STAT3 mutations identified in human hematologic neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model

Abstract

STAT3 protein phosphorylation is a frequent event in various hematologic malignancies and solid tumors. Acquired STAT3 mutations have been recently identified in 40% of patients with T-cell large granular lymphocytic leukemia, a rare T-cell disorder. In this study, we investigated the mutational status of STAT3 in a large series of patients with lymphoid and myeloid diseases. STAT3 mutations were identified in 1.6% (4 of 258) of patients with T-cell neoplasms, in 2.5% (2 of 79) of patients with diffuse large B-cell lymphoma but in no other B-cell lymphoma patients (0 of 104) or patients with myeloid malignancies (0 of 96). Functional in vitro assays indicated that the STAT3Y640F mutation leads to a constitutive phosphorylation of the protein. STA21, a STAT3 small molecule inhibitor, inhibited the proliferation of two distinct STAT3 mutated cell lines. Using a mouse bone marrow transplantation assay, we observed that STAT3Y640F expression leads to the development of myeloproliferative neoplasms with expansion of either myeloid cells or megakaryocytes. Together, these data indicate that the STAT3Y640F mutation leads to constitutive activation of STAT3, induces malignant hematopoiesis in vivo, and may represent a novel therapeutic target in some lymphoid malignancies.

Figure 1.

(A). STAT3 phosphorylation analysis by flow cytometry of YT1 and FEPD (STAT3 mutated cell lines) as compared to K562 (STAT3 WT cell line). Control represents the unstained cells. Analysis was performed in normal conditions and after serum starvation. (B). STAT3 phosphorylation analysis by Western Blotting of YT1 and FEPD (STAT3 mutated cell lines) as compared to K562 (STAT3 WT cell line). (C). Proliferation assay of YT1, FEPD (STAT3 mutated cell lines) and K562 (STAT3 WT cell line) after treatment with the STAT3 inhibitor STA-21 (25 μM) (left panel) as compared to DMSO treatment (middle panel). The right panel shows the relative cell counts after 72-h of STA-21 treatment normalized to the number of DMSO-treated cells for each cell line. (D). STAT3 phosphorylation analysis by Western blotting of transduced murine Ba/F3 cells with empty (MSCV), wild-type (WT) STAT3 or STAT3 Y640F retroviruses.

Figure 2.

(A). White blood cells (WBC) count in peripheral blood samples obtained from MSCV, STAT3 WT and STAT3 Y640F mice. The x-axis values represent the number of months after bone marrow transplantation. (B). Hemoglobin level in peripheral blood samples obtained from MSCV, STAT3 WT and STAT3 Y640F mice. The x-axis values represent the number of months after bone marrow transplantation. (C). Platelet count in peripheral blood samples obtained from MSCV, STAT3 WT and STAT3 Y640F mice. The x-axis values represent the number of months after bone marrow transplantation. (D). Spleen weight of MSCV, STAT3 WT and STAT3 Y640F mice. (E). Representative flow cytometrical analysis of the mature myeloid cells (left), erythroid (center) and megakaryocytic (right) lineages in the bone marrow of STAT3 Y640F mice with myeloid and thrombocytosis diseases, as compared to MSCV and STAT3 WT mice. The percentages of GFP-positive cells are indicated.