A leucemia linfoide aguda (LLA) é uma neoplasia hematológica agressiva, caracterizada pela expansão clonal de progenitores linfoides e ativação exacerbada de vias de sinalização. A via de sinalização de IGF1R/IRS1 inicia-se pela ligação do ligante IGF1 ao seu receptor transmembrana IGF1R, e subsequente ativação de seu substrato IRS1, que transmite sinais mitogênicos e antiapoptóticos, principalmente através da modulação das vias de sinalização PI3K/AKT/mTOR e MAPK . Estas vias de sinalização desempenham uma importante função na proliferação, sobrevivência e migração de células de leucêmicas. Dessa forma, o objetivo do nosso trabalho foi investigar a participação da via de sinalização IGF1R/IRS1 na LLA. Linhagens celulares Jurkat, MOLT-4, Namalwa e Raji foram tratadas ou não com inibidor de IGF1R/IRS1-2, NT157, ou com inibidor de IGF1R/IR, OSI-906, e submetidas à avaliação da viabilidade celular, apoptose, proliferação, ciclo celular, migração e expressão/ativação gênica e proteica. Células mononucleares de pacientes com LLA e de doadores saudáveis foram submetidas aos ensaios de viabilidade e apoptose, após tratamento com NT157 e OSI-906. O efeito do NT157 in vivo foi avaliado utilizando modelo de xenotransplante de células CEM em camundongos NSG. O estudo foi aprovado pelo Comitê de Ética em Pesquisa e Comitê de Ética no Uso dos Animais da Instituição. A análise estatística foi realizada pelo teste ANOVA e teste t de Student. O tratamento com NT157 reduziu a viabilidade e a proliferação, induziu apoptose e modulou o ciclo celular em todas as linhagens testadas (p<0,05). Similarmente, OSI- 906 reduziu a viabilidade e a proliferação, modulou o ciclo celular, porém não foi capaz de induzir apoptose nas linhagens de LLA (p<0,05). Os tratamentos com NT157 e com OSI-906 diminuíram significativamente a migração de Jurkat em fibronectina, porém não modularam a migração de Namalwa. Em um contexto molecular, a exposição ao NT157 resultou em inibição da fosforilação de proteínas da via de sinalização PI3K/AKT/mTOR e modulou a expressão de 25 genes relacionados com a via de sinalização MAPK, dentre eles CDKN1A (p21), FOS e JUN (p<0,05). OSI-906modulou a ativação das proteínas da via de sinalização PI3K/AKT/mTOR e a expressão gênica de p21, FOS e JUN, porém de uma forma diferente da modulação encontrada pelo tratamento com NT157 (p<0,05). Em células mononucleares de pacientes com LLA, NT157 induziu uma resposta heterogênea na viabilidade e induziu apoptose, e OSI-906 reduziu a viabilidade, porém não foi capaz de induzir apoptose nestes pacientes (p<0,05). Os tratamentos com NT157 e OSI-906 não apresentaram citotoxicidade em células de doadores saudáveis. Adicionalmente, o tratamento in vivo com veículo ou NT157 na dose de 50mg/kg/dia, via intraperitoneal, em modelos de xenotransplante com células CEM em camundongos NSG (n=5 para cada grupo) não apresentou efeitos antineoplásicos. Em conclusão, a inibição farmacológica de IGF1R/IRS1-2, por NT157, e de IGF1R/IR, por OSI-906, apresentaram efeitos antineoplásicos significativos em modelos de linhagens celulares e amostras primárias de pacientes com LLA. Os resultados dos estudos in vivo em modelos de xenotransplante indicam a necessidade de estudos de farmacocinética e farmacodinâmica para o NT157. Nossos resultados revelaram que NT157 exerce um efeito citotóxico nas células de LLA, enquanto que OSI-906 tem um efeito predominantemente citostático. Estes dados indicam que o inibidor de IGF1R/IRS1-2, NT157, obteve resultados antineoplásicos mais atrativos e a inibição direta de IRS1 pode ser um potencial alvo terapêutico em LLA. / Acute lymphoid leukemia (ALL) is an aggressive hematological neoplasm, characterized by clonal expansion of lymphoid progenitors and exacerbated activation of signaling pathways. The IGF1R/IRS1 signaling pathway initiated through binding of the ligand IGF1 to its transmembrane receptor IGF1R, and the subsequent activation of its substrate IRS1, which transmit mitogenic and antiapoptotic signals, mainly through the modulation of the PI3K/AKT/mTOR and MAPK signaling pathways. These signaling pathways play an important function in cell proliferation, survival and migration of leukemia cells. Therefore, the objective of our study was to investigate the participation of the IGF1R/IRS1 signaling pathway in ALL. Jurkat, MOLT-4, Namalwa