A mutação recorrente JAK2V617F é a lesão molecular com maior impacto na fisiopatologia das neoplasias mieloproliferativas (NMP) BCR ABL1 negativas. A ausência de resposta clínica completa ao inibidor seletivo de JAK1/2, ruxolitinibe, indica a necessidade de novas abordagens terapêuticas. Dados recentes sugerem que IGF1R/IRS representa um potencial alvo de inibição para o tratamento das NMP: (i) o substrato do receptor de insulina 2 (IRS2) coopera com JAK2V617F na transformação maligna em NMP; (ii) a desregulação da via de sinalização de IGF1R induz NMP. O composto NT157 foi desenvolvido para inibir IRS1/2 e apresentou efeitos antineoplásicos em neoplasias sólidas. Os objetivos deste trabalho foram avaliar os efeitos celulares e moleculares do tratamento com o inibidor de IRS1/2, NT157, isolado e em combinação com ruxolitinibe, em NMP JAK2V617F. Células HEL e SET2 JAK2V617F foram tratadas com veículo, NT157 e/ou ruxolitinibe e submetidas à avaliação da viabilidade celular, apoptose, proliferação, clonogenicidade, ciclo celular, expressão gênica e/ou expressão/ativação proteica. Células primárias de pacientes com policitemia vera foram submetidos a tratamento com NT157 e avaliação de formação espontânea de colônias eritroides. O efeito do NT157 in vivo foi avaliado utilizando modelo de xenotransplante de células HEL em camundongos NSG. A análise estatística foi realizada através do teste ANOVA ou t de Student. Em células HEL e/ou SET2 JAK2V617F, o tratamento com NT157 promoveu redução da viabilidade, clonogenicidade e proliferação celular, aumentou a apoptose e resultou em parada do ciclo celular em G2/M (p?0,05). Exposição ao NT157 resultou em inibição da fosforilação de STAT3, STAT5 e ERK e na modulação da expressão de 23 oncogenes (CCND1, MYB e WT1) e genes supressores tumorais (CDKN1A, JUN e FOS) em células HEL (p?0,05). O tratamento combinado com ruxolitinibe não apresentou efeito potencializador, sendo que a redução da viabilidade nas condições de combinação corresponde ao efeito das monoterapias nas linhagens celulares avaliadas. Em células primárias de pacientes com policitemia vera (n=3), NT157 reduziu a formação espontânea de colônias eritroides (p?0,05). O tratamento in vivo com veículo ou NT157 na dose de 70mg/kg, 3 vezes por semana, via intraperitoneal, em modelos de xenotransplante com células HEL em camundongos NSG (n=5 para cada grupo) não apresentou efeitos antineoplásicos. Em conclusão, a inibição farmacológica de IRS1/2 apresentou efeitos antineoplásicos significativos em modelos de linhagens celulares e amostras primárias de pacientes com NMP JAK2V617F. A inibição farmacológica combinada de IRS1/2 e JAK1/2 não potencializou o efeito antineoplásico das monoterapias nos processos celulares investigados. Os resultados dos estudos in vivo em modelos de xenotransplante indicam a necessidade de estudos de farmacocinética e farmacodinâmica para o NT157. Os efeitos moleculares identificados permitiram uma melhor compreensão sobre os mecanismos de ação da droga NT157 em NMP. / The recurrent V617F mutation in JAK2 is a major contributor to the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need for targeting other signaling pathways that contribute to JAK2. Recent data indicate that IGF1R/IRS is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates to malignant transformation induced by JAK2V617F, (ii) IGF1R signaling upregulation induces MNP phenotype. NT157 is a synthetic compound designed as IRS1/2 inhibitor and was able to induce anti-neoplastic effects in solid tumors. We, herein, aimed to characterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2V617F. HEL and SET2 JAK2V617F cells were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability assay, apoptosis, proliferation, clonogenicity, cell cycle, gene expression and protein expression/activation. Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 treatment and evaluated for erythropoietin-independent colony formation. NT157 effects in vivo were evaluated in a xenograft model of leukemogenesis induced by HEL cells in NSG mice. Statistical analysis was performed using ANOVA or Student\'s t test. In MPN cell lines, NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation, increased apoptosis and cell cycle arrest in G2/M (all p<0.05). NT157 exposure resulted in inhibition of STAT3, STAT5 and ERK phosphorylation. NT157 also modulated the expression of 23 oncogenes (CCND1, MYB and WT1) and suppressor tumor genes (CDKN1A, FOS and JUN) in HEL cells (p?0.05). In both cell lines, the combined treatment, NT157 plus ruxolitinib, did not potentiate the effects of monotherapies. In primary cells from polycythemia vera patients, NT157 exposition reduced spontaneous erythroid colony formation (all p<0.05). In vivo treatment with vehicle or NT157 (70mg/kg intraperitoneal), three times a week, showed no antineoplastic effects in NSG mice transplanted with HEL cells (n = 5 for each group). In summary, the IRS1/2 pharmacological inhibitor NT157 displayed remarkable antineoplastic effects in JAK2V617F cells lines and MPN primary cells. The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapy. The results of in vivo treatment using a xenograft model highlight the need for pharmacokinetic and pharmacodynamic studies for the NT157 compound. The molecular effects identified allowed a better understanding about the mechanisms of NT157 action in MPNs.

A mutação recorrente JAK2V617F é a lesão molecular com maior impacto na fisiopatologia das neoplasias mieloproliferativas (NMP) BCR ABL1 negativas. A ausência de resposta clínica completa ao inibidor seletivo de JAK1/2, ruxolitinibe, indica a necessidade de novas abordagens terapêuticas. Dados recentes sugerem que IGF1R/IRS representa um potencial alvo de inibição para o tratamento das NMP: (i) o substrato do receptor de insulina 2 (IRS2) coopera com JAK2V617F na transformação maligna em NMP; (ii) a desregulação da via de sinalização de IGF1R induz NMP. O composto NT157 foi desenvolvido para inibir IRS1/2 e apresentou efeitos antineoplásicos em neoplasias sólidas. Os objetivos deste trabalho foram avaliar os efeitos celulares e moleculares do tratamento com o inibidor de IRS1/2, NT157, isolado e em combinação com ruxolitinibe, em NMP JAK2V617F. Células HEL e SET2 JAK2V617F foram tratadas com veículo, NT157 e/ou ruxolitinibe e submetidas à avaliação da viabilidade celular, apoptose, proliferação, clonogenicidade, ciclo celular, expressão gênica e/ou expressão/ativação proteica. Células primárias de pacientes com policitemia vera foram submetidos a tratamento com NT157 e avaliação de formação espontânea de colônias eritroides. O efeito do NT157 in vivo foi avaliado utilizando modelo de xenotransplante de células HEL em camundongos NSG. A análise estatística foi realizada através do teste ANOVA ou t de Student. Em células HEL e/ou SET2 JAK2V617F, o tratamento com NT157 promoveu redução da viabilidade, clonogenicidade e proliferação celular, aumentou a apoptose e resultou em parada do ciclo celular em G2/M (p?0,05). Exposição ao NT157 resultou em inibição da fosforilação de STAT3, STAT5 e ERK e na modulação da expressão de 23 oncogenes (CCND1, MYB e WT1) e genes supressores tumorais (CDKN1A, JUN e FOS) em células HEL (p?0,05). O tratamento combinado com ruxolitinibe não apresentou efeito potencializador, sendo que a redução da viabilidade nas condições de combinação corresponde ao efeito das monoterapias nas linhagens celulares avaliadas. Em células primárias de pacientes com policitemia vera (n=3), NT157 reduziu a formação espontânea de colônias eritroides (p?0,05). O tratamento in vivo com veículo ou NT157 na dose de 70mg/kg, 3 vezes por semana, via intraperitoneal, em modelos de xenotransplante com células HEL em camundongos NSG (n=5 para cada grupo) não apresentou efeitos antineoplásicos. Em conclusão, a inibição farmacológica de IRS1/2 apresentou efeitos antineoplásicos significativos em modelos de linhagens celulares e amostras primárias de pacientes com NMP JAK2V617F. A inibição farmacológica combinada de IRS1/2 e JAK1/2 não potencializou o efeito antineoplásico das monoterapias