Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

BACKGROUND: Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. AIMS: We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. RESULTS: In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40 mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. CONCLUSIONS: Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

NT157, an IGF1R-IRS1/2 inhibitor, exhibits antineoplastic effects in pre-clinical models of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

IRS1/ß-Catenin Axis Is Activated and Induces MYC Expression in Acute Lymphoblastic Leukemia Cells.

Insulin-like growth factor 1 (IGF1) and its receptor IGF1R regulate normal cell growth and contribute to cell transformation through activation of downstream signaling pathways. In fibroblast cells, insulin receptor substrate 1 (IRS1), through IGF1 signaling, was found to be the key protein for nuclear translocation of ß-catenin and MYC transcription activation. We herein investigated the IRS1/ß-catenin axis in acute lymphoblastic leukemia (ALL) cells. Samples were obtained from 45 patients with ALL and 13 healthy donors. ALL cell lines were used. Gene expression was measured by quantitative PCR. Protein expression, associations, and cellular localization were evaluated by immunoprecipitation, subcellular fractionation, and confocal microscopy. Cells were submitted to IGF1 stimulation and/or IGF1R pharmacological inhibition (OSI-906). IRS1, ß-catenin, and MYC mRNA expression were significantly elevated in ALL patients, compared to normal controls. MYC mRNA expression positively correlated with ß-catenin and IRS1. Increased age and MYC expression negatively affected overall survival by univariate analysis. Total and phospho-IGF1R and IRS1, MYC and ß-catenin protein expression were higher in ALL cells, compared to normal peripheral blood mononuclear cells (PBMC). IRS1 and ß-catenin were found to be colocalized in the nuclei and the cytoplasm of ALL cell lines, whereas both proteins were only slightly detected in the cytoplasm of normal PBMC. In Jurkat cells, a constitutive IRS1 and ß-catenin protein interaction were observed; OSI-906 treatment decreased IGF1R tyrosine phosphorylation, IRS1 expression and phosphorylation, nuclear translocation of ß-catenin, IRS1 and ß-catenin association, and MYC protein expression. In conclusion, the IRS1/ß-catenin axis is activated in ALL cells. J. Cell. Biochem. 118: 1774-1781, 2017. © 2016 Wiley Periodicals, Inc.

Differential cytotoxic activity of pharmacological inhibitors of IGF1R-related pathways in JAK2V617F driven cells.

Myeloproliferative neoplasms (MPN) belong to a group of clonal diseases of hematopoietic stem cells characterized by aberrant proliferation of mature myeloid lineages. The constitutive activation of the JAK2/STAT signaling pathway is now well established to play a central role in MPN pathogenesis; however, accumulating evidence now indicates that the IGF1R-mediated signaling pathway contributes to the maintenance of the malignant phenotype. Studies using inhibitors of IGF1-mediated signaling have reported cytotoxic effects in cellular and murine models of MPN, but no consensus has been reached regarding the potency and efficacy of inhibitors of the IGF1R-related pathway in this context. In the present study, we compared the potency and efficacy of three inhibitors of IGF1R-related pathways in a JAK2V617F-driven cellular model. These inhibitors (NT157, OSI-906, and NVP-AEW54) present antineoplastic activity with similar efficacy in Ba/F3 JAK2V617F cells, with NT157 showing the greatest potency. Both the induction of apoptosis and reduction in cell proliferation were associated with the observed reduction in cell viability. Downregulation of JAK2/STAT signaling was an advantageous off-target effect of all three inhibitors. These preclinical studies reinforce the potential of the IGF1R-related pathway as a therapeutic target in MPN.

NT157 has antineoplastic effects and inhibits IRS1/2 and STAT3/5 in JAK2V617F-positive myeloproliferative neoplasm cells.

Recent data indicate that IGF1R/IRS signaling is a potential therapeutic target in BCR-ABL1-negative myeloproliferative neoplasms (MPN); in this pathway, IRS2 is involved in the malignant transformation induced by JAK2V617F, and upregulation of IGF1R signaling induces the MPN phenotype. NT157, a synthetic compound designed as an IGF1R-IRS1/2 inhibitor, has been shown to induce antineoplastic effects in solid tumors. Herein, we aimed to characterize the molecular and cellular effects of NT157 in JAK2V617F-positive MPN cell lines (HEL and SET2) and primary patient hematopoietic cells. In JAK2V617F cell lines, NT157 decreased cell viability, clonogenicity, and cell proliferation, resulting in increases in apoptosis and cell cycle arrest in the G2/M phase (p < 0.05). NT157 treatment inhibited IRS1/2, JAK2/STAT, and NFκB signaling, and it activated the AP-1 complex, downregulated four oncogenes (CCND1, MYB, WT1, and NFKB1), and upregulated three apoptotic-related genes (CDKN1A, FOS, and JUN) (p < 0.05). NT157 induced genotoxic stress in a JAK2/STAT-independent manner. NT157 inhibited erythropoietin-independent colony formation in cells from polycythemia vera patients (p < 0.05). These findings further elucidate the mechanism of NT157 action in a MPN context and suggest that targeting IRS1/2 proteins may represent a promising therapeutic strategy for MPN.

IGF1R/IRS1 targeting has cytotoxic activity and inhibits PI3K/AKT/mTOR and MAPK signaling in acute lymphoblastic leukemia cells.