and Raji cell lines were treated or not with IGF1R/IRS1-2 inhibitor, NT157, or with IGF1R/IR inhibitor, OSI-906, and evaluated for cell viability, apoptosis, proliferation, cell cycle, migration, gene and protein expression/activation. Mononuclear cells from patients with ALL and from healthy donors were submitted to the viability and apoptosis assays, after treatment with NT157 and OSI-906. The NT157 effect in vivo was evaluated using CEM cell line xenograft model in NSG mice. The study was approved by the Research Ethics Committee and the Ethics Committee on the Use of Animals of the Institution. Statistical analysis was performed by ANOVA and Student t test. Treatment with NT157 reduced viability and proliferation, induced apoptosis, and modulated the cell cycle in all cell lines tested (p<0.05). Similarly, OSI-906 reduced viability and proliferation, modulated the cell cycle, but was not able to induce apoptosis in ALL cell lines (p<0.05). Treatments with NT157 and OSI-906 significantly decreased the migration of Jurkat in fibronectin, but did not modulate the Namalwa migration. In a molecular context, the NT157 exposure resulted in inhibition of the PI3K/AKT/mTOR signaling pathway protein phosphorylation and modulated the expression of 25 genes related to the MAPK signaling pathway, including CDKN1A (p21), FOS and JUN (p<0,05). OSI-906 modulated the activation of the PI3K/AKT/mTOR signaling pathway proteins and the p21, FOS and JUN geneexpression, but in a different way from the modulation found by treatment with NT157 (p<0.05). In mononuclear cells of patients with ALL, NT157 induced a heterogeneous response in viability and induced apoptosis, and OSI-906 reduced viability, but was not able to induce apoptosis in these patients (p<0.05). Treatments with NT157 and OSI- 906 did not show cytotoxicity in healthy donor cells. Additionally, in vivo treatment with vehicle or NT157 at the dose of 50 mg/kg/day, intraperitoneally, in xenotransplantation models with CEM cells in NSG mice (n=5 for each group) showed no antineoplastic effects. In conclusion, the pharmacological inhibition of IGF1R/IRS1- 2, by NT157, and IGF1R/IR, by OSI-906, showed significant antineoplastic effects in cell line models and in primary samples of patients with ALL. The results of in vivo studies in xenotransplantation models indicate the need for pharmacokinetic and pharmacodynamic studies for NT157. In conclusions, our results revealed that NT157 exerts a cytotoxic effect on ALL cell lines and primary ALL cells, whereas OSI-906 has a predominantly cytostatic effect. These data indicate that the IGF1R/IRS1-2 inhibitor, NT157, obtained more attractive antineoplastic results and the direct inhibition of IRS1 may be a potential therapeutic to target in ALL.

A leucemia linfoide aguda (LLA) é uma neoplasia hematológica agressiva, caracterizada pela expansão clonal de progenitores linfoides e ativação exacerbada de vias de sinalização. A via de sinalização de IGF1R/IRS1 inicia-se pela ligação do ligante IGF1 ao seu receptor transmembrana IGF1R, e subsequente ativação de seu substrato IRS1, que transmite sinais mitogênicos e antiapoptóticos, principalmente através da modulação das vias de sinalização PI3K/AKT/mTOR e MAPK . Estas vias de sinalização desempenham uma importante função na proliferação, sobrevivência e migração de células de leucêmicas. Dessa forma, o objetivo do nosso trabalho foi investigar a participação da via de sinalização IGF1R/IRS1 na LLA. Linhagens celulares Jurkat, MOLT-4, Namalwa e Raji foram tratadas ou não com inibidor de IGF1R/IRS1-2, NT157, ou com inibidor de IGF1R/IR, OSI-906, e submetidas à avaliação da viabilidade celular, apoptose, proliferação, ciclo celular, migração e expressão/ativação gênica e proteica. Células mononucleares de pacientes com LLA e de doadores saudáveis foram submetidas aos ensaios de viabilidade e apoptose, após tratamento com NT157 e OSI-906. O efeito do NT157 in vivo foi avaliado utilizando modelo de xenotransplante de células CEM em camundongos NSG. O estudo foi aprovado pelo Comitê de Ética em Pesquisa e Comitê de Ética no Uso dos Animais da Instituição. A análise estatística foi realizada pelo teste ANOVA e teste t de Student. O tratamento com NT157 reduziu a viabilidade e a proliferação, induziu apoptose e modulou o ciclo celular em todas as linhagens testadas (p<0,05). Similarmente, OSI- 906 reduziu a viabilidade e a proliferação, modulou o ciclo celular, porém não foi capaz de induzir apoptose nas linhagens de LLA (p<0,05). Os tratamentos com NT157 e com OSI-906 diminuíram significativamente a migração de Jurkat em fibronectina, porém não modularam a migração de Namalwa. Em um contexto molecular, a exposição ao NT157 resultou em inibição da fosforilação de proteínas da via de sinalização PI3K/AKT/mTOR e modulou a expressão de 25 genes relacionados com a via de sinalização MAPK, dentre eles CDKN1A (p21), FOS e JUN (p<0,05). OSI-906modulou a ativação das proteínas da via de sinalização PI3K/AKT/mTOR e a expressão gênica de p21, FOS e JUN, porém de uma forma diferente da modulação encontrada pelo tratamento com NT157 (p<0,05). Em células mononucleares de pacientes com LLA, NT157 induziu uma resposta heterogênea na viabilidade e induziu apoptose, e OSI-906 reduziu a viabilidade, porém não foi capaz de induzir apoptose nestes pacientes (p<0,05). Os tratamentos com NT157 e OSI-906 não apresentaram citotoxicidade em células de doadores saudáveis. Adicionalmente, o tratamento in vivo com veículo ou NT157 na dose de 50mg/kg/dia, via intraperitoneal, em modelos de xenotransplante com células CEM em camundongos NSG (n=5 para cada grupo) não apresentou efeitos antineoplásicos. Em conclusão, a inibição farmacológica de IGF1R/IRS1-2, por NT157, e de IGF1R/IR, por OSI-906, apresentaram efeitos antineoplásicos significativos em modelos de linhagens celulares e amostras primárias de pacientes com LLA. Os resultados dos estudos in vivo em modelos de xenotransplante indicam a necessidade de estudos de farmacocinética e farmacodinâmica para o NT157. Nossos resultados revelaram que NT157 exerce um efeito citotóxico nas células de LLA, enquanto que OSI-906 tem um efeito predominantemente citostático. Estes dados indicam que o inibidor de IGF1R/IRS1-2, NT157, obteve resultados antineoplásicos mais atrativos e a inibição direta de IRS1 pode ser um potencial alvo terapêutico em LLA. / Acute lymphoid leukemia (ALL) is an aggressive hematological neoplasm, characterized by clonal expansion of lymphoid progenitors and exacerbated activation of signaling pathways. The IGF1R/IRS1 signaling pathway initiated through binding of the ligand IGF1 to its transmembrane receptor IGF1R, and the subsequent activation of its substrate IRS1, which transmit mitogenic and antiapoptotic signals, mainly through the modulation of the PI3K/AKT/mTOR and MAPK signaling pathways. These signaling pathways play an important function in cell proliferation, survival and migration of leukemia cells. Therefore, the objective of our study was to investigate the participation of the IGF1R/IRS1 signaling pathway in ALL. Jurkat, MOLT-4, Namalwa and Raji cell lines were treated or not with IGF1R/IRS1-2 inhibitor, NT157, or with IGF1R/IR inhibitor, OSI-906, and evaluated for cell viability, apoptosis, proliferation, cell cycle, migration, gene and protein expression/activation. Mononuclear cells from patients with ALL and from healthy donors were submitted to the viability and apoptosis assays, after treatment with NT157 and OSI-906. The NT157 effect in vivo was evaluated using CEM cell line xenograft model in NSG mice. The study was approved by the Research Ethics Committee and the Ethics Committee on the Use of Animals of the Institution. Statistical analysis was performed by ANOVA and Student t test. Treatment with NT157 reduced viability and proliferation, induced apoptosis, and modulated the cell cycle in all cell lines tested (p<0.05). Similarly, OSI-906 reduced viability and proliferation, modulated the cell cycle, but was not able to induce apoptosis in ALL cell lines (p<0.05). Treatments with NT157 and OSI-906 significantly decreased the migration of Jurkat in fibronectin, but did not modulate the Namalwa migration. In a molecular context, the NT157 exposure resulted in inhibition of the PI3K/AKT/mTOR signaling pathway protein phosphorylation and modulated the expression of 25 genes related to the MAPK signaling pathway, including CDKN1A (p21), FOS and JUN (p<0,05). OSI-906 modulated the activation of the PI3K/AKT/mTOR signaling pathway proteins and the p21, FOS and JUN geneexpression, but in a different way from the modulation found by treatment with NT157 (p<0.05). In mononuclear cells of