Reproduction de : Thèse doctorat : Pharmacie. Biochimie et biologie moléculaire : Reims : 2004. / Bibliogr.

<p>The Abl family of protein tyrosine kinases regulates diverse cellular processes by coordinating cytoskeletal rearrangements. Recent data indicate that pharmacological inhibition of Abl kinases reduces inflammation in preclinical models and in the clinic. While a previous role for Abl kinases in lymphocytes had been described, it remained unclear if Abl kinases regulate innate immune function. To explore this possibility, we generated a myeloid-specific conditional Abl knockout mouse. Using a combination of molecular, genetic, and pharmacological approaches, we demonstrate a role for Abl kinases in regulating the efficiency of macrophage phagocytosis and inflammatory responses. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Abl kinases are present at the phagocytic cup and are activated by Fcgamma receptor engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the Syk kinase. Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to Fcgamma receptor ligation. The link between Abl family kinases and Syk may be direct as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis.</p><p>Our studies also revealed a role for Abl kinases in macrophage and cancer cell invasion. Inhibition of Abl kinases suppressed cell invasion in vitro, whereas overexpression of Abl kinases enhanced extracellular matrix degradation. We found that partial loss of Abl kinase expression in myeloid cells reduced macrophage infiltration into tumors in a mouse model of breast cancer. Furthermore, pharmacological inhibition of Abl kinases reduced myeloid cell infiltration and slowed tumor growth in subcutaneous tumor models. We also found that Abl expression and activity are elevated in subsets of human tumor samples. Taken together, our results suggest Abl kinases have an important role in cancer and inflammation, and represent important therapeutic targets for their treatment.</p> / Dissertation

Cancer patient mortality is caused by the ability of tumor cells to invade the extracellular matrix and metastasize. Our lab was the first to identify the role of Abl family of non-receptor tyrosine kinases (c-Abl and Arg) in the progression of solid tumor cancers. In our previous studies, we showed that high c-Abl/Arg activity promotes proliferation, invasion, and metastasis in melanoma and breast cancer cells lines. Here, we demonstrate that our previous findings are clinically relevant by showing increased c-Abl/Arg kinase activity in primary melanoma tumor tissue in comparison to low activity as compared to benign nevi. Additionally, in breast cancer tissue, we found aggressive tumor subtypes (triple-negative and high-grade breast cancer) had increased c-Abl/Arg activity as compared to less aggressive subtypes. To define the mechanism by which c-Abl and Arg promote melanoma and breast cancer metastasis, we searched for novel pathways by which c-Abl and Arg promote invasion, a key step in metastasis. Significantly, we found that c-Abl and Arg decrease the expression of non-metastatic protein, NM23-H1, a metastasis suppressor that is lost during metastatic progression. We demonstrate that NM23-H1 is localized and degraded within the lysosome via proteases, cathepsins L and B. Moreover, we show that c-Abl and Arg upregulate cathepsin mRNA levels and activate the cathepsins, which in-turn degrade NM23-H1. We demonstrate that this pathway is functionally significant as c-Abl and Arg require the downregulation of NM23-H1 to promote invasion in melanoma and breast cancer cell lines. We show that the pathway is clinically significant as c-Abl/Arg activity is inversely correlated with NM23-H1 expression in mouse lung metastases, as well as in human primary melanoma and primary breast cancer tissue. In summary, we are the first to demonstrate novel crosstalk between oncogenic and metastasis suppressor signaling pathways, and provide evidence that pharmacological inhibition of Abl family kinases in melanoma and breast cancer patients may prevent metastatic progression by stabilizing a metastasis suppressor.

