was processed for cytokine–chemokine array analyses with the Mouse Cytokine Antibody Array Kit following the instructions provided by the manufacturer. Representative results from one pair of the mice are shown. b, MSPCs (CD45Ter-119CD31CD140α+Sca-1+) were freshly isolated from paired Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice at 7–8 months old by FACS. Total RNA was extracted and processed for RNA-sequencing analyses as described in Methods. The correlation coefficient between the two groups was 0.954, verifying that the method was accurate (left). Genes with more than 2.0 fold increased (in red) or decreased (in green) mRNA levels are shown on the right. Secreted protein factors are indicated. c, HSCs sorted from wild-type C57BL/6 mice were cultured in the presence of IL-1β (10 ng ml−1), CCL3 (20 ng ml−1), CCL4 (20 ng ml−1), or CCL12 (20 ng ml−1). Six days later, cells were collected and analysed for Mac-1+ myeloid cells, F4/80+ macrophages, and CD115+ monocytes by FACS. Data presented are mean ± s.d. of four independent experiments; ***P < 0.001. Source Data for this figure are available online.

Source data

Extended Data Figure 7 Ptpn11E76K/+ mutation increases MSPC proliferation by enhancing cell signalling activities.

a, Seven–ten-month-old Ptpn11E76K/+Nestin-Cre+ mice and Ptpn11+/+Nestin-Cre+ littermates were analysed. Femurs were processed for immunofluorescence staining with the indicated antibodies (n = 3 mice per group). Representative images are shown. b, BM cells (2 × 106 cells) freshly collected from the indicated mouse lines (n = 3 mice per group) were assessed by the CFU-F assay, as detailed in Methods. Statistical significance was determined between the indicated cell-type-specific Ptpn11E76K/+ knock-in mice and Ptpn11+/+Nestin-Cre+ control mice. c, d, MSPCs were enriched from Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice (n = 4 mice per group) as described in Methods. MSPCs were analysed for growth rates (c, left) and expression levels of Shp2 (c, right), and cell cycle distributions (d). e, Confluent MSPCs (n = 3 mice per group) were starved in serum and growth factor-free medium for 48 h and then stimulated with basic fibroblast growth factor (bFGF, 50 ng ml−1) for the indicated periods of time. Whole-cell lysates were prepared and examined for Erk, Akt, c-Src, and S6 activities by immunoblotting with anti-phospho-Erk, anti-phospho-Akt, anti-phospho-c-Src Y416, and anti-phospho-S6 antibodies. Blots were stripped and reprobed with anti-pan-Erk, anti-pan-Akt, anti-c-Src, anti-S6, and anti-Shp2 antibodies to check protein loading and Shp2 levels. Densitometric analyses were performed to determine phosphorylation levels of the indicated proteins and normalized against protein loading levels (arbitrary units). Data shown in be are mean ± s.d. of all mice examined. *P < 0.05; **P < 0.01; ***P < 0.001. Source Data for this figure are available online.

Source data

Extended Data Figure 8 Ptpn11E76K/+ MSPCs recruit monocytes, but not macrophages, to the vicinity.

a, Bone sections (one section per femur or tibia) prepared from Ptpn11E76K/+Prx1-Cre+ (n = 4 mice) and Ptpn11+/+Prx1-Cre+ mice (n = 3 mice) at 6–7 months old were immunostained with the indicated antibodies and counterstained with DAPI. bd, Bone sections (one section per femur or tibia) prepared from Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice at 7–10 months old were immunostained with the indicated antibodies and counterstained with DAPI (n = 5 mice per group) (b). The distance of HSCs (LinCD48CD41CD150+) from closest CD31+CD144+ endothelial cells was determined (n = 8 mice per group) (c). The spatial relationship between HSCs (LinCD41CD150+) and megakaryocytes (CD41+) was examined. HSCs within <8 μm of megakaryocytes were considered as close to megakaryocytes (n = 5 mice per group) (d). Representative images are shown in all panels.

Source data

Extended Data Figure 9 Administration of CCL3 receptor antagonists reverses MPN phenotypes in Ptpn11E76K/+Osx1-Cre+ mice.

Ptpn11E76K/+Osx1-Cre+ mice at 6–7 months old were treated daily with the CCR1 antagonist BX471 and the CCR5 antagonist Maraviroc or vehicle control for 23 days as described in Methods. Myeloid cells (Mac-1+Gr-1+) in the peripheral blood were determined at the indicated time points (n = 5 mice per group, each line represents one mouse) (a). Mice were euthanized at the end of the experiments. Monocytes (CD115+Gr-1+) (n = 5 and 6 mice for the antagonist and vehicle groups, respectively) in the BM, spleen, and peripheral blood were determined (b). BM cells were assayed by multiparameter FACS analyses to determine the pool size (c) of HSCs (LinSca-1+c-Kit+CD150+CD48Flk2) (n = 5 and 6 mice for the antagonist and vehicle groups, respectively). Data shown in b, c are mean ± s.d. of all mice examined; ***P < 0.001. Source Data for this figure are available online.

Source data

Extended Data Figure 10 Administration of CCL3 receptor antagonists mitigates MPN in Ptpn11E76K/+Mx1-Cre+ mice.

Ptpn11E76K/+Mx1-Cre+ mice (4 weeks after pI–pC administration; n = 5 mice per group) were treated daily with the CCR1 and CCR5 antagonists or vehicle as described above. Mice were euthanized, and spleens were photographed and weighted (a). White blood cell counts (b) and myeloid cells (Mac-1+Gr-1+) (c) in the peripheral blood were determined at the indicated time points. Mac-1+Gr-1+ myeloid cells (d) and CD115+Gr-1+ monocytes (e) in the BM, spleen, peripheral blood, and liver were determined at the end of the experiments. Data shown in all panels are mean ± s.d. of all mice examined; *P < 0.05; **P < 0.01; ***P < 0.001. Source Data for this figure are available online.

Source data

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Supplementary Data

This file contains the source data for Extended Data Figures 7b, 7d. (PDF 333 kb)

Supplementary Data

This file contains the original data from three independent experiments for Extended Data Figure 5. (XLSX 12 kb)

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Dong, L., Yu, WM., Zheng, H. et al. Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment. Nature 539, 304–308 (2016). https://doi.org/10.1038/nature20131

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