Shp2 and Pten have antagonistic roles in myeloproliferation but cooperate to promote erythropoiesis in mammals

Abstract

Previous data suggested a negative role of phosphatase and tensin homolog (Pten) and a positive function of SH2-containing tyrosine phosphatase (Shp2)/Ptpn11 in myelopoiesis and leukemogenesis. Herein we demonstrate that ablating Shp2 indeed suppressed the myeloproliferative effect of Pten loss, indicating directly opposing functions between pathways regulated by these two enzymes. Surprisingly, the Shp2 and Pten double-knockout mice suffered lethal anemia, a phenotype that reveals previously unappreciated cooperative roles of Pten and Shp2 in erythropoiesis. The lethal anemia was caused collectively by skewed progenitor differentiation and shortened erythrocyte lifespan. Consistently, treatment of Pten-deficient mice with a specific Shp2 inhibitor suppressed myeloproliferative neoplasm while causing anemia. These results identify concerted actions of Pten and Shp2 in promoting erythropoiesis, while acting antagonistically in myeloproliferative neoplasm development. This study illustrates cell type-specific signal cross-talk in blood cell lineages, and will guide better design of pharmaceuticals for leukemia and other types of cancer in the era of precision medicine.

Keywords: Pten; Shp2; anemia; erythropoiesis; myeloproliferative neoplasm.

Fig. 1.

Shp2 ablation neutralizes development of myeloid proliferative neoplasm induced by Pten loss. (A) A Pten and Shp2 DKO mouse line was generated by breeding Mx1-Cre⁺:Pten^fl/fl:Shp2^fl/fl with Mx1-Cre⁻:Pten^fl/fl:Shp2^fl/fl mice and injection of poly-I:C. (B) Excision of the floxed DNA sequences was detected by PCR analysis of BM cells isolated 1 wk after final poly-I:C injection. (C) WBC, lymphocyte (LYM), monocyte (MON), and granulocyte (GRA) count in the peripheral blood was performed 1 wk after final poly-I:C injection (n = 5–8). (D) Representative FACS plots for Mac1 and Gr1 staining of BM cells. (E) Shp2 ablation suppresses Pten deletion-mediated accumulation of Mac1⁺Gr1⁺ BM cells (n = 3–5). (F) Shp2 removal restrains overproliferation of splenic Gr1⁺ cells induced by Pten deficiency (n = 3–4). (G) Representative H&E staining of spleen sections. Images were scanned by ScanScope Digital Slide Scanners with 100× magnification. (H) Representative chloroacetate esterase staining of liver sections shows myeloid infiltration in PKO but not DKO liver. Arrows point to the myeloid cells. Images were scanned by ScanScope Digital Slide Scanners with 100× magnification. (***P < 0.001, **P < 0.01, *P < 0.05; data are presented as means ± SEM.)

Fig. 2.

Inhibition of Pten^−/− myeloid progenitor expansion and MPN engraftment by additional Shp2 ablation. (A–D) The frequency of GMPs (A), CMPs (B), MEPs (C), and LSK cells (D) was determined in the BM. GMPs were gated as Lin⁻Sca-1⁻Kit⁺FcgR^hiCD34⁺, CMPs as Lin⁻Sca-1⁻Kit⁺FcgR^loCD34⁺, MEPs as Lin⁻Sca-1⁻Kit⁺FcgR^loCD34⁻, and LSK cells as Lin⁻Sca-1⁺Kit⁺ (n = 4–5). (E and F) In vitro CFU assays were performed for nucleated BM (E) or splenic cells (F), by seeding 20,000 BM or 100,000 splenic cells in MethoCultGF M3434 medium (StemCell Technologies) with cytokines for 14 d before colony enumeration (n = 3–4). (G) Myeloid progenitors accumulate and MPN develops in recipients engrafted with PKO but not DKO BM cells. The representative flow cytometric plots denote the Gr1 and Mac1 staining of BM cells. In the reconstitution experiments, 5 × 10⁵ BMNCs were injected to recipients that received lethal irradiation. BMNCs from control or mutant animals (CD45.2) were mixed with 2 × 10⁵ BMNCs from CD45.1 mice for radioprotection in the transplantation assay. (H) Mice that received DKO BM cells survived longer than those transplanted with PKO BM cells. (I) MPN development was examined in recipients as shown, with 1 × 10⁶ or 2.5 × 10⁵ to 5 × 10⁵ BMNCs used for transplantation. BMNCs from control or mutant animals (CD45.2) were mixed with 2 × 10⁵ BMNCs from CD45.1 mice for radioprotection in the transplantation assays. (***P < 0.001, **P < 0.01,*P < 0.05; ns, not significant; data are presented as means ± SEM).

