Metformin Suppress Cellular and Molecular Processes Related to Maintenance and Proliferation of Myeloproliferative Neoplasm Stem Cell

Background: Preclinical rationale of metformin treatment for myeloproliferative neoplasms (MPN) has recently been demonstrated (Cell Death Dis. 2018;9(3):311). Current MPN treatment options are mainly directed to symptoms and have not greatly improve clinical outcomes. It is a consensus that potential curative approaches should affects MPN stem cell function. Aims: To interrogate the metformin action in MPN stem cell function according to frequency and ex vivo differentiation capacity, as well as to underlying molecular mechanisms related to the treatment. Material and Methods: For induction of PV phenotype, 5×106 total bone marrow cells from Jak2V617F conditional mice (Jak2WT/V617F-CD45.2) were transplanted into lethally irradiated) Pep/boy mice (CD45.1-700cGy X-Ray). After 4 weeks, peripheral blood chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry and mice were randomized for treatment by intraperitoneal injection of vehicle (PBS, n=6) or metformin (125 mg/kg/day, n=4) for 6 weeks according to chimerism and body weight. Erythroid progenitors (CD71 and Ter119) were evaluated in bone marrow (BM) and spleen. Hematopoietic stem and progenitor cells (LSK: Lin-Sca-1+C-KitHi/long-term HSC[LT-HSC]:LSKCD48-CD150+/ short-term HSC [ST-HSC]:LSKCD48+CD150+/multipotent progenitor [MPP]: LSKCD48+CD150-), myeloid progenitors (LK:Lin-Sca-1+C-KitHi/myelo-erythroid progenitor [MEP]: LKCD34-CD16/32-/common myeloid progenitors [CMP]: LKCD34+CD16/32-/granulocytic progenitors [GMP]: LKCD34+CD16/32+) were evaluated in BM by flow cytometry. BM stroma was evaluated according to frequencies of macrophages (F4/80+CD11b+), inflammatory macrophages (M1) (F4/80+CD11b+CD11c+), arterioles (Cd45/Ter119-CD31+Sca-1+), osteoblasts (Cd45/Ter119-CD51+Sca-1-) and mesenchymal stem cell (Cd45/Ter119-CD51+Sca-1+). BM c-Kit cells were enriched using AutoMacs separator with anti-CD117 magnetics beads (Miltenyi) and used for RNA extraction and PCR array for cell cycle and apoptosis (PAMM-020Z and PAMM-012Z, respectively-Qiagen). Genes with fold-change ≥2 in both directions were selected. All experiments were approved by the Animal Ethics Committee. For comparisons, unpaired T-test or Mann-Whitney test were used as appropriated. All P-values were two-sided with a significance level of 0.05. Results: At randomization, mice of both groups were similar according PB chimerism, body weight and blood counts (all P>0.05). After 6 weeks of metformin treatment mice had