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

Irs2 Silencing Increases Apoptosis And Potentiates The Effects Of Ruxolitinib In Jak2v617f-positive Myeloproliferative Neoplasms.

The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2V617F HEL cells, but not in the JAK2WT U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2V617F-positive but not JAK2WT specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34+ cells from essential thrombocythemia patients compared to healthy donors, and in JAK2V617F MPN patients when compared to JAK2WT. Our data indicate that IRS2 is a binding partner of JAK2V617F in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN.

Investigation of the effects of IGF1R-IRS1/2 pharmacological inhibition on the phenotype of BCR-ABL1+ leukemic cells

Leucemia mieloide crônica (LMC) é uma neoplasia hematológica maligna associada à atividade tirosinoquinase da oncoproteína BCR-ABL1. A maioria dos casos de LMC é tratada com sucesso com inibidores tirosinoquinase de BCR-ABL1, mas uma porcentagem significativa de pacientes desenvolve resistência ao fármaco. Estudos recentes indicam que a célula-tronco leucêmica é resistente ao tratamento com imatinibe. A identificação de outras proteínas que cooperam com as vias de sinalização BCR-ABL1 podem indicar novos alvos terapêuticos. Os substratos do receptor de insulina (IRS) têm emergido como proteínas importantes na fisiopatologia de neoplasias sólidas e hematológicas. Um inibidor farmacológico de IGF1R-IRS1/2, NT157, foi desenvolvido e mostrou resultados promissores em estudos pré-clínicos com tumores sólidos. A associação constitutiva de IRS1 com BCRABL1 e o efeito antineoplásico resultante do silenciamento espefício de IRS1 em células K562 BCR-ABL1+ suportam a hipótese deste trabalho. O objetivo do presente estudo foi investigar o efeito da inibição farmacológica de IGF1R-IRS1/2 no fenótipo de células hematopoéticas leucêmicas BCR-ABL1+ utilizando células primárias, células K562 e modelos murinos. IRS1, mas não IRS2, apresentou-se menos expresso em amostras de medula óssea de pacientes com diagnóstico de LMC quando comparadas às amostras de células hematopoéticas normais (p<0,0001). NT157 reduziu a formação de colônias de células primárias de pacientes com LMC, mas não de células hematopoéticas de indivíduos saudáveis. Em células K562, o tratamento com o inibidor farmacológico de IGF1R-IRS1/2, NT157, reduziu a viabilidade e proliferação celular e induziu apoptose (p<0,05), inibiu a fosforilação de IGF1R, STAT3, STAT5, 4EBP1, P70S6K e ERK1/2, aumentou a expressão dos genes supressores de tumor CDKN1A, FOS e JUN e reduziu a expressão dos oncogenes MYC e BCL2 (p<0,05); a inibição específica de IRS1 através de lentivírus, mas não de IRS2, reduziu a viabilidade celular (p<0,05). Em células murinas Ba/F3 BCR-ABL1 e BCRABL1T315I, o inibidor farmacológico de IGF1R-IRS1/2, NT157, induziu apoptose e reduziu ativação de ERK1/2 in vitro. Na dose de 50mg/kg/dia, NT157 intraperitoneal e/ou imatinibe por gavagem falharam em reduzir o crescimento tumoral in vivo em modelo de tumor alográfico induzido por células Ba/F3 BCR-ABL1 e BCR-ABL1T315I. Em modelo animal de leucemia induzido por transplante de células hematopoéticas transduzidas com BCR-ABL1: (i) o tratamento com NT157 100mg/kg intraperitoneal, 3 vezes por semana, combinado com imatinibe 100mg/kg/dia por gavagem, reduziu significativamente o peso do baço, e preveniu a perda de peso comparado com veículo (p<0,05); apenas a monoterapia com imatinibe prolongou a sobrevida dos animais, (ii) o tratamento com NT157 70mg/kg intraperitoneal, 3 vezes por semana, e/ou imatinibe 70mg/kg/dia por gavagem não teve impacto no peso do baço e na variação do peso corporal; o