Abstract
The recurrent gain-of-function JAK2V617F 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 exerts selective antineoplastic activity in hematological malignancies. In the present study, we investigate and compare effects of metformin and ruxolitinib alone and in combination on cell signaling and cellular functions in JAK2V617F-positive cells. In JAK2V617F-expressing cell lines, metformin treatment significantly reduced cell viability, cell proliferation, clonogenicity, and cellular oxygen consumption and delayed cell cycle progression. Metformin reduced cyclin D1 expression and RB, STAT3, STAT5, ERK1/2 and p70S6K phosphorylation. Metformin plus ruxolitinib demonstrated more intense reduction of cell viability and induction of apoptosis compared to monotherapy. Notably, metformin reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice and spontaneous erythroid colony formation in primary cells from polycythemia vera patients. In conclusion, metformin exerts multitarget antileukemia activity in MPN: downregulation of JAK2/STAT signaling and mitochondrial activity. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action and provides insights for development of alternative/complementary therapeutic strategies for MPN.
Highlights
Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), are characterized by excessive myeloidOfficial journal of the Cell Death Differentiation AssociationMachado-Neto et al Cell Death and Disease (2018)9:311hematocrit, reduces the spleen volume, and improves symptoms[5]
Proliferation, clonogenicity and cell cycle progression in HEL and SET2 cells To characterize the potential efficacy of metformin in human JAK2V617F-positive cells, we first investigated the effects of metformin treatment on cell viability in HEL and SET2 cells
Our results show that metformin reduces cell viability, proliferation, cell cycle progression and clonogenicity in JAK2V617F cells
Summary
Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), are characterized by excessive myeloidOfficial journal of the Cell Death Differentiation AssociationMachado-Neto et al Cell Death and Disease (2018)9:311hematocrit, reduces the spleen volume, and improves symptoms[5]. ***p < 0.0001 for metformin-treated cells vs untreated cells; ANOVA test and Bonferroni post-test, all pairs were analyzed and statistically significant differences are indicated. B Cell viability was determined by MTT assay in HEL or SET2 cells treated, or not, with the indicated concentrations of ruxolitinib and/or metformin for 48 h and normalized to corresponding untreated cells. Bar graphs represent the mean ± SD of at least four independent experiments. D Bar graphs represent the mean ± SD of at least four independent experiments quantifying apoptotic cell death. *p < 0.05 for metformin-treated and/or ruxolitinib-treated cells vs untreated cells, #p < 0.05 for metformin-treated or ruxolitinib-treated cells vs combination treatment at the corresponding doses; ANOVA test and Bonferroni post-test, all pairs were analyzed and statistically significant differences are indicated metabolic syndromes. Preclinical studies testing the combination of chemotherapeutic agents with metformin have appeared promising in the treatment of some solid tumors[11]
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