Metformin Reduces Tumor Burden and Splenomegaly in JAK2^V617F Murine Models

Introduction : The recurrent gain-of-function JAK2^V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). Metformin (1,1-dimethylbiguanide) is a biguanide widely prescribed for the treatment of type II diabetes and metabolic syndromes. In recent years, studies using cancer cell lines and murine models have provided evidence for potential anticancer activity of metformin. Previous results from our research group indicate that metformin reduces cell viability, proliferation, clonogenicity and JAK2/STAT activation in human JAK2^V617F cell lines and spontaneous erythroid colony formation in primary cells from patients with polycythemia vera (Machado-Neto et al. ASH Annual Meeting Abstracts #1960, 2016). Objectives : To investigate the effects of metformin treatment in tumor formation and MPN phenotype using Ba/F3 JAK2^V617F cells and Jak2^V617F knockin-induced MPN mice. Methods : For tumor formation, experimental groups consisted of 8-10 week-old NSG female mice that received a 100 µL (50 µL PBS plus 50 µL matrigel) dorsal subcutaneous injection of 2×10⁶ Ba/F3 JAK2^V617F cells and daily treatment by intraperitoneal injection of vehicle (PBS, n=9) or metformin (125 mg/kg, n=9) for 13 days after initial tumor growth (7 days). Tumor volume (V) was obtained using the formula (V = W² × L × 0.52), where W and L are the smaller and larger diameters, respectively. For MPN phenotype induction, 5×10⁶ bone marrow cells from Jak2^V617F knockin (Jak2^WT/V617F) mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry in peripheral blood. Mice with >70% CD45.2 cells were randomized and treated daily by intraperitoneal injection of vehicle (PBS, n=4) or metformin (125 mg/kg, n=5) for 6 weeks. At the conclusion of the experiment, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen were evaluated by CD71 and Ter119 markers (Becton-Dickinson) by flow cytometry. All experiments were approved by the Animal Ethics Committee. For comparisons, Mann-Whitney test or ANOVA test and Bonferroni post-test was used. A p value <0.05 was considered as statistically significant. Results : In NSG mice inoculated with Ba/F3 JAK2^V617F, metformin treatment (125 mg/kg/day) was able to significantly reduce the tumor burden after 13 days of treatment. At the endpoint, the mean±SD of tumor volume for vehicle (n=4) and metformin (n=4) treated mice was 1120.7±289.1 mm³ and 510.3±244.4 mm³, respectively (p <0.001). A second independent experiment including five mice in each group presented similar results (vehicle versus metformin: 1307.7±511.9 mm³versus 725.1±380.6 mm³; p <0.01). In Jak2^V617F knockin-induced MPN animals, metformin treatment (125 mg/kg/day) was well-tolerated, reduced splenomegaly (p=0.02) and improved splenic architecture. Erythoid progenitors in the spleen and bone marrow did not differ between control and metformin-treated groups (p >0.05), but the smaller spleens presented with a very low percentage of erythroid progenitors. Hemoglobin and hematocrit were not modulated by metformin treatment. Conclusions : Metformin treatment at a dose of 125 mg/kg/day effectively reduced tumor burden in Ba/F3 JAK2^V617F allograft and splenomegaly in Jak2^V617F knockin-induced MPN murine models. In humans, this dose represents a pharmacological dose similar to that which is used for type II diabetes treatment. Our results provide additional insights for metformin in preclinical models of MPN.