tratamento com imatinibe em monoterapia ou combinado com NT157 prolongou a sobrevida dos animais (p<0,05). Em conclusão, o inibidor farmacológico de IGF1R-IRS1/2 representa uma droga potencialmente eficaz no tratamento da LMC, especialmente em casos de resistência com mutação BCR-ABL1 T315I. A avaliação da eficácia do inibidor farmacológico NT157 em modelos animais de LMC exige ajustes nos modelos murinos utilizados no presente estudo, melhor entendimento da farmacodinâmica e farmacocinética do composto e ajustes no esquema terapêutico utilizado.Chronic myeloid leukemia (CML) is a hematological malignancy associated with the tyrosine kinase activity of the BCR-ABL1 oncoprotein. Most cases of CML are successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Recent studies indicate that leukemic stem cell is resistant to imatinib treatment. The identification of other proteins that cooperate with the BCR-ABL1 signaling pathway may indicate novel therapeutic targets. Insulin receptor substrates (IRS) have emerged as important proteins in the pathophysiology of solid and hematological neoplasms. A pharmacological inhibitor of IGF1R-IRS1/2, NT157, has been developed and shown promising results in preclinical studies with solid tumors. The constitutive association of IRS1 with BCR-ABL1, and the antineoplastic effects resulting from IRS1-specific silencing in K562 BCR-ABL1+ cells, support the hypothesis of this work. The aim of the present study was to investigate the effect of pharmacological inhibition of IGF1R-IRS1/2 on the phenotype of BCR-ABL1+ leukemia cells, using primary cells, K562 cell line and murine models. IRS1, but not IRS2, was downregulated in bone marrow samples from CML patients compared to bone marrow cells from healthy donors (p<0.0001). NT157 reduced colony formation of primary cells from CML patients but not from healthy donors. In K562 cells, treatment with the pharmacological inhibitor IGF1R-IRS1/2, NT157, reduced cell viability and proliferation, induced apoptosis (p<0.05), inhibited the phosphorylation of IGF1R, STAT3, STAT5, 4EBP1, P70S6K and ERK1/2, increased expression of tumor suppressor genes CDKN1A, FOS and JUN, and reduced expression of oncogenes MYC and BCL2 (p<0.05); IRS1 silencing mediated by lentivirus, but not IRS2, reduced cell viability (p<0.05). In murine Ba/F3 BCRABL1 and Ba/F3 BCR-ABL1T315I cells, the pharmacological inhibitor of IGF1R-IRS1/2, NT157, induced apoptosis and reduced ERK1/2 activation in vitro. At 50mg/kg/day, NT157 intraperitoneally and/or imatinib orally, failed to reduce tumor burden in vivo in an allographic tumor model induced by Ba/F3 BCR-ABL1 and Ba/F3 BCR-ABL1T315I cells . In an animal leukemia model induced by transplantation of BCR-ABL1-transduced hematopoietic cells : (i) treatment with NT157 100mg/kg intraperitoneally, 3 times per week, combined with imatinib 100mg/kg/day per gavage, significantly reduced spleen weight, and prevented weight loss compared to vehicle (p<0.05); only imatinib monotherapy prolonged survival (p<0.05), (ii) treatment with NT157 70 mg/kg intraperitoneally, 3 times per week, and/or imatinib 70 mg/kg/day per gavage had no impact on spleen weight and body weight; treatment with imatinib alone or combined with NT157 prolonged survival (p<0.05). In conclusion, the pharmacological inhibitor of IGF1R-IRS1/2 represents a potential effective drug in CML therapy, especially in cases of resistant BCR-ABL1 T315I mutation. The evaluation of the NT157 pharmacological efficacy in CML animal models requires adjustments in the murine models used in the present study, better understanding of the pharmacodynamics and pharmacokinetics of the compound and adjustments in the therapeutic scheme used

