Advanced search
Start date
Betweenand
Related content


Investigation of the antineoplastic effects of different classes of IGF1R-IRS1/2 inhibitors in myeloproliferative neoplasm

Full text
Author(s):
Jaqueline Cristina Fernandes
Total Authors: 1
Document type: Doctoral Thesis
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Fabíola Traina; Fernando Chahud; Mariana Lazarini; Fernanda Maris Peria
Advisor: Fabíola Traina; João Agostinho Machado Neto
Abstract

The IGF1R-IRS1/2 signaling pathway is a potential target in myeloproliferative neoplasms (MPN). Insulin receptor substrate 2 (IRS2) silencing reduced viability and increased apoptosis in JAK2V617F cells. The Igf1r haploinsufficiency proved a remarkable reversal of the malignant phenotype in a knockin murine model of JAK2V617F. NT157, OSI-906 and NVP-AEW54 are inhibitors of the IGF1R-IRS1/2 pathway with antineoplastic effect in a variety of tumor cell lines, however the best pharmacological strategy for MPN and the molecular consequences of these inhibitors, remains undetermined. The aim of this work was to investigate the antineoplastic potential of different classes of IGF1R-IRS1/2 pathway inhibitors in JAK2V617F myeloproliferative neoplasms. Ba/F3 JAK2WT and Ba/F3 JAK2V617F cells were subjected to pharmacological inhibition of IGF1R using NT157 (0.2, 0.4, 0.8, 1.6 and 3.2 &mu;M), OSI-906 (2.5, 5, 10, 20 and 40 &mu;M) and NVP-AEW541 (2, 4, 6, 8 and 10 &mu;M) and cell viability (MTT), apoptosis (annexin V/PI and caspase 3), cell proliferation (Ki-67 ) and protein expression/activation (Western blot) were evaluated. NT157 and OSI-906 were tested in a knockin murine model of JAK2V617F MPN in vivo and ex vivo and in samples from patients with primary myelofibrosis. Statistical analysis was performed using Student\'s t-test or Mann-Whitney test. In Ba/F3 JAK2V617F cells, NT157 at a dose &ge;0.4 &mu;M, significantly reduced cell viability and proliferation and induced apoptosis at all treatment times. OSI-906 reduced the viability of Ba/F3 JAK2V617F cells from the dose of 10 &mu;M in 24 and 48 hours; and induced apoptosis only at the highest dose (40 &mu;M). NVP-AEW54 reduced viability and increased apoptosis from the dose of 2 &mu;M at all treatment times (p <0.05). Similar results were found in caspase 3 activation in Ba/F3 JAK2V617F cells by western blotting. In Ba/F3 JAK2WT cells, with the presence of Wehi-3B conditioned medium, all three inhibitors affected growth and induced apoptosis in ways like the mutated cell. NT157, OSI-906 and NVP-AEW54 reduced phosphorylation/activation of Irs1/2, Igf1r, Jak2, Stat3, Stat5 and P70s6k in Ba/F3 cells JAK2V617F; NT157 increased phosphorylation of Erk1/2 and did not modulate Akt1/2/3; NVP-AEW541 has not modulated Erk1/2. OSI-906 induced autophagy from the dose of 5uM in Ba/F3 cells JAK2V617F. NT157 (6.4&mu;M), OSI-906 (&ge;20 &mu;M) and NVP-AEW54 (&ge;6 &mu;M) ex vivo reduced the viability of murine cells expressing the mutated (V617F) or wild form of the JAK2 protein. NT157 and OSI-906 reduced colony formation and inhibited phosphorylation of Igf1r and Stat3; NT157 increased the phosphorylation of Akt1/2/3 and Erk1/2 in murine cells expressing the mutated form (V617F) of the JAK2 protein. NT157 did not modulate the hematimetric parameters or the spleen size of the knockin murine model of JAK2V617F MPN. Treatment with OSI-906 in the knockin murine model of JAK2V617F MPN resulted in a reduction in erythroid hematopoietic progenitors and an increase in bone marrow myeloid progenitors but did not modulate hematimetric parameters. OSI-906 reduced tumor growth in the murine model induced by Ba/F3 cells JAK2V617F. NT157 and OSI-906 reduced colony formation in peripheral blood samples from patients with myelofibrosis. In conclusion, OSI-906, NVP-AEW54 and NT157 have similar antineoplastic effects in the JAK2V617F NMP model, however variation of cellular and molecular effects were observed according to the inhibitor tested. The results of preclinical studies with the tested inhibitors reinforce the implication of the IGF1R/IRS pathway in the pathogenesis of JAK2V617F MPN while indicating the multi-target effect of the drugs. (AU)

FAPESP's process: 16/14049-0 - Investigation of mutations in IGF1R/IRS pathway in Myeloproliferative Neoplasm and the effect of pharmacological inhibitors of this signaling pathway in JAK2V617F knockin murine model
Grantee:Jaqueline Cristina Fernandes
Support Opportunities: Scholarships in Brazil - Doctorate