patients with ALL, NT157 induced a heterogeneous response in viability and induced apoptosis, and OSI-906 reduced viability, but was not able to induce apoptosis in these patients (p<0.05). Treatments with NT157 and OSI- 906 did not show cytotoxicity in healthy donor cells. Additionally, in vivo treatment with vehicle or NT157 at the dose of 50 mg/kg/day, intraperitoneally, in xenotransplantation models with CEM cells in NSG mice (n=5 for each group) showed no antineoplastic effects. In conclusion, the pharmacological inhibition of IGF1R/IRS1- 2, by NT157, and IGF1R/IR, by OSI-906, showed significant antineoplastic effects in cell line models and in primary samples of patients with ALL. The results of in vivo studies in xenotransplantation models indicate the need for pharmacokinetic and pharmacodynamic studies for NT157. In conclusions, our results revealed that NT157 exerts a cytotoxic effect on ALL cell lines and primary ALL cells, whereas OSI-906 has a predominantly cytostatic effect. These data indicate that the IGF1R/IRS1-2 inhibitor, NT157, obtained more attractive antineoplastic results and the direct inhibition of IRS1 may be a potential therapeutic to target in ALL.

Résumé : L’autophagie est un mécanisme constitutif et inductible de dégradation des composants cytoplasmiques afin de maintenir l’homéostasie cellulaire. Elle est souvent modulée par les virus car il s’agit également d’un mécanisme de défense antiviral. Elle peut avoir un rôle proviral quand elle est détournée et régulée par les virus. Nous avons précédemment observé au laboratoire que le cytomégalovirus humain (HCMV) stimule la formation des autophagosomes de manière précoce indépendamment de l’expression des protéines virales, puis qu’il entraine un blocage de l’autophagie aux temps tardifs. Dans ce travail, nous avons montré que ce virus a développé des stratégies impliquant la synthèse de deux protéines virales, IRS1 et TRS1, pour inhiber l’autophagie. De façon surprenante, nous avons également mis en évidence un rôle proviral de l’autophagie aux temps tardifs de l’infection par le HCMV. Nous avons pu montrer par des techniques de biochimie et d’imagerie cellulaire que l’expression aussi bien de TRS1 que d’IRS1 est capable de bloquer la formation des autophagosomes dans les cellules. Nous avons identifié le mécanisme d’action de ces protéines. Il est indépendant de la protéine kinase PKR mais nécessite une interaction avec Beclin 1, une protéine de la machinerie autophagique. Nous avons localisé le site d'interaction de Beclin 1 avec IRS1 et TRS1 (BBD pour Beclin 1 binding domain) au niveau de leur région N-terminale. Ce domaine, conservé entre les deux protéines, est nécessaire pour l’inhibition de l’autophagie. Le site d’interaction d’IRS1 a été identifié dans le domaine en superhélice (coiled-coil domain) CCD de Beclin 1. Nous avons caractérisé le rôle de TRS1 et IRS1 dans la modulation de l’autophagie dans le contexte de l’infection virale, en utilisant différents virus mutants : des virus dans lesquels on a supprimé soit le gène IRS1, soit le gène TRS1 et un virus dans lequel il manque les deux gènes IRS1 et TRS1. Les résultats obtenus suggèrent qu’IRS1 et TRS1 sont effectivement toutes les deux impliquées dans ce processus. Afin de mieux comprendre le rôle de l’interaction de ces protéines avec Beclin 1, nous avons étudié le phénotype d’un virus mutant qui n’exprime pas IRS1 et qui contient une délétion de la région BBD de TRS1. Nous avons montré que ce virus mutant ne se lie pas à Beclin 1 et qu’il ne bloque pas l’autophagie. De manière surprenante, il n’a pas de défaut de production virale, suggérant que l’inhibition de l'autophagie ne serait pas essentielle pour la réplication virale. Nous avons développé d’autres approches, comme l’utilisation de modulateurs pharmacologiques de l’autophagie ou de lentivirus hébergeant des shRNA, qui montrent que l’inhibition de l’autophagie est capable de diminuer la production virale et au contraire que sa stimulation l’augmente. Ces derniers résultats suggèrent que l’autophagie pourrait être bénéfique au HCMV dans certaines conditions. / Abstract: Autophagy is a constitutive and inducible mechanism of degradation of cytoplasmic components, in order to maintain the cellular homeostasis. Autophagy is often modulated by viruses, because it is also considered as an