<p>The vasculature has a crucial function in normal physiology, enabling the transport of oxygen and nutrients to cells throughout the body. In turn, endothelial cells, which form the inner-most lining of blood vessels, are key regulators of vascular function. In addition to forming a barrier which separates the circulation from underlying tissues, endothelial cells respond to diverse extracellular cues and produce a variety of biologically-active mediators in order to maintain vascular homeostasis. Disruption of normal vascular function is a prominent feature of a variety of pathological conditions. Thus, elucidating the signaling pathways regulating endothelial function is critical for understanding the role of endothelial cells in both normal physiology and pathology, as well as for potential development of therapeutic interventions.</p><p>In this dissertation, we use a combination of pharmacological inhibition and knockdown studies, along with generation of endothelial conditional knockout mice, to demonstrate an important role of the Abelson (Abl) family of non-receptor tyrosine kinases (Abl and Arg) in vascular function. Specifically, loss of endothelial expression of the Abl kinases leads to late-stage embryonic and perinatal lethality in conditional knockout mice, indicating a crucial requirement for Abl/Arg kinases in normal vascular development and function. Endothelial <italic>Abl</italic>/<italic>Arg</italic>-null embryos display focal regions of vascular loss and tissue damage, as well as increased endothelial cell apoptosis. An important pro-survival function for the Abl kinases is further supported by our finding that either microRNA-mediated <italic>Abl</italic>/<italic>Arg</italic> depletion or pharmacological inhibition of the Abl kinases increases endothelial cell susceptibility to stress-induced apoptosis <italic>in vitro</italic>. The Abl kinases are activated in response to treatment with the pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We show that both VEGF- and bFGF-mediated endothelial cell survival is impaired following Abl kinase inhibition.</p><p>These studies have uncovered a previously unappreciated role for the Abl kinases in the regulation of the angiopoietin/Tie2 signaling pathway, which functions to support endothelial cell survival and vascular stability. Loss of Abl/Arg expression leads to reduced mRNA and protein levels of the Tie2 receptor, resulting in impaired activation of intracellular signaling pathways by the Tie2 ligand angiopoietin-1 (Angpt1), as well as decreased Angpt1-mediated endothelial cell survival following serum-deprivation stress. Notably, we found that the Abl kinases are activated following Angpt1 stimulation, suggesting a unique dual role for Abl and Arg in Angpt/Tie2 signaling, potentially modulating Tie2 downstream signaling responses, as well as regulating Tie2 receptor expression.</p><p>Further, we show an important contribution of the Abl family kinases to the regulation of endothelial permeability responses both <italic>in vitro</italic> and <italic>in vivo</italic>. The Abl kinases are activated in response to a diverse group of permeability-inducing factors, including VEGF and the inflammatory mediators thrombin and histamine. We show that inhibition of Abl kinase activity, using either the ATP-competitive inhibitor imatinib or the allosteric inhibitor GNF-2, protects against disruption of endothelial barrier function by the permeability-inducing factors <italic>in vitro</italic>. VEGF-induced vascular permeability similarly is decreased in conditional knockout mice lacking endothelial Abl expression, as well as following treatment with Abl kinase inhibitors <italic>in vivo</italic>. Mechanistically, we show that loss of Abl kinase activity is accompanied by activation of the barrier-stabilizing GTPases (guanosine triphosphatases) Rac1 and Rap1, as well as inhibition of agonist-induced Ca<super>2+</super> mobilization and generation of acto-myosin contractility.</p><p>Taken together, these results demonstrate involvement of the Abl family kinases in the regulation of endothelial cell responses to a broad range of pro-angiogenic and permeability-inducing factors, as well as a critical requirement for the endothelial Abl kinases in normal vascular development and function <italic>in vivo</italic>. These findings have implications for the clinical use of Abl kinase inhibitors.</p> / Dissertation

CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

Around 50% of chronic myeloid leukaemia (CML) patients who remain on imatinib treatment for more than 5 years will achieve a complete molecular response (CMR), defined by undetectable BCR-ABL mRNA in a sensitive reverse transcriptase real-time quantitative PCR (RQ-PCR) assay. Given the increasing importance of CMR on imatinib therapy the primary aim of this study was to improve the accuracy and sensitivity of MRD detection to allow a more accurate estimation of relapse risk when therapy is withdrawn. Firstly, we investigated ways of improving the sensitivity of RT-PCR methods for the detection of BCR-ABL mRNA. Secondly, we investigated the use of the patient-specific BCR-ABL gene for the detection of MRD. Thirdly, we conducted a multi-centre clinical trial of imatinib withdrawal in selected CML patients in a stable CMR. This clinical trial provided patient samples that could be used to test our optimized MRD assays, and provided clinical data on the risk and patterns of relapse after withdrawal of imatinib therapy. The trial is ongoing, but an interim analysis of the study data was performed. In 22 patients the estimated probability of molecular relapse after imatinib withdrawal was 54%, and 60% of relapses occurred within the first 4 months. The average detection limit of BCR-ABL mRNA by RQ-PCR is estimated at around 4.5 log below the level of BCR-ABL prior to commencing treatment. The number of leukaemic cells at diagnosis is around 10¹ ², so the number of residual leukaemic cells in CMR might vary from zero to over a million. We hypothesized that the amount of residual leukaemia in CMR is variable between patients, and that this heterogeneity is a determinant of the risk of relapse when treatment is withdrawn. We developed more sensitive methods for the detection of BCR-ABL and tested these methods in samples from our study patients. We showed that random pentadecamer (15-mer) primers improved the efficiency of reverse transcriptase PCR (RT-PCR), and resulted in a lower detection limit of BCR-ABL mRNA. We also developed a novel nested RT-PCR method using real-time PCR for the second round of the reaction, and this resulted in a lower detection limit of BCR-ABL in patient samples. The utility of this nested RT-PCR method was limited by a false positive rate of 2-3% in the HeLa cell line that we used as our negative control. Consequently, we examined the detection of the patient-specific genomic BCR-ABL sequence as an alternative to RT-PCR. Breakpoints in BCR and ABL1 in CML patients are widely dispersed over 3 kb and 150 kb, respectively. Therefore, the BCR-ABL genomic sequence is essentially unique to each patient. We sequenced the genomic breakpoints of 43 CML patients. We showed that the distribution of breakpoints in BCR and ABL1 was non-random, but we were unable to identify any genomic feature that determined the specific location of individual breakpoints. We developed a novel BCR-ABL DNA Q-PCR method for 12 of the study patients, and in 11 of the patients BCR-ABL DNA was detected when the patient was in a CMR, confirming that this method was more sensitive than RQ-PCR. Contrary to our hypothesis, the detection of BCR-ABL DNA was not predictive of relapse. In most patients who relapsed there was a significant increase in BCR-ABL DNA prior to mRNA relapse. Two patients had stable levels of BCR-ABL DNA measurable on multiple occasions, but remained in remission after 6 months and 15 months, respectively. We have shown that a stable CMR after the withdrawal of imatinib therapy does not necessarily indicate the eradication of leukaemia. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 124-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Biochemistry) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 137-161). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Introdução: A leucemia mielóide crônica (LMC) é resultado de uma proliferação clonal de uma célula tronco hematopoética. A doença é caracterizada por três fases distintas: fase crônica, fase acelerada e fase blástica, e também pela presença do cromossomo Philadelphia (Ph), uma anormalidade genética resultante da translocação recíproca entre os cromossomos t(9;22)(q34;q11) que leva ao surgimento do gene híbrido BCRABL, cuja oncoproteína de mesmo nome, tem alta atividade tirosina quinase e sem controle. Os tratamentos para a LMC incluem a hidroxiuréia (HU), o transplante de medula óssea, considerado o único curativo, o alfa-interferona (IFN-alfa) e, mais recentemente, o mesilato de imatinibe (MI), que revolucionou o tratamento para LMC por sua baixa toxicidade e efeitos terapêuticos. Quanto maior a complexidade dos novos tratamentos disponíveis, maior o risco de problemas relacionados com medicamentos (PRMs).As reações adversas aos medicamentos (RAMs) do câncer podem acarretar baixa adesão ao tratamento ou conseqüências graves originárias da alta toxicidade da terapia. Esses problemas de saúde, causa de morbidade e mortalidade, vinculados ou suspeitos de estar relacionados ao tratamento, podem vir a interferir nos resultados e na qualidade de vida do paciente.Objetivos: Este estudo tem por objetivos (1) avaliar a efetividade do tratamento com mesilato de imatinibe em uma coorte de pacientes com leucemia mielóide crônica, (2) descrever e classificar as taxas de reações adversas ao MI, (3) relacionar as freqüências de RAMs com as doses do MI,(4) avaliar a resposta ao tratamento e, (5) avaliar a qualidade de vida dos pacientes. Métodos: esta coorte não controlada, contemporânea, incluiu 50 pacientes com LMC, tratados ambulatorialmente com MI e em acompanhamento farmacêutico em um hospital universitário no sul do Brasil. Os pacientes foram acompanhados por 12 meses para identificar suspeitas de reações adversas a medicamentos (RAMs) durante o tratamento. Os tipos de reações foram classificados e a relação de causalidade foi estabelecida através do algoritmo de Naranjo. A qualidade de vida foi avaliada através do questionário WHOQOL-bref.9 Resultados: O estudo acompanhou 50 pacientes com doses e ≤ 400mg (35) e >400mg (15). As RAMs mais comuns foram câimbra (66%), náuseas (58%), cefaléia (30%), edema periorbital (30%), fadiga (20%) e diarréia (20%). Dos pacientes em tratamento ≤400 mg, 30 (85,7%) atingiram remissão citogenética maior (RCGM) e dos 15 pacientes tratados com >400 mg, 6 (40%) atingiram a RCGM. Não houve diferença estatística significativa na qualidade de vida medida no início, após 6 meses e após 12 meses de tratamento. Conclusão: os pacientes com LMC tratados com MI no ambulatório do HCPA apresentaram boa resposta ao tratamento, com baixo índice de reações adversas graves, e boa qualidade de vida, semelhantes às descritas na literatura.

MAIA FILHO, P. A. Genotoxicidade e mutagenicidade em pacientes com leucemia mieloide crônica tratados com inibidores de tirosino-quinase. 2017. 64 f. Dissertação (Mestrado em Patologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-03-24T13:51:01Z No. of bitstreams: 1 2017_dis_pamaiafilho.pdf: 1620056 bytes, checksum: b8fde4a5c189ab98dfc52d58490d9eb1 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-03-24T13:51:12Z (GMT) No. of bitstreams: 1 2017_dis_pamaiafilho.pdf: 1620056 bytes, checksum: b8fde4a5c189ab98dfc52d58490d9eb1 (MD5) / Made available in DSpace on 2017-03-24T13:51:12Z (GMT). No. of bitstreams: 1 2017_dis_pamaiafilho.pdf: 1620056 bytes, checksum: b8fde4a5c189ab98dfc52d58490d9eb1 (MD5) Previous issue date: 2017-02-15 / Chronic myelogenous leukemia (CML) is a myeloproliferative disease of hematopoietic stem cells, characterized by the presence of the Philadelphia (Ph) chromosome, originating from a reciprocal translocation between the long arms of chromosomes 9 and 22, forming the gene BCR-ABL, which encodes a BCR-ABL oncoprotein with constitutive tyrosine kinase activity. The clinical course of CML is often divided into three phases: chronic, accelerated, and blast. The treatment of choice for the chronic phase is the first-generation tyrosine kinase inhibitor (TKI), imatinib mesylate, and for refractory patients, second-generation TKIs (dasatinib or nilotinib) are used. Studies have shown that residual leukemia may persist even in the best responders to TKI, since therapy is not curative. In this context, the present study aimed to evaluate the genotoxicity and mutagenicity of TKI in patients with CML followed at the hematology clinic of the Walter Cantídio University Hospital (HUWC). It is a cross-sectional study with 44 patients with clinical and molecular diagnosis of CML. Patients were stratified into three groups: diagnosis (CML D) (n = 5), use of first generation TKI (CML) (n = 31) and use of second generation TKI (CML) (n = 8). The control group (CG) consisted of apparently healthy individuals. Genotoxicity and mutagenicity were analyzed by the