Fig. S1.

Shp2 removal abolishes the increase of G-CSF, M-CSF, or GM-CSF responsive colony-forming cells. In vitro CFU-G, CFU-M, or CFU-GM assays for BMNCs (A) or splenic cells (B) were performed, by seeding 10,000 BM (A) or 100,000 spleen (B) cells in MethoCultGF M3234 medium (StemCell Technologies) containing 10 ng/mL G-CSF, M-CSF, or GM-CSF, respectively, for 7 d before colony enumeration (n = 4). Data are presented as means ± SEM (***P < 0.001, **P < 0.01,*P < 0.05; ns, not significant).

Fig. S2.

Additional Shp2 deletion alleviates the hyperphosphorylation of AKT and Erk in PKO myeloid progenitors. (A) Representative flow cytometry plots of phospho-AKT(S473) staining in Gr1⁺Mac1⁺ myeloid progenitors. (B) Statistical analysis of median phospho-AKT staining fluorescence intensity (n = 3–4). (C) Representative flow cytometry plots of phospho-Erk1/2(T202/Y204) staining in Gr1⁺Mac1⁺ myeloid progenitors. (D) Statistical analysis of median phospho-Erk1/2(T202/Y204) staining fluorescence intensity (n = 3–4). Data are presented as means ± SEM (***P < 0.001, **P < 0.01, *P < 0.05).

Fig. 3.

Dual deletion of Pten and Shp2 causes lethal anemia, expansion of erythroid progenitors and abortive differentiation of RBCs. (A) Survival curve of WT, PKO, SKO, and DKO mice after final injection of poly-I:C (*P = 0.028 between PKO and DKO, **P = 0.0029 between SKO and DKO, n = 9). (B) DKO mouse shows pale toe and tiny RBC pellet after centrifugation of whole peripheral blood. (C) Severe anemic DKO mice show hematocrit as low as 25% of normal count (n = 3–4). (D) Representative FACS plots for CD71 and Ter119 staining of BM cells. (E) Quantification of CD71^highTER119^low (gate I), CD71^highTER119⁺ (gate II), CD71^medTER119⁺ (gate III), and CD71^lowTER119⁺ (gate IV) frequency in BM shows expansion of CD71^highTER119^low, CD71^highTER119⁺, and CD71^medTER119⁺ erythroid progenitors in DKO mice. In contrast, CD71^lowTER119⁺ erythroid cells were significantly reduced in DKO BM (n = 3–5). (F) In vitro BFU-E assay shows much higher BFU-E for nucleated BM cells of DKO animals (n = 3–4). BFU-E assays were performed by seeding 20,000 BM cells in MethoCultGF M3434 medium (StemCell Technologies) for 14 d (n = 3–4). (G) In vitro CFU-E assay indicates dramatically reduced CFU-E for nucleated BM cells of DKO mice. CFU-E was assayed for nucleated BM cells in MethoCultGF M3234 medium supplemented by 6 units per milliliter recombinant human Epo (n = 3–4). (H) Serum EPO concentrations were measured by an ELISA Kit (R&D). (n = 3–5). (***P < 0.001, **P < 0.01, *P < 0.05, data are presented as means ± SEM.)

Fig. S3.

Accumulation of erythroblasts in DKO hematopoietic organs. (A) Erythroblasts were detected in the DKO peripheral blood. Representative pictures were shown for Wright–Giemsa staining of blood smears. At least four animals in each group were examined in the experiment, and arrows point to the erythroblasts. (B and C) Comparable B₁₂ (B) and folic acid (C) concentrations in WT, SKO, PKO, and DKO sera (n = 3–5). (D–F) Enlarged spleen and liver in DKO and PKO mice (n = 3–4). (G and H) Accumulation of erythroblasts in DKO BM and spleen. Wright–Giemsa staining of BM (G) and spleen cytospin specimens (H) was performed. At least four animals in each group were examined, with representative pictures shown. Arrows point to the erythroblasts. A, G, and H were captured with 1,000× magnification using a Nikon light microscope. (I) Representative FACS plots for nucleated RBCs in BM samples. (J) Statistical data of nucleated RBCs in BM samples (n = 3–4). Data are presented as means ± SEM (**P < 0.01, *P < 0.05).

Fig. 4.