antiviral defense mechanism. It can have a beneficial role, when it is hijacked and regulated by viruses. We have previously observed in our laboratory that the human cytomegalovirus (HCMV) stimulates autophagosome formation, at the early stage of infection, independently of viral protein expression then, later on, it blocks autophagy. In this work, we showed that this virus has developed strategies involving the synthesis of several viral proteins, such as IRS1 and TRS1, to inhibit autophagy. Surprisingly, we also demonstrated a proviral role of autophagy at late stages of infection with HCMV. We showed, through biochemical and cellular imaging technologies, that expression of both TRS1 and IRS1 is able to block the formation of autophagosomes. We identified the mechanism of action of these proteins. It is independent of the protein kinase PKR but requires interaction with Beclin 1, a protein of the autophagic machinery. We mapped the interaction site of Beclin 1 with IRS1 and TRS1 in their N-terminal region and called it BBD for Beclin 1-binding domain. This domain (BBD)is conserved between the two proteins and essential to inhibit autophagy. We also identified the site of interaction of IRS1 in the coiled-coil domain (CCD) of Beclin 1. We characterized the role of IRS1 and TRS1 in the modulation of autophagy, in the context of viral infection, using different mutant viruses: viruses in which either the IRS1 or the TRS1 gene has been removed and a mutant virus lacking both IRS1 and TRS1 genes. Our results suggest that both IRS1 and TRS1 are involved in the regulation of this process. To better understand the role of the interaction of these proteins with Beclin 1, we studied the phenotype of a mutant virus that does not express IRS1 and which contains a deletion of the N-terminal region of TRS1. We showed that this mutant does not bind to Beclin 1 and is not able to block autophagy. Surprisingly, it has no defects in viral production, suggesting that inhibition of autophagy is not essential for viral replication. We developed other approaches, including the use of pharmacological modulators of autophagy or shRNA knockdown, which show that the inhibition of autophagy is able to reduce viral production and, on the contrary, that its stimulation increases it. These results suggest that autophagy may be beneficial to HCMV in certain conditions.

The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.

A leucemia linfoide aguda (LLA) compreende um grupo heterogêneo de neoplasias caracterizadas por proliferação anormal e acúmulo de células imaturas na medula óssea, o que prejudica a produção de eritrócitos, leucócitos e plaquetas. O fator de crescimento semelhante à insulina 1 (insulin-like growth factor 1; IGF1) e seu receptor (IGF1R) regulam o crescimento celular normal e contribuem para a transformação e crescimento de células malignas através da ativação de vias de sinalização intracelular. A via de sinalização do IGF1 é iniciada através da ativação de seu receptor seguido da ativação de seus substratos, incluindo o substrato 1 do receptor de insulina (insulin receptor substrate 1; IRS1). IRS1 é uma proteína predominantemente citosólica envolvida na transdução de sinal, e também desempenha um papel na transformação maligna, sendo altamente expresso em muitos tipos de câncer. Em fibroblastos de camundongos, Irs1, através da sinalização do Igf1, foi descoberto como uma proteína chave para a translocação nuclear da ?-catenina e ativação da transcrição de seus genes alvos, como o Myc e a ciclina D1. MYC e ciclina D1 podem atuar como oncogenes, contribuindo para o desenvolvimento de diversas neoplasias, inclusive as hematopoéticas. Deste modo, o objetivo deste projeto de pesquisa foi investigar a participação do IRS1 nuclear na via da ?-catenina em LLA. Foram utilizadas no estudo linhagens celulares de LLA (Jurkat, MOLT4, Raji e Namalwa) e células hematopoéticas primárias de doadores normais (n=13) e de pacientes adultos com LLA (n=45) atendidos em nossa Instituição. Estudos de expressão gênica (PCRq), expressão, associação proteica (western blotting, imunoprecipitação) e localização celular (fracionamento subcelular e microscopia confocal) foram utilizados. Células da linhagem Jurkat foram submetidas à estimulação com IGF1 e/ou inibição farmacológica de IGF1R (OSI-906). Observamos elevada expressão gênica relativa de IRS1, ?