Investigation of TET2 and DNMT3A expression in myelodysplastic syndrome and acute myeloid leukemia

Orientador: Fabíola TrainaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: As neoplasias mieloides compreendem um grupo heterogêneo de doenças hematológicas que se originam de um precursor mieloide comum, em diferentes fases de diferenciação. As alterações celulares que levam ao desenvolvimento de neoplasias podem ocorrer através de mecanismos epigenéticos ou de alterações genéticas. DNMT3A codifica metiltransferases que adicionam grupamentos metil a resíduos de citosina do DNA e TET2 promove a hidroxilação da citosina metilada, o que os caracteriza como elementos importantes no controle epigenético. DNMT3A e TET2 encontram-se frequentemente mutados em neoplasias mieloides, mas o impacto prognóstico destas mutações ainda é controverso. A consequência funcional da mutação de DNMT3A em neoplasias mieloides ainda não foi definida, mas o silenciamento da proteína em células progenitoras murinas favorece a autorrenovação e compromete a diferenciação celular. A mutação de TET2 tem como consequência a perda de função do gene e participa da transformação neoplásica das células mieloides, favorecendo a proliferação da série mielomonocítica. Entretanto, a expressão de TET2 e DNMT3A nestas doenças ainda é pouco elucidada. Assim, os objetivos deste estudo foram (1) investigar a expressão de TET2 e DNMT3A em células hematopoéticas de indivíduos normais e pacientes com síndrome mielodisplásica (SMD) e leucemia mieloide aguda (LMA), (2) correlacionar a expressão de TET2 e DNMT3A com o fenótipo clínico e sobrevida de pacientes com SMD; (3) investigar a expressão de TET2 e DNMT3A durante a diferenciação celular hematopoética e (4) avaliar o efeito do silenciamento de DNMT3A no fenótipo de linhagens celulares leucêmicas. No presente estudo, verificamos redução na expressão de TET2 em células provenientes de pacientes com SMD e LMA quando comparada à expressão em controles normais (p<.001), e redução em SMD alto risco quando comparada à SMD baixo risco (p=.02). Os resultados em amostras sequenciais de cinco pacientes com SMD indicaram redução da expressão de TET2 no momento da progressão da doença. A análise univariada evidenciou que fatores clínicos tiveram impacto tanto na sobrevida livre de evento como sobrevida global, incluindo a classificação de risco pela OMS 2008 (alto vs. baixo, p<.0001), IPSS (int-2/alto vs. baixo/int-1, p<.0001), hemoglobina (<10 vs. ? 10, p<.05), contagem de leucócitos (< 3 vs. ? 3 x109/L, p<.05), contagem absoluta de neutrófilos (< 1.5 vs. ? 1.5, p<.05) e porcentagem de blastos na medula óssea (? 5 vs. <5 ou ? 10 vs. <10, p<.0004). Além disso, a baixa expressão de TET2 teve impacto negativo na sobrevida livre de evento (HR: 6.51 [2.42-17.49], p=.0002) e na sobrevida global (HR: 7.25 [2.77-18.99], p<.0001). A análise multivariada indicou que a baixa expressão de TET2 (p <.0001), IPSS alto/intermediate-2 (p <.0001), e hemoglobina <10 g/dL (P<.03) são fatores prognósticos para menor sobrevida livre de evento e sobrevida global. Durante a diferenciação eritroide de células CD34+ de indivíduos normais e pacientes com SMD, observamos um aumento significativo da expressão de TET2 (p=. 03). Na avaliação da diferenciação celular de linhagens leucêmicas, observamos aumento significativo na expressão de TET2 durante as diferenciações granulocítica (p=.04) e megacariocítica (p=.03); e um aumento não significativo durante a diferenciação eritrocítica. A expressão de DNMT3A foi semelhante entre pacientes com LMA, SMD e controles normais, e não teve impacto significativo na sobrevida dos pacientes com SMD. A expressão de DNMT3A não foi modulada durante a diferenciação eritroide de células CD34+ de indivíduos normais e pacientes com SMD. Nos modelos de diferenciação celular de linhagens leucêmicas, observamos aumento significativo da expressão de DNMT3A durante a diferenciação granulocítica, mas não durante a diferenciação eritrocítica e megacariocítica. A redução na expressão de DNMT3A não resultou em alteração significativa na apoptose, na proliferação e no ciclo celular em linhagens leucêmicas HL60 e U937. A expressão gênica e proteica de PTEN não foi modulada em células leucêmicas submetidas à inibição de DNMT3A. Os achados aqui descritos sugerem que, similarmente à presença de mutação no TET2, a baixa expressão de TET2 pode participar do processo de transformação celular em SMD de alto risco e LMA; estudos clínicos deveriam considerar a investigação da expressão gênica de TET2 em conjunto com a pesquisa de mutação TET2 na definição de prognóstico. Os resultados de expressão e função de DNMT3A sugerem que a mutação, e não a expressão, deva ser o principal mecanismo pelo qual o DNMT3A participa da transformação neoplásica e que a função de DNMT3A pode depender da linhagem celular estudadaAbstract: Myeloid neoplasms comprise a heterogeneous group of hematologic malignancies that originate from a common myeloid precursor at different stages of differentiation. Cellular changes that lead to development of malignancies may occur through epigenetic mechanisms or genetic alterations. DNMT3A encodes methyltransferases that add methyl groups to cytosine residues in DNA, TET2 promotes hydroxylation of methylated cytosine, and both proteins are important elements in epigenetic control. TET2 and DNMT3A are recurrently mutated