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-Kit^Hi/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-Kit^Hi/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 no evidence of hematological toxicity or weight loss. Metformin treated mice presented lower platelets counts in PB (mean for vehicle vs. metformin: 4370×10³/uL vs. 3451×10³/uL; P=0.04) and reduction of splenomegaly (P=0.02). Metformin reduced CD45.2 chimerism (92.7% vs. 88.9%; P=0.03), and showed a trend in reducing pro-erythroblasts (0.91% vs. 0.53%; P=0.06) and early erythroid progenitors (37.4% vs. 32.4%; P=0.11), while increased late erythroid progenitors (16.2% vs. 27.2%; P=0.008) in spleen. Metformin significantly increased ST-HSC frequency (0.06% vs. 0.29%; P=0.001) compared to vehicle. Conversely, bone marrow mononuclear cells from metformin treated mice showed decreased clonogenic capacity (2.5 colonies/1000 BMMC vs. 8.6 colonies/1000 BMMC; P=0.001). Competitive secondary transplant into lethally irradiated mice was performed and results will be show at meeting. Metformin treatment did not modulate the frequency of erythroid progenitors, LT-HSC, MPP, MEP, GMP and CMP in BM nor of the stromal cells evaluated. In c-Kit enriched cells, PCR-array identified 31 downregulated and 64 upregulated genes by metformin treatment. Metformin reduced expression of anti-apoptotic genes (Bcl2 [Fold-change: 0.33] and Xiap [0.48]), cell cycle regulators (Cdkn2a [0.21], Ccnd1 [0.27], Ccn1a [0.42]), while increased expression of TP53 family members (Trp63 [4.1] and Trp53 [2.1]), pro-apoptotic (Bid [2.5] and Bcl10 [1.9]) and cellular-stress response genes (Atm [66.9] and Cdkn2a [3.1]). Conclusions: In a conditional Jak2V617F-induced MPN murine model, daily treatment with metformin reduced tumor burden, extramedullary hematopoiesis, and clonogenic capacity of HSPC by acting on molecular processes related to maintenance and proliferation of MPN stem cell.