nos processos celulares investigados. Os resultados dos estudos in vivo em modelos de xenotransplante indicam a necessidade de estudos de farmacocinética e farmacodinâmica para o NT157. Os efeitos moleculares identificados permitiram uma melhor compreensão sobre os mecanismos de ação da droga NT157 em NMP. / The recurrent V617F mutation in JAK2 is a major contributor to the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need for targeting other signaling pathways that contribute to JAK2. Recent data indicate that IGF1R/IRS is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates to malignant transformation induced by JAK2V617F, (ii) IGF1R signaling upregulation induces MNP phenotype. NT157 is a synthetic compound designed as IRS1/2 inhibitor and was able to induce anti-neoplastic effects in solid tumors. We, herein, aimed to characterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2V617F. HEL and SET2 JAK2V617F cells were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability assay, apoptosis, proliferation, clonogenicity, cell cycle, gene expression and protein expression/activation. Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 treatment and evaluated for erythropoietin-independent colony formation. NT157 effects in vivo were evaluated in a xenograft model of leukemogenesis induced by HEL cells in NSG mice. Statistical analysis was performed using ANOVA or Student\'s t test. In MPN cell lines, NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation, increased apoptosis and cell cycle arrest in G2/M (all p<0.05). NT157 exposure resulted in inhibition of STAT3, STAT5 and ERK phosphorylation. NT157 also modulated the expression of 23 oncogenes (CCND1, MYB and WT1) and suppressor tumor genes (CDKN1A, FOS and JUN) in HEL cells (p?0.05). In both cell lines, the combined treatment, NT157 plus ruxolitinib, did not potentiate the effects of monotherapies. In primary cells from polycythemia vera patients, NT157 exposition reduced spontaneous erythroid colony formation (all p<0.05). In vivo treatment with vehicle or NT157 (70mg/kg intraperitoneal), three times a week, showed no antineoplastic effects in NSG mice transplanted with HEL cells (n = 5 for each group). In summary, the IRS1/2 pharmacological inhibitor NT157 displayed remarkable antineoplastic effects in JAK2V617F cells lines and MPN primary cells. The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapy. The results of in vivo treatment using a xenograft model highlight the need for pharmacokinetic and pharmacodynamic studies for the NT157 compound. The molecular effects identified allowed a better understanding about the mechanisms of NT157 action in MPNs.

<p>Hematopoiesis is the continual process of blood cell generation that primarily occurs in the bone marrow of adult animals. Hematologic neoplasms can also occur in the bone marrow and often result from dysregulation of signal transduction pathways. One example is the activation of the Ras oncogene, which has been linked to a variety of different cancers, including hematologic neoplasms. Ras is located proximal to the cell membrane and can activate many downstream effector pathways, thus it is difficult to determine which downstream pathway is mediating oncogenic Ras function. My thesis work focused on the effect of inappropriate activation of MEK/ERK, a downstream Ras effector pathway, in the hematopoietic system.</p><p>Using a retroviral transduction system, we expressed a constitutively active form of MEK1 in hematopoietic stem cells (HSCs). Mice transplanted with HSCs expressing active MEK developed a lethal myelodysplastic syndrome/myeloproliferative disease (MDS/MPN) characterized by the expansion of granulocytes/macrophages (GM) at the expense of lymphoid cell development. Transplantation of active MEK-induced MDS/MPNs into naïve mice did not result in further disease, suggesting that the MDS/MPN is not a frank leukemia.