-catenina e MYC nos pacientes com LLA quando comparada aos controles normais (p<0,05), mas não houve diferença na expressão gênica de ciclina D1 e IGF1R entre os dois grupos. Observamos uma correlação positiva entre a expressão gênica de ?-catenina e MYC (p=0.0004; r=0.50), e entre a expressão de IRS1 e MYC (p=0.001; r=0.45) na coorte de pacientes com LLA. Na análise univariada, idade e expressão de MYC correlacionaram-se negativamente com a sobrevida global de pacientes com LLA; idade foi fator independente de prognóstico para a sobrevida. Em linhagens celulares de LLA (Jurkat, MOLT4, Raji e Namalwa), observamos co-localização de IRS1 e ?-catenina no núcleo e no citoplasma. Em células primárias de doador normal, IRS1 e ?-catenina localizaram-se predominantemente no citoplasma. Em células da linhagem celular Jurkat, observamos interação entre IRS1 e ?- catenina e o estímulo com IGF1 provocou o aumento da fosforilação em tirosina de IRS1. O tratamento com OSI-906 diminuiu a fosforilação em tirosina de IGF1R, a translocação nuclear de ?-catenina e a expressão proteica de MYC em células Jurkat. Em conclusão, nossos dados suportam uma relação entre a via de sinalização IGF1R/IRS1 e a ativação da ?- catenina em leucemia linfoide aguda, o que pode representar um importante eixo de sinalização envolvido na fisiopatologia da doença. / The acute lymphoblastic leukemia (ALL) is a heterogeneous group of malignancies characterized by abnormal proliferation and accumulation of immature cells in the bone marrow, which impairs the production of erythrocytes, leukocytes and platelets. Insulin-like growth factor 1 (IGF1) and its receptor (IGF1R) regulate normal cell growth and contribute to transformation and growth of malignant cells through activation of downstream signaling pathways. The IGF1 signaling pathway is initiated through activation of its receptor followed by activation of its substrates, including insulin receptor substrate 1 (IRS1). IRS1 is well known as a cytosolic protein involved in signal transduction, but also plays a role in malignant transformation, being highly expressed in many cancers. In mouse fibroblasts, Irs1, through Igf1 signaling, was found to be the key protein for nuclear translocation of ?-catenin and transcription activation of its target genes, such as Myc and cyclin D1. MYC and cyclin D1 may act as oncogenes, contributing to the development of cancers, including hematopoietic neoplasm. Thus, the aims of this study were to investigate the role of nuclear IRS1 in the ?-catenin pathway in LLA. We used in the study ALL cell lines (Jurkat, MOLT-4, Namalwa and Raji) and primary hematopoietic cells from healthy donors (n=13) and from adult patients with ALL (n=45) treated at our Institution. Studies of gene expression (qPCR), protein expression, association (Western blotting and immunoprecipitation) and cell location (subcellular fractionation and confocal microscopy) were used. Jurkat cells were submitted to IGF1 stimulation and/or IGF1R pharmacological inhibition (OSI-906). IRS1, ?-catenin and MYC relative gene expression were significantly elevated in ALL patients compared to normal controls (p<0.05), but there was no difference in gene expression of cyclin D1 and IGF1R between the two groups. A positive correlation between ?-catenin and MYC relative expression (p=0.0004; r=0.50) and between IRS1 and MYC expression (p=0.001; r=0.45) was found. Univariate analysis revealed that increasing age and elevated expression of MYC are factors that adversely affect the overall survival; age was an independent prognostic factor for survival. IRS1 and ?-catenin co-localized in the nucleus and cytoplasm of ALL cell lines (Jurkat, MOLT4, Raji e Namalwa). In primary cell of normal donor, IRS1 and ?-catenin were found predominantly in the cytoplasm. In Jurkat cells, a constitutive IRS1 and ?-catenin protein interaction was observed and IGF1 stimulation increased IRS1 tyrosine phosphorylation. OSI-906 treatment decreased IGF1R tyrosine phosphorylation, nuclear translocation of ?-catenin and MYC protein expression in Jurkat cells. In conclusion, our data support a link between the signaling pathway IGF1R/IRS1 and activation of ?-catenin in acute lymphoblastic leukemia, which may represent an important axis involved in the pathophysiology of the disease.