comet assay and micronucleus test. Statistical analysis of the data was performed using the GraphPad Prism 6.0 program using the Kruskal-Wallis or ANOVA, Mann Whitney or T-student tests, depending on the normality of the data and the level of significance was 5% (p < 0.05). Patients with CML had a statistically higher ADN damage index (DI) compared to CG (p < 0.0001). When the patients were stratified, a progressive increase of the DNA ID was verified in the groups: CML D, CML G1 and CML G2, respectively, relative to GC (p < 0.05). Patients with CML had a statistically higher micronucleus index (NMI), nucleoplasmic bridge index (NPI) and nuclear bud index (NBI) compared to the CG (p < 0.05). By stratifying patients with CML, it was found that patients in the G1 and G2 CML groups had statistically higher NMI and NPI compared to CG (p <0.001). NMI was also elevated in the CML G2 group in relation to the patients in the CML D group (p <0.01). The nuclear bud index (NBI) did not present statistical difference in the analyzes performed after the stratification of the groups. The TKI revolutionized CML therapy, improving patient survival. However, these results point to the relevance of studies that evaluate the possible genotoxic and mutagenic effects of this therapy in the long term. The mechanisms involved should be elucidated for the purpose of improving treatment as well as assessing the clinical impact this harm may cause. / A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa das células-tronco hematopoéticas, caracterizada pela presença do cromossomo Philadelphia (Ph), originado a partir de uma translocação recíproca entre os braços longos dos cromossomos 9 e 22, formando o gene BCR-ABL, que codifica uma oncoproteína BCR-ABL com atividade tirosino-quinase constitutiva. O curso clínico da LMC é frequentemente dividido em três fases: crônica, acelerada e blástica. O tratamento de escolha para a fase crônica é o inibidor de tirosino-quinase (ITK) de primeira geração, mesilato de imatinibe, e para os pacientes refratários, utiliza-se os ITK de segunda geração (dasatinibe ou nilotinibe). Estudos têm demonstrado que a leucemia residual pode persistir mesmo nos melhores respondedores aos ITK, uma vez que a terapia não é curativa. Nesse contexto, o presente estudo objetivou avaliar a genotoxicidade e mutagenicidade dos ITK em pacientes com LMC acompanhados no ambulatório de hematologia do Hospital Universitário Walter Cantídio (HUWC). Trata-se de um estudo transversal com 44 pacientes com diagnóstico clínico e molecular de LMC. Os pacientes foram estratificados em três grupos: ao diagnóstico (LMC D) (n=5), em uso de ITK de primeira geração (LMC G1) (n=31) e em uso de ITK de segunda geração (LMC G2) (n=8). O grupo controle (GC) foi composto por indivíduos aparentemente saudáveis. A genotoxicidade e mutagenicidade foram analisadas através do ensaio cometa e teste de micronúcleos. A análise estatística dos dados foi realizada através do programa GraphPad Prism 6.0 utilizando-se os testes de Kruskal–Wallis ou ANOVA, Mann Whitney ou T-student, dependendo da normalidade dos dados e o nível de significância foi de 5% (p < 0,05). Pacientes com LMC apresentaram índice de dano (ID) no DNA estatisticamente mais elevado em comparação ao GC (p < 0,0001). Quando os pacientes foram estratificados, foi verificado um aumento progressivo do ID no DNA nos grupos: LMC D, LMC G1 e LMC G2, respectivamente, em relação ao GC (p < 0,05). Pacientes com LMC apresentaram índice de micronúcleos (IMN), índice de pontes nucleoplasmáticas (IPN) e índice de buds nucleares (IBN) estatisticamente mais elevados em comparação com o GC (p < 0,05). Ao se estratificar os pacientes com LMC, foi verificado que pacientes dos grupos LMC G1 e G2 apresentaram IMN e IPN estatisticamente mais elevados em comparação ao GC (p < 0,001). O IMN também foi elevado no grupo LMC G2 em relação aos pacientes do grupo LMC D (p < 0,01). O índice de bud nuclear (IBN) não apresentou diferença estatística nas análises realizadas após a estratificação dos grupos. Os ITK revolucionaram a terapia da LMC, melhorando a sobrevida dos pacientes. No entanto esses resultados alertam para a relevância de estudos que avaliem os possíveis efeitos genotóxicos e mutagênicos dessa terapia a longo prazo. Os mecanismos envolvidos devem ser elucidados com a finalidade de aprimorar o tratamento, bem como avaliar o impacto clínico que esse dano pode causar.