SHP2 inhibition reduces leukemogenesis in models of combined genetic and epigenetic mutations

Abstract

In patients with acute myeloid leukemia (AML), 10% to 30% with the normal karyotype express mutations in regulators of DNA methylation, such as TET2 or DNMT3A, in conjunction with activating mutation in the receptor tyrosine kinase FLT3. These patients have a poor prognosis because they do not respond well to established therapies. Here, utilizing mouse models of AML that recapitulate cardinal features of the human disease and bear a combination of loss-of-function mutations in either Tet2 or Dnmt3a along with expression of Flt3ITD, we show that inhibition of the protein tyrosine phosphatase SHP2, which is essential for cytokine receptor signaling (including FLT3), by the small molecule allosteric inhibitor SHP099 impairs growth and induces differentiation of leukemic cells without impacting normal hematopoietic cells. We also show that SHP099 normalizes the gene expression program associated with increased cell proliferation and self-renewal in leukemic cells by downregulating the Myc signature. Our results provide a new and more effective target for treating a subset of patients with AML who bear a combination of genetic and epigenetic mutations.

Keywords: Epigenetics; Hematology; Leukemias; Oncology.

Figure 1. Effect of SHP099 treatment on leukemic Tet2^–/–Flt3^ITD cells.

Bone marrow cells from Boy/J (CD45.1⁺) and Tet2^–/–Flt3^ITD (CD45.2⁺) mice were transplanted in irradiated F1 (CD45.1⁺CD45.2⁺) mice followed by treatment with vehicle (n = 5) or SHP099 (n = 6) for 4 weeks. (A) Number of WBCs, neutrophils, and monocytes in the PB. (B) Percentage CD45.2⁺CD45.1^– cells in PB. (C) Representative flow cytometric profiles of Gr1 and CD11b expression in PB cells gated on CD45.1^–. (D) Quantification of Gr1^–CD11b⁺ and Gr1⁺CD11b⁺ cells within CD45.1^– gate. (E) Image of spleens from treated mice. (F) Quantification of spleen weight. (G) Representative H&E-stained images from spleen and femur of treated mice, acquired at ×20. (H) Representative flow plots of CD11b and c-KIT expression in CD45.1^–-gated spleen cells. (I) Representative flow plots of CD48 and CD150 expression in CD45.1^–lin^–Sca1⁺C-KIT⁺ (leukemic LSK) cells. (J) Quantification of HPC1 (CD48⁺CD150^–) and HPC2 (CD48⁺CD150⁺) cells therein. Data points are values from individual mice in each group from 1 of the 2 representative experiments. Median value for each group is indicated with interquartile range. *P < 0.05, **P < 0.01; Student’s t test with Welch’s correction for unequal variance.

Figure 2. Effect of SHP099 treatment on leukemic Dnmt3a^+/–Flt3^ITD cells.

Bone marrow cells from Boy/J (CD45.1⁺) and Dnmt3a^+/–Flt3^ITD (CD45.2⁺) mice were transplanted in irradiated Boy/J (CD45.1⁺) mice and treated with vehicle (n = 5) or SHP099 (n = 4). (A) WBC, neutrophil, and monocyte counts in PB. (B) Percentage of CD45.2⁺ cells in PB. (C) Image of spleen. (D) Quantification of spleen weight. (E) Percent CD11b^–c-KIT⁺ cells in spleen within CD45.2⁺ gate. (F) Image of liver. (G) Quantification of liver weight. (H) Percentage of Gr1^–CD11b⁺ and Gr1⁺CD11b⁺ cells in the BM in CD45.2⁺ gate. Data points shown are values from individual mice in each group. Median value for each group is indicated with the interquartile range. *P < 0.05, **P < 0.01, ****P < 0.0001; Student’s t test with Welch’s correction for unequal variance.

Figure 3. Effect of SHP099 treatment on bone marrow stem cells in Dnmt3a^+/–Flt3^ITD-induced AML model.

Analysis of bone marrow cells from mice transplanted with Dnmt3a^+/–Flt3^ITD (CD45.2⁺) and Boy/J (CD45.1⁺) cells and treated with vehicle or SHP099 as in Figure 2. (A) Representative flow plots showing the analysis strategy (top, vehicle-treated mice; bottom, SHP099-treated mice). Quantification of (B) percentage of CD45.2⁺ cells, (C) percentage of lin^– cells, (D) percentage of LSK cells in the leukemic (CD45.2⁺) compartment and (E) percentage of HPC1 (lin^–Sca1⁺KIT⁺CD48⁺CD150^–) within the CD45.2⁺LSK gate. Data points shown are values from individual mice in each group. Median value for each group is indicated with the interquartile range. *P < 0.05, **P < 0.01; Student’s t test with Welch’s correction for unequal variance.

Figure 4. Effect of SHP099 treatment on gene expression in Tet2^–/–Flt3^ITD (TF) cells.

Lineage-depleted bone marrow cells from individual mice (n = 3) were expanded in cytokine cocktail (SCF, IL-3, and IL-6) and treated with SHP099 or vehicle for 24 hours. RNA was isolated and subjected to RNA-seq analysis. (A) Heatmap of differentially expressed genes regulating proliferation. (B) GSEA of genes regulating proliferation in TF vehicle-treated vs. WT cells. (C) GSEA analysis of genes regulating proliferation in TF vehicle- vs. SHP099-treated cells. (D) Heatmap of differentially expressed genes in the MYC pathway. (E) GSEA analysis of MYC signature in TF vehicle vs. WT treatment. (F) GSEA analysis of MYC signature in TF vehicle- vs. SHP099-treated cells. (G) Expression of genes that regulate self-renewal. Individual values from 3 biological replicates are shown. Columns indicate the median value and error bars denote the range. The color code for the heatmap is indicated.