A leucemia linfoide aguda (LLA) compreende um grupo heterogêneo de neoplasias caracterizadas por proliferação anormal e acúmulo de células imaturas na medula óssea, o que prejudica a produção de eritrócitos, leucócitos e plaquetas. O fator de crescimento semelhante à insulina 1 (insulin-like growth factor 1; IGF1) e seu receptor (IGF1R) regulam o crescimento celular normal e contribuem para a transformação e crescimento de células malignas através da ativação de vias de sinalização intracelular. A via de sinalização do IGF1 é iniciada através da ativação de seu receptor seguido da ativação de seus substratos, incluindo o substrato 1 do receptor de insulina (insulin receptor substrate 1; IRS1). IRS1 é uma proteína predominantemente citosólica envolvida na transdução de sinal, e também desempenha um papel na transformação maligna, sendo altamente expresso em muitos tipos de câncer. Em fibroblastos de camundongos, Irs1, através da sinalização do Igf1, foi descoberto como uma proteína chave para a translocação nuclear da ?-catenina e ativação da transcrição de seus genes alvos, como o Myc e a ciclina D1. MYC e ciclina D1 podem atuar como oncogenes, contribuindo para o desenvolvimento de diversas neoplasias, inclusive as hematopoéticas. Deste modo, o objetivo deste projeto de pesquisa foi investigar a participação do IRS1 nuclear na via da ?-catenina em LLA. Foram utilizadas no estudo linhagens celulares de LLA (Jurkat, MOLT4, Raji e Namalwa) e células hematopoéticas primárias de doadores normais (n=13) e de pacientes adultos com LLA (n=45) atendidos em nossa Instituição. Estudos de expressão gênica (PCRq), expressão, associação proteica (western blotting, imunoprecipitação) e localização celular (fracionamento subcelular e microscopia confocal) foram utilizados. Células da linhagem Jurkat foram submetidas à estimulação com IGF1 e/ou inibição farmacológica de IGF1R (OSI-906). Observamos elevada expressão gênica relativa de IRS1, ?-catenina e MYC nos pacientes com LLA quando comparada aos controles normais (p<0,05), mas não houve diferença na expressão gênica de ciclina D1 e IGF1R entre os dois grupos. Observamos uma correlação positiva entre a expressão gênica de ?-catenina e MYC (p=0.0004; r=0.50), e entre a expressão de IRS1 e MYC (p=0.001; r=0.45) na coorte de pacientes com LLA. Na análise univariada, idade e expressão de MYC correlacionaram-se negativamente com a sobrevida global de pacientes com LLA; idade foi fator independente de prognóstico para a sobrevida. Em linhagens celulares de LLA (Jurkat, MOLT4, Raji e Namalwa), observamos co-localização de IRS1 e ?-catenina no núcleo e no citoplasma. Em células primárias de doador normal, IRS1 e ?-catenina localizaram-se predominantemente no citoplasma. Em células da linhagem celular Jurkat, observamos interação entre IRS1 e ?- catenina e o estímulo com IGF1 provocou o aumento da fosforilação em tirosina de IRS1. O tratamento com OSI-906 diminuiu a fosforilação em tirosina de IGF1R, a translocação nuclear de ?-catenina e a expressão proteica de MYC em células Jurkat. Em conclusão, nossos dados suportam uma relação entre a via de sinalização IGF1R/IRS1 e a ativação da ?- catenina em leucemia linfoide aguda, o que pode representar um importante eixo de sinalização envolvido na fisiopatologia da doença. / The acute lymphoblastic leukemia (ALL) is a heterogeneous group of malignancies characterized by abnormal proliferation and accumulation of immature cells in the bone marrow, which impairs the production of erythrocytes, leukocytes and platelets. Insulin-like growth factor 1 (IGF1) and its receptor (IGF1R) regulate normal cell growth and contribute to transformation and growth of malignant cells through activation of downstream signaling pathways. The IGF1 signaling pathway is initiated through activation of its receptor followed by activation of its substrates, including insulin receptor substrate 1 (IRS1). IRS1 is well known as a cytosolic protein involved in signal transduction, but also plays a role in malignant transformation, being highly expressed in many cancers. In mouse fibroblasts, Irs1, through Igf1 signaling, was found to be the key protein for nuclear translocation of ?-catenin and transcription activation of its target genes, such as Myc and cyclin D1. MYC and cyclin D1 may act as oncogenes, contributing to the development of cancers, including hematopoietic neoplasm. Thus, the aims of this study were to investigate the role of nuclear IRS1 in the ?