in myeloid malignancies, but the prognostic consequence of TET2 and DNMT3A mutation is still controversial. The functional consequences of DNMT3A mutation has not been defined, but the protein silencing in murine progenitor cells promotes self-renewal and reduces cell differentiation. TET2 mutation results in loss of function and participates in the neoplastic transformation of myeloid cells, favoring the proliferation of granulomonocytic cells. However, the expression of TET2 and DNMT3A in these diseases has been rarely addressed. Then, the aims of this study were (1) to evaluate TET2 and DNMT3A gene expression in hematopoietic cells from healthy individuals and from patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); (2) to correlate TET2 and DNMT3A expression with clinical phenotype and outcomes of MDS patients; (3) to investigate TET2 and DNMT3A expression during hematopoietic cell differentiation; and (4) to evaluate the effect of DNMT3A silencing in the phenotype of leukemia cell lines. In this study, the expression of TET2 was decreased in cells from patients with MDS and AML compared to healthy donors (p<.001) and reduced high-risk MDS compared to low risk MDS (p=.02). The results in sequential samples from five patients with MDS indicate reduced expression of TET2 at the time of disease progression. By univariate analysis, clinical factors that significantly affected both event free survival (EFS) and overall survival (OS) included risk stratification by WHO 2008 (high vs. low, p<.0001), IPSS (int-2/high vs. low/int-1, p <.0001), hemoglobin (<10 vs. ? 10, p<.05), white blood cell counts (< 3 vs. ? 3 x109/L, p<.05), absolute neutrophil counts (< 1.5 vs. ? 1.5, p<.05) and bone marrow blast percentage (? 5 vs. <5 or ? 10 vs. <10, p<.0004). Furthermore, low TET2 expression negatively impacted both EFS (HR: 6.51 [2.42-17.49], p=.0002) and OS (HR: 7.25 [2.77-18.99], p<.0001). Multivariate analyses indicated that low TET2 expression (p <.0001), along with IPSS high/intermediate-2 risk group (p <.0001), and hemoglobin <10 g/dL (p<.03) were independently prognostic for worse EFS and OS. During erythroid differentiation of CD34+ cells from normal individuals and patients with low-risk MDS, we observed an increased expression of TET2 (p=.03). During cell differentiation of leukemic cell lines, we observed a significantly increase in the expression of TET2 during granulocytic and megakaryocytic differentiation (p=.04 and p=.03, respectively); there was also an increased expression during erythrocytic differentiation, but this was not statistically significant. The expression of DNMT3A was similar between patients with AML, MDS and healthy donors, and it did not impact survival outcomes in MDS patients. DNMT3A expression was not modulated during erythroid differentiation of CD34+ cells from normal individuals and patients with MDS. In leukemic cell lines models of differentiation, we observed a significantly increase in the DNMT3A expression during granulocytic differentiation, but not in erythrocytic and megakaryocytic differentiation. The DNMT3A silencing did not result in significant changes in apoptosis, proliferation and cell cycle in leukemic cell lines HL60 and U937. PTEN gene and protein expression was not modulated in leukemic cell lines submitted to inhibition of DNMT3A. The findings reported here suggest that, similarly to the presence of TET2 mutations, the low expression of TET2 can participate in the process of cell transformation in high risk MDS and AML. Clinical studies should consider the investigation of TET2 expression together with the studies of TET2 mutation to defining prognosis. Our results of expression and function suggest that DNMT3A mutation, instead of the expression, should be the main mechanism by which DNMT3A participates in neoplastic transformation and that DNMT3A function may vary according to the cell line studiedMestradoClinica MedicaMestra em Ciência

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

Increased levels of cyclin D1 negatively impacts on acute lymphoblastic leukemia overall survival

Abstract Background Cyclin D1 is a protein essential for transition from G1 to S phase during cell cycle progression, which has an oncogenic potential and is highly expressed in several human malignancies. However, in view of the heterogeneity of the findings in the literature, the prognostic value of cyclin D1 expression still needs to be validated in different cohorts of adult acute lymphoblastic leukemia (ALL) patients. Methods Bone marrow samples from 13 healthy donors and 45 adult patients with acute lymphoblastic leukemia were included. Cyclin D1 gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Fisher’s exact test, Chi-squared test and Cox regression were used, as appropriate. All p values were two-sided with a significance level of 5%. Results Cyclin D1 mRNA levels were similar between primary cells from ALL patients and healthy donors. In ALL patients, high cyclin D1 expression was associated with older age at the diagnosis, presence of BCR-ABL1, and lower white blood cell counts. Importantly, increased cyclin D1 expression was an independent factor that predicted worse overall survival in our adult ALL cohort. Conclusion Increased levels of cyclin D1 negatively impacted on ALL survival outcome, suggesting that this gene is involved in the malignant phenotype of ALL