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Introduction:Pharmacological inhibition of insulin receptor substrate 1 (IRS1) protein has been demonstrated to promote antineoplastic effects in hematological disorders, including myeloproliferative neoplasms, chronic myeloid leukemia and acute lymphoblastic leukemia. However, the role of IRS1 in normal hematopoiesis has not yet been elucidated. In this scenario, using a murine Irs1 knockout model represents an interesting tool to evaluate the role of this gene in normal hematopoiesis. B6.129S2-Irs1smlamouse carries a spontaneous nonsense mutation in serine 57 (Irs1S57X), which in homozygosis produces a knockout animal for Irs1, characterized by a small size. Throughout the development of the study, we noticed that the successive mating between Irs1smlaheterozygous animals did not result in homozygous mice among the offspring. In view of these findings, the present study aimed to compare the hematological parameters of wild type and heterozygous mice for the Irs1S57Xmutation and to describe the fetal lethality of homozygous mice.

Introduction: The recurrent gain-of-function JAK2V617Fmutation 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 JAK2V617Fcell 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 JAK2V617Fcells and Jak2V617Fknockin-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 2×106Ba/F3 JAK2V617Fcells 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, 5×106bone marrow cells from Jak2V617Fknockin (Jak2WT/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 pvalue <0.05 was considered as statistically significant. Results: In NSG mice inoculated with Ba/F3 JAK2V617F, 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 mm3and 510.3±244.4 mm3, respectively (p<0.001). A second independent experiment including five mice in each group presented similar results (vehicle versusmetformin: 1307.7±511.9 mm3versus725.1±380.6 mm3; p<0.01). In Jak2V617Fknockin-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 JAK2V617Fallograft and splenomegaly in Jak2V617Fknockin-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.

Background:Acute lymphoblastic leukemia (ALL) is an aggressive cancer of immature progenitors that shows aberrant activation of signaling pathways. The IGF1/IGF1R signaling pathway is initiated through binding of the ligand (IGF1) to its transmembrane receptor (IGF1R), and subsequent activation of IRS1 and IRS2 proteins activating PI3K/Akt/mTOR and MAPK pathways, which are important signaling pathways reported to contributes to pathogenesis of ALL. However, the therapeutic potential of IGF1R/IRS signaling has not been previously studied in ALL cells.