-catenin pathway in LLA. We used in the study ALL cell lines (Jurkat, MOLT-4, Namalwa and Raji) and primary hematopoietic cells from healthy donors (n=13) and from adult patients with ALL (n=45) treated at our Institution. Studies of gene expression (qPCR), protein expression, association (Western blotting and immunoprecipitation) and cell location (subcellular fractionation and confocal microscopy) were used. Jurkat cells were submitted to IGF1 stimulation and/or IGF1R pharmacological inhibition (OSI-906). IRS1, ?-catenin and MYC relative gene expression were significantly elevated in ALL patients compared to normal controls (p<0.05), but there was no difference in gene expression of cyclin D1 and IGF1R between the two groups. A positive correlation between ?-catenin and MYC relative expression (p=0.0004; r=0.50) and between IRS1 and MYC expression (p=0.001; r=0.45) was found. Univariate analysis revealed that increasing age and elevated expression of MYC are factors that adversely affect the overall survival; age was an independent prognostic factor for survival. IRS1 and ?-catenin co-localized in the nucleus and cytoplasm of ALL cell lines (Jurkat, MOLT4, Raji e Namalwa). In primary cell of normal donor, IRS1 and ?-catenin were found predominantly in the cytoplasm. In Jurkat cells, a constitutive IRS1 and ?-catenin protein interaction was observed and IGF1 stimulation increased IRS1 tyrosine phosphorylation. OSI-906 treatment decreased IGF1R tyrosine phosphorylation, nuclear translocation of ?-catenin and MYC protein expression in Jurkat cells. In conclusion, our data support a link between the signaling pathway IGF1R/IRS1 and activation of ?-catenin in acute lymphoblastic leukemia, which may represent an important axis involved in the pathophysiology of the disease.

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.

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.

This thesis is comprised of two parts. In the first part we study the influence of four frequently disputed genes on the susceptibility for developing prostate cancer, and in the second part we attempt to establish a basic understanding of the molecular genetic events in penile cancer. In a prostate cancer cohort we have investigated the relation of prostate cancer risk and single nucleotide polymorphisms (SNPs) in four different genes coding for the androgen receptor (AR), the vitamin D receptor (VDR), insulin (INS) and insulin receptor substrate 1 (IRS1). Despite strong biological indications of an involvement of these genes in prostate carcinogenesis, the results from different studies are contradictory and inconclusive. The action of the AR varies between individuals in part owing to a repetitive CAG sequence (polyglutamine) in the first exon of the AR gene. The results presented in this thesis show that in our cohort of prostate cancer patients the average number of repeats is 20.1, which is significantly (p&lt;0.001) fewer repeats compared to healthy control individuals, where the average is 22.5 repeats. We find a 4.94 fold (p=0.00003) increased risk of developing prostate cancer associated with having short repeat lengths (≤19 repeats), compared with long repeats (≥23 repeats). In paper I we also study the TaqI polymorphism in the VDR gene, and find that it does not modify the risk of prostate cancer. In the INS gene we study the +1127 PstI polymorphism and find no overall effect on the risk of prostate cancer. However, we do find that the CC genotype is associated with low grade disease defined as having a Gleason score ≤6 (OR=1.46; p=0.018). In the IRS1 gene we study the G972R polymorphism and observe that the R allele is significantly associated with a 2.44 fold increased prostate cancer risk (p=0.010). The knowledge of molecular genetic events in penile cancer is very scarce and to date very few genes have been identified to be involved in penile carcinogenesis. We chose therefore to analyse the penile cancer samples using genome-wide high-density SNP arrays. We find major regions of frequent copy number gain in chromosome arms 3q, 5p and 8q, and slightly less frequent in 1p, 16q and 20q. The chromosomal regions of most frequent copy number losses are 3p, 4q, 11p and 13q. We suggest four candidate genes residing in these areas, the PIK3CA gene (3q26.32), the hTERT gene (5p15.33), the MYC gene (8q24.21) and the FHIT gene (3p14.2). The mutational status of the PIK3CA and PTEN genes in the PI3K/AKT pathway and the HRAS, KRAS, NRAS and BRAF genes in the RAS/MAPK pathway was assessed in the penile cancer samples. We find the PIK3CA, HRAS and KRAS genes to be mutated in 29%, 7% and 3% of the cases, respectively. All mutations are mutually exclusive. In total the PI3K/AKT and RAS/MAPK pathways were found to be activated through mutation or amplification in 64% of the cases, indicating the significance of these pathways in the aetiology of penile cancer.

No description available.