The IGF1R/IRS1 signaling is activated in acute lymphoblastic leukemia (ALL) and can be targeted by the pharmacological inhibitors NT157 (IGF1R-IRS1/2 inhibitor) and OSI-906 (IGF1R/IR inhibitor). Here we investigate the cellular and molecular effects of NT157 and OSI-906 in ALL cells. NT157 and OSI-906 treatment reduced viability, proliferation and cell cycle progression in ALL cell lines. Similarly, in primary samples of patients with ALL, both OSI-906 and NT157 reduced viability, but only NT157 induced apoptosis. NT157 and OSI-906 did not show cytotoxicity in primary samples from healthy donor. NT157 and OSI-906 significantly decreased Jurkat cell migration, but did not modulate Namalwa migration. Consistent with the more potent effect of NT157 on cells, NT157 significantly modulated expression of 25 genes related to the MAPK signaling pathway in Jurkat cells, including oncogenes and tumor suppressor genes. Both compounds inhibited mTOR and p70S6K activity, but only NT157 inhibited AKT and 4-EBP1 activation. In summary, in ALL cells, NT157 has cytotoxic activity, whereas OSI-906 is cytostatic. NT157 has a stronger effect on ALL cells, and thus the direct inhibition of IRS1 may be a potential therapeutic target in ALL.

Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis.

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic proteins that integrate and coordinate the transmission of signals from the extracellular to the intracellular environment via transmembrane receptors, thus regulating cell growth, metabolism, survival and proliferation. The PI3K/AKT/mTOR and MAPK signaling pathways are the best-characterized downstream signaling pathways activated by IRS signaling (canonical pathways). However, novel signaling axes involving IRS proteins (noncanonical pathways) have recently been identified in solid tumor and hematologic neoplasm models. Insulin receptor substrate-1 (IRS1) and insulin receptor substrate-2 (IRS2) are the best-characterized IRS proteins in hematologic-related processes. IRS2 binds to important cellular receptors involved in normal hematopoiesis (EPOR, MPL and IGF1R). Moreover, the identification of IRS1/ABL1 and IRS2/JAK2V617F interactions and their functional consequences has opened a new frontier for investigating the roles of the IRS protein family in malignant hematopoiesis. Insulin receptor substrate-4 (IRS4) is absent in normal hematopoietic tissues but may be expressed under abnormal conditions. Moreover, insulin receptor substrate-5 (DOK4) and insulin receptor substrate-6 (DOK5) are linked to lymphocyte regulation. An improved understanding of the signaling pathways mediated by IRS proteins in hematopoiesis-related processes, along with the increased development of agonists and antagonists of these signaling axes, may generate new therapeutic approaches for hematological diseases. The scope of this review is to recapitulate and review the evidence for the functions of IRS proteins in normal and malignant hematopoiesis.

Metformin exerts multitarget antileukemia activity in JAK2V617F-positive myeloproliferative neoplasms.

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.

Paclitaxel induces Stathmin 1 phosphorylation, microtubule stability and apoptosis in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by abnormal proliferation and accumulation of lymphoblasts in the hematopoietic system. Stathmin 1 is a proliferation marker for normal lymphocytes, which has been described as highly expressed in ALL patients and functionally important for leukemia phenotype. In the present study, we expand our previous observations and aim to investigate Stathmin 1 expression and its impact on laboratory features and clinical outcomes in an independent cohort of ALL patients, and to verify the effects of paclitaxel treatment on Stathmin 1 phosphorylation and cell viability in ALL cell lines. In ALL patients, Stathmin 1 expression was significantly increased, associated with lower age onset and positively correlated with white blood cell counts, but did not impact on clinical outcomes. Functional assays revealed that paclitaxel induces Stathmin 1 phosphorylation at serine 16 (an inhibitory site), microtubule stability and apoptosis in Jurkat and Namalwa cell lines. Paclitaxel treatment did not modulate cell viability of normal peripheral blood leukocytes. In conclusion, our data confirm increased levels of Stathmin 1 in ALL patients and that therapeutic doses of paclitaxel inhibits Stathmin 1 function and promote microtubule stability and apoptosis in ALL cells.

Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic proteins that integrate and coordinate the transmission of signals from the extracellular to the intracellular environment via transmembrane receptors, thus regulating cell growth, metabolism, survival and proliferation. The PI3K/AKT/mTOR and MAPK signaling pathways are the best-characterized downstream signaling pathways activated by IRS signaling (canonical pathways). However, novel signaling axes involving IRS proteins (noncanonical pathways) have recently been identified in solid tumor and hematologic neoplasm models. Insulin receptor substrate-1 (IRS1) and insulin receptor substrate-2 (IRS2) are the best-characterized IRS proteins in hematologic-related processes. IRS2 binds to important cellular receptors involved in normal hematopoiesis (EPOR, MPL and IGF1R). Moreover, the identification of IRS1/ABL1 and IRS2/JAK2V617F interactions and their functional consequences has opened a new frontier for investigating the roles of the IRS protein family in malignant hematopoiesis. Insulin receptor substrate-4 (IRS4) is absent in normal hematopoietic tissues but may be expressed under abnormal conditions. Moreover, insulin receptor substrate-5 (DOK4) and insulin receptor substrate-6 (DOK5) are linked to lymphocyte regulation. An improved understanding of the signaling pathways mediated by IRS proteins in hematopoiesis-related processes, along with the increased development of agonists and antagonists of these signaling axes, may generate new therapeutic approaches for hematological diseases. The scope of this review is to recapitulate and review the evidence for the functions of IRS proteins in normal and malignant hematopoiesis.