Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2021.tde-08092021-135736
Document
Author
Full name
Jaqueline Cristina Fernandes
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2021
Supervisor
Committee
Traina, Fabíola (President)
Chahud, Fernando
Lazarini, Mariana
Peria, Fernanda Maris
Title in Portuguese
Investigação do potencial antineoplásico de diferentes classes de inibidores da via IGF1R-IRS1/2 em neoplasia mieloproliferativa
Keywords in Portuguese
Neoplasia mieloproliferativa
NT157
OSI-906
Vias de sinalização
Abstract in Portuguese
A via de sinalização de IGF1R-IRS1/2 é um alvo potencial em neoplasias mieloproliferativas (NMP). O silenciamento do substrato 2 do receptor de insulina (IRS2) reduziu a viabilidade e aumentou a apoptose em células JAK2V617F. A haploinsuficiência de Igf1r provou uma reversão notável do fenótipo maligno em um modelo de camundongo knockin JAK2V617F. NT157, OSI-906 e NVP-AEW54 são inibidores da via IGF1R-IRS1/2 com efeito antineoplásico em uma variedade de linhagens tumorais, entretanto a melhor estratégia farmacológica para NMP e as consequências moleculares desses inibidores permanece indeterminada. O objetivo deste trabalho foi investigar o potencial antineoplásico de diferentes classes de inibidores da via IGF1R-IRS1/2 em neoplasia mieloproliferativa JAK2V617F. Células Ba/F3 JAK2WT e Ba/F3 JAK2V617F foram submetidas à inibição farmacológica de IGF1R usando NT157 (0,2, 0,4, 0,8, 1,6 e 3,2 μM), OSI-906 (2,5, 5, 10, 20 e 40 μM) e NVP-AEW541 (2, 4, 6, 8 e 10 μM) e a viabilidade celular (MTT), apoptose (anexina V/PI e caspase 3), proliferação celular (Ki-67) e expressão/ativação proteica (Western blot) foram avaliadas. NT157 e OSI-906 foram testados em modelo murino de NMP knockin para JAK2V617F in vivo e ex vivo e em amostras de pacientes com mielofibrose primária. A análise estatística foi realizada pelo teste t de Student ou teste de Mann-Whitney. Em células Ba/F3 JAK2V617F, NT157 na dose ≥0,4 μM, reduziu significativamente a viabilidade e proliferação celular e induziu apoptose em todos os tempos de tratamento. OSI-906 reduziu a viabilidade de células Ba/F3 JAK2V617F a partir da dose de 10 μM em 24 e 48 horas; e induziu a apoptose apenas na dose mais alta (40 μM). NVP-AEW54 reduziu a viabilidade e aumentou a apoptose a partir da dose de 2 μM em todos os tempos de tratamento (p<0,05). Resultados semelhantes foram encontrados na ativação da caspase 3 em células Ba/F3 JAK2V617F por western blotting. Em células Ba/F3 JAK2WT, com a presença de meio condicionado com IL3, todos os três inibidores afetaram o crescimento e induziram apoptose de maneira semelhante a célula mutada. NT157, OSI-906 e NVP-AEW54 reduziram a fosforilação/ativação de Irs1/2, Igf1r, Jak2, Stat3, Stat5 e P70s6k em células Ba/F3 JAK2V617F; NT-157 aumentou a fosforilação de Erk1/2 e não modulou Akt1/2/3; NVP-AEW541 não modulou Erk1/2. OSI-906 induziu autofagia a partir da dose de 5uM em células Ba/F3 JAK2V617F. NT157 (6,4μM), OSI-906 (≥20 μM) e NVP-AEW54 (≥6 μM) ex vivo reduziram a viabilidade de células de camundongo expressando a forma mutada (V617F) ou selvagem da proteína JAK2. NT157 e OSI-906 reduziram a formação de colônia e inibiram a fosforilação de Igf1r e Stat3; NT157 aumentou a fosforilação de Akt1/2/3 e Erk1/2 em células de camundongo expressando a forma mutada (V617F) da proteína JAK2. NT157 não modulou os parâmetros hematimétricos e o tamanho do baço do modelo murino de NMP JAK2 V617F knockin. O tratamento com OSI-906 no modelo murino de NMP knockin para JAK2V617F resultou em redução dos progenitores hematopoéticos eritroides e aumento dos progenitores mieloides da medula óssea, mas não modulou os parâmetros hematimétricos. OSI-906 reduziu o crescimento tumoral no modelo de camundongo induzido por células Ba/F3 JAK2V617F. NT157 e OSI-906 reduziram a formação de colônia em amostras de sangue periférico de pacientes com mielofibrose. Em conclusão, OSI-906, NVP-AEW54 e NT157 apresentam efeitos antineoplásicos semelhantes em modelo de NMP JAK2V617F, porém foi observado variação dos efeitos celulares e moleculares de acordo com o inibidor testado. Os resultados dos estudos pré-clínicos com os inibidores testados reforçam a implicação da via IGF1R/IRS na patogênese da NMP JAK2V617F ao mesmo tempo que indicam o efeito multialvo das drogas.
Title in English
Investigation of the antineoplastic effects of different classes of IGF1R-IRS1/2 inhibitors in myeloproliferative neoplasm
Keywords in English
Myeloproliferative neoplasia
NT157
OSI-906
Signaling pathways
Abstract in English
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 μM), OSI-906 (2.5, 5, 10, 20 and 40 μM) and NVP-AEW541 (2, 4, 6, 8 and 10 μM) 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 ≥0.4 μM, 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 μM in 24 and 48 hours; and induced apoptosis only at the highest dose (40 μM). NVP-AEW54 reduced viability and increased apoptosis from the dose of 2 μM 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 (6.4μM), OSI-906 (≥20 μM) and NVP-AEW54 (≥6 μM) 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.
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Publishing Date
2021-10-01