Multitarget Antileukemic Effects of Metformin in Myeloproliferative Neoplasm Cells: Inhibition of JAK2/STAT Signaling and Mitochondrial Activity

Background: The recurrent JAK2^V617F gain-of-function mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that inhibits the mitochondrial complex I and metformin antineoplastic activity has been described in leukemia, lymphoma and multiple myeloma. More importantly, metformin antineoplastic effects appear to act selectively in cancer cells, since cytotoxicity was not observed for normal hematopoeitic cells. Considering that metformin has been widely used in clinical practice, preclinical studies in an attempt to determine its effects, alone or in combination with ruxolitinib, in MPN, may be of interest, since these findings have a translational potential. Objectives: To investigate the effects of metformin and/or ruxolitinib on cell signaling and cellular functions of JAK2^V617F cells. Metformin and/or ruxolitinib effects were also evaluated on spontaneous erythroid colony formation from polycythemia vera (PV) patients. Materials and methods: HEL, SET2 (JAK2^V617F) and Ba/F3 JAK2^V617F cell lines treated or not with metformin (5 and 10 mM) and/or ruxolitinib (300 nM) were submitted for cell viability (MTT), clonogenicity (colony formation), cell cycle (flow cytometry), cell proliferation (Ki-67 staining), apoptosis (annexin-V/PI and caspase 3/PARP1 cleavage), PCR array for PI3K/AKT-related genes and oxygen consumption (high-resolution respirometry). For spontaneous erythroid colony formation assays, mononuclear cells from peripheral blood or bone marrow of PV patients (n=3) were cultured on methylcellulose supplied with hematopoietic growth factors except EPO, in the presence or not of metfomin (2.5 mM) and/or ruxolitnib (50 nM). Gene and protein expressions were evaluated by qPCR and Western blot. Statistical analyses were performed by ANOVA test or Student's t-test. Results: In HEL, SET2 and Ba/F3 JAK2^V617F cells, metformin or ruxoltinib treatment significantly reduced cell viability and clonogenicity (p<0.05). Ruxolitinib plus metformin significantly decreased cell viability and induced apoptosis when compared with monotherapy (p<0.05). Metformin and/or ruxolitinib delay cell cycle progression (p<0.05). In HEL cells, PCR-array identified 11, 14 and 17 genes modulated by metformin, ruxolitinib, and combined treatment, respectively. Reduced cyclin D1 (CCND1) and upregulated p27 (CDKN1B) were validated in HEL and SET2 cells (p<0.05). We also observed downregulation of retinoblastoma (RB) phosphorylation, a cyclin D1 target and a key cell cycle progression-related protein, in both JAK2^V617F cell lines upon all treatment conditions. Ruxolitinib treatment reduced STAT3, STAT5, ERK1/2, 4EBP1 and P70S6K phosphorylation. Metformin also reduced activation of same proteins, but combined treatment presented more intense inhibition of ERK1/2 and P70S6K activation compared to metformin or ruxolitinib monotherapy. A strong reduction of cellular oxygen consumption supported by exogenous substrates in culture media (ROUTINE state), at the non-phosphorylating state (LEAK) and at the maximum respiratory capacity (ETS state) was observed in metformin- and metformin plus ruxolitinib-treated HEL, SET2 and Ba/F3 JAK2^V617F cells (p<0.05). Ruxolitinib significantly reduced oxygen consumption at routine, leak and ETS states only in SET2 and Ba/F3 JAK2^V617F cells and at LEAK state in HEL cells. In primary cells from PV patients, a reduction of 71±21%, 94±4%, 98±1% was observed on spontaneous erythroid colony formation upon metformin, ruxolitinib and metformin plus ruxolitinib treatment, respectively. Conclusion: Metformin exerts an anticancer activity and downregulates JAK2/STAT and MAPK signaling in JAK2^V617F cells. PCR-array identified cyclin D1 and p27 as target of metformin and ruxolitinib, corroborating cell cycle and proliferation findings. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action on JAK2^V617F aberrant signaling and provides insights for development of alternative/complementary therapeutic strategies for MPN.