</p><p>Bcl-2 is an anti-apoptotic molecule that has been shown to play a role in leukemia development and maintenance. Coupling expression of active MEK and Bcl-2 resulted in MDS/MPNs that were phenotypically identical and had very similar disease onset compared to active MEK-induced MDS/MPNs. However, transplantation of Bcl-2/active MEK-induced MDS/MPNs did not result in a myeloid disease; rather, it resulted in the development of T-acute lymphoblastic leukemia (T-ALL) that was marked by activated Notch signaling. </p><p>These results led us to conclude that activation of MEK/ERK was sufficient to cause a pre-leukemic myeloid disease; however, additional oncogenic factors, such as Bcl-2 and Notch, were necessary for frank leukemia development. Moreover, additional oncogenic factors can alter the disease phenotype and disease course. Future analysis of the interplay between oncogenic factors will help shed light on disease development and aid in the development of more effective cancer treatments.</p> / Dissertation

Les syndromes myéloprolifératifs (SMP) non leucémie myéloïde chronique (LMC) sont des hémopathies myéloïdes chroniques affectant la cellule souche hématopoïétique, pouvant évoluer en leucémie aigüe myéloïde (LAM). Les SMP non LMC incluent la polyglobulie de Vaquez (PV), la thrombocytémie essentielle (TE) et la myélofibrose (MF). La mutation JAK2V617F est retrouvée dans 97% des cas de PV et dans 50% des cas de TE et MF ; elle n'est pas indispensable à la physiopathologie des SMP car JAK2 n'est pas muté à 100%. Afin de progresser dans la compréhension de la physiopathologie des SMP et afin d'identifier de nouveaux marqueurs moléculaires pour le diagnostic, le suivi et le pronostic; nous avons étudié des échantillons de PV, TE, MF ainsi que des LAM post-SMP. Nous avons utilisé des approches moléculaires complémentaires: séquençage, hybridation génomique comparative (CGH-array) et profils d'expression génique. Nous avons identifié des mutations de gènes régulateurs de l'épigénétique (ASXL1, TET2, DNMT3A, SUZ12) et des gènes de la machinerie de l'épissage de l'ARN (SF3B1, SRSF2). Nous avons également identifié que les co-mutations des gènes JAK2 et ASXL1 étaient associées à un mauvais pronostic. Au sein du sous-type MF, nous avons identifié par CGH-array des aberrations du nombre de copies des gènes. Celles-ci contiennent plusieurs gènes candidats susceptibles de participer à la physiopathologie des MF et à l'évolution en LAM (délétion 20q, NF1, ETV6). Nos travaux sur la caractérisation moléculaire des SMP contribuent à l'évolution vers une classification moléculaire avec l'objectif d'une médecine de précision où chaque SMP sera traité en fonction de ses altérations. / Myeloproliferative neoplasms (MPN) are chronic and clonal stem cell myeloid disorders, which can evolve to acute myeloid leukemia (AML). MPN non-chronic myeloid leukemia (CML) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) (primary or secondary to PV/ET). JAK2V617F mutation is found in 97% of PV and in around half of patients with ET or MF. Nevertheless, this mutation is not essential for MPN physiopathology, because in half of ET/MF cases, JAK2 is not mutated. To progress in the knowledge of MPN physiopathology and in particular of MF; and to find new molecular markers for MPN diagnosis, disease course, and prognosis, we studied several samples of PV, ET, MF and post-AML. We used gene sequencing, array-Comparative Genomic Hybridization (aCGH) and gene expression analyses. We identified several mutations in genes implicated in Epigenetic regulation (ASXL1, TET2, DNMT3A, SUZ12) and in genes implicated in the RNA splicing machinery (SF3B1, SRSF2). We also found that JAK2 and ASXL1 co-mutation is associated with a poor prognosis. In MF, we found by aCGH several copy number aberrations that involve potential leukemogenic genes. Our gene expression data support the hypothesis that PV, ET and MF are a continuum of the same pathology. Our results on molecular characterization help establish a new molecular classification of MPNs with the objective personalized treatment where each MPN will be treated depending on the alterations present in the myeloid cell genome.