Accumulation of ROS and reduced RBC lifespan contributes to the severe anemia in DKO mice. (A) RBC lifespan was measured by sulfo-NHS-biotin labeling (n = 3–4). Significantly shortened lifespan was observed for PKO and DKO RBCs. (B) ROS staining was done on freshly collected peripheral blood cells by staining with 25 μm CM-H₂DCFDA (Life Technologies) for 30 min at 37 °C, and analyzed by flow cytometry. (Upper) Representative FACS plots for CM-H₂DCFDA staining. (Lower) Median fluorescence intensity (MFI) for CM-H₂DCFDA staining of erythrocytes. (C) Antioxidant treatment greatly improved the gross appearance and pale toes of DKO animals. (D) Partial but significant restoration of RBC number and hematocrit in DKO mice subjected to the antioxidant administration (n = 3). (E) Suppression of ROS accumulation in DKO erythroblasts by the antioxidant treatment. MFI for CM-H₂DCFDA staining of erythrocytes is shown (Right) (n = 3). (**P < 0.01, *P < 0.05, data are presented as means ± SEM.)

Fig. S4.

Shortened lifespan of DKO RBCs was not a result of destructive environment. Untreated BMNCs from different donors were transplanted into lethally irradiated mice. One month after transplantation, three doses of poly-I:C were administered to induce gene deletion. The RBC lifespan was then measured by sulfo-NHS-biotin labeling in transplanted recipient mice. Significantly decreased lifespan of Shp2/Pten-deficient RBCs was detected as in primary DKO animals (comparing Fig. 4A to Fig. S4) (*P < 0.05, data are presented as mean ± SEM, n = 3–5).

Fig. S5.

Unaltered ROS levels in DKO myeloid progenitors. Elevated ROS concentration was observed in PKO but not DKO Gr1⁺Mac1⁺ myeloid progenitors (three to four animals in each group were examined and representative FACS plots are shown). Statistical analysis is shown (Right) (**P < 0.01, data are presented as mean ± SEM).

Fig. S6.

Hyperphosphorylation of AKT is detected in PKO erythroblasts. (A) Representative flow cytometry plots of phospho-AKT(S473) staining in Ter119⁺ BMNCs. (B) Statistical analysis of median phospho-AKT staining fluorescence intensity (n = 3–4). (***P < 0.001, data are presented as mean ± SEM.)

Fig. S7.

Antioxidant treatment leads to elongated life span of DKO erythrocytes but does not affect the skewed differentiation of DKO erythroblasts. (A) Antioxidant treatment increases the life span of DKO erythrocytes (n = 6). (B) Representative FACS plots for CD71 and Ter119 staining of BM cells from vehicle and antioxidant-treated WT or DKO mice (three animals in each group were examined in the experiment and representative FACS plots are shown). Statistical analysis is shown in the right panel (***P < 0.001, **P < 0.01, *P < 0.05, data are presented as mean ± SEM).

Fig. 5.

A Shp2 inhibitor suppresses myeloproliferation while inducing anemia in PKO mice. (A) Shp2 inhibitor 11a-1 ameliorated excessive myeloproliferation in the BM driven by homozygous deletion of Pten (Veh, vehicle; Inh, inhibitor; n = 3–4, representative FACS plots are shown). (B) Shp2 inhibitor treatment suppressed accumulation of granulocytes in the peripheral blood of PKO mice (n = 3–4). (C and D) PKO mice developed anemia upon treatment of the Shp2 inhibitor. Reduced hematocrit and RBC count in PKO mice treated with 11a-1 (n = 3–4). (E) Accumulation of erythroblasts in the BM of Shp2 inhibitor-treated PKO mice. Representative FACS plots for CD71 and Ter119 staining of BM cells are shown (three to four mice in each group were examined in the experiment). (*P < 0.05, data are presented as means ± SEM.)

Fig. S8.

MEK inhibitor Trametinib, a new anticancer drug, suppresses myeloproliferation but induces anemia in PKO mice. (A) Trametinib treatment ameliorated the accumulation of granulocytes in the peripheral blood of PKO mice (n = 3–4). Veh, Vehicle; Tra, Trametinib. (B) Decreased RBC count in PKO mice treated with Trametinib (n = 3–4). (C) Increased early erythroblasts in the BM from Trametinb-treated PKO mice. Representative FACS plots for CD71 and Ter119 staining of BM cells are shown (three to four animals in each group were examined in the experiment). Statistical analysis is shown in the right panel (**P < 0.01, *P < 0.05, data are presented as mean ± SEM).