Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cancer is a global health catastrophe, with neuroblastoma, the most common solid childhood tumor, and glioblastoma, a deadly brain tumor, being aggressive and unresponsive to current treatment modalities. These tumors are known to utilize uncontrollable cell proliferative capabilities as a mechanism for tumor survival. Therefore, malignant cell growth can be mitigated by targeting the essential proteins that regulate cell growth, such as receptor tyrosine kinases (RTKs). Under normal physiological conditions, RTKs bind with varying affinity to mitogenic stimuli such as growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) which, in turn, leads to receptor phosphorylation and activation.

Non-small cell lung cancer (NSCLC) makes up the majority of lung cancers, which remains the leading cause of cancer mortality worldwide. NSCLC with mutant epidermal growth factor receptor (EGFR) is currently treated with tyrosine kinase inhibitors (TKIs). TKIs have proven effective in improving survival until resistance is conferred, mostly by way of the exon 20, threonine 790 to methionine (T790M) point mutation in EGFR. The mechanism by which this point mutation arises is poorly understood. Herein we report a possible pathway by which the C to T transition that leads to T790M comes about. We show that activation-induced cytidine deaminase (AID) mRNA expression is induced upon treatment with EGFR TKIs in mutant-EGFR human lung cancer cell lines but not in control cell lines. We also show that stable expression of AID is sufficient to produce resistance to one such TKI, erlotinib, and is sufficient to produce T790M itself. We also report that B-cell lymphoma 6 (BCL6) may precede AID in this pathway. Our results show that BCL6 is upregulated in these cell lines treated with EGFR TKIs but not in normal bronchial cells. We then treated human lung cancer cell lines with EGFR TKIs in combination with BCL6 inhibitors. Our results show that AID is dependent upon BCL6 expression. Finally, we report on results from a transient BCL6 overexpression which lead us to believe that AID mRNA receives input from at least one alternate pathway in addition to BCL6. We also performed these experiments on a family of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, that show they may be involved in this pathway downstream of AID. Taken together, our results suggest a potential pathway involving BCL6, AID, and APOBEC cytidine deaminases that lead to the C to T transition that produces T790M, thereby conferring resistance to EGFR TKIs in mutant-EGFR NSCLC. They also provide potential new targets for treatment should further study confirm our results. / 2021-10-07T00:00:00Z

Chronic myeloid leukemia is characterized by a unique reciprocal translocation between chromosomes 9 and 22 resulting in deregulated tyrosine kinase activity. Tyrosine kinase inhibitors, such as imatinib, dasatinib, and nilotinib have revolutionized treatment of Chronic myeloid leukemia. However, tyrosine kinase inhibitors? use has presented new challenges in managing both acute and chronic toxicities, particularly ?off-target? toxicities like pleural effusion. Pleural effusions are seen less often with imatinib and very rarely with nilotinib. A 66-year-old male presented to emergency department with complaints of mild chest pain and dyspnea of 3 days duration with progressive worsening, including dyspnea at rest. Patient was currently taking nilotinib after failing imatinib for chronic myeloid leukemia. Nilotinib was put on hold. After exclusion of cardiac and pulmonary etiologies patient was treated for community acquired pneumonia with minimal improvement. Despite the very low incidence of pleural effusion with nilotinib (<1%), he was started on 20?mg of prednisone PO for 3 days. Patient had a dramatic improvement within 48?h after beginning prednisone. This treatment approach suggests that pleural effusions associated with nilotinib can be successfully treated in the same way as pleural effusions associated with dasatinib.

No description available.

Cytochrome P450 (CYP) enzymes play a crucial role in the human body. These enzymes are responsible for the synthesis of steroid hormones and cholesterol, as well as the metabolism of external substances such as medications. While more than 50 CYP enzymes have been identified, just 6 are credited with metabolizing most drugs. Of note is CYP 3A4, which metabolizes ~34% of medications that use the CYP enzyme system. CYP enzymes are polymorphic, meaning there are different versions of the same enzyme; therefore there is variability from individual to individual in their ability to metabolize medications. In the oncology field tyrosine kinase has been identified as an important target controlling cell regulatory functions and proliferation. Thus, tyrosine kinase inhibitors have become widely used for a variety of malignancies. Many of these tyrosine kinase inhibitors rely on CYP 3A4 for metabolism and are subject to variable toxicities based on an individual patient’s genome. A 62-year-old female was diagnosed with Renal Cell Carcinoma (RCC). After undergoing a left nephrectomy, a surveillance scan 21 months after diagnosis was concerning for metastatic disease, which was then confirmed through biopsy. The patient was started on sunitinib 50 mg on days 1-28 of a six-week cycle for metastatic RCC. The patient suffered from Grade 3 myelosupression/mucositis within two weeks of the initiation of therapy. The early onset and severity of the toxicity lead to CYP 3A4 pharmacogenetic testing. She was subsequently found to have a 3A4 polymorphism (*1/*28). The dose of sunitinib was reduced to 25 mg followed by a further reduction to 12.5 mg due to toxicity. Eighteen months after starting sunitinib, a CT scan showed disease progression and therapy was changed to pazopanib. Due to her 3A4 polymorphism, the starting pazopanib dose was empirically reduced by 50% and was started at 400 mg/daily. Pazopanib was held for episodes of severe diarrhea and was further reduced to 200 mg/daily. Nine months after starting pazopanib, new imaging showed lesions in the patient’s liver, confirming disease progression. The patient was subsequently started on nivolumab but quickly progressed. She was then started on cabozantinib at a dose reduced 20 mg/daily. This initial dosing was tolerated well by the patient, so a decision was made to alternate between 20 and 40 mg/daily to increase to an average of 30 mg/daily. She is currently tolerating the 30 mg/daily and continues treatment for metastatic RCC. As described above, CYP 3A4 polymorphisms can result in severe toxicities that present earlier in the treatment course than traditionally expected. Moving forward there may be a role in testing for these polymorphisms to determine an individual’s optimal dose before initiating therapy with tyrosine kinase inhibitors. The advantage of performing such testing would be to limit the severity of toxicities experienced by this patient population, while retaining the overall benefit of these medications.

No description available.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

No description available.

ADP-ribosylation factor-1 (ARF1) est une petite GTPase principalement connue pour son rôle dans la formation de vésicules au niveau de l’appareil de Golgi. Récemment, dans des cellules de cancer du sein, nous avons démontré qu’ARF1 est aussi un médiateur important de la signalisation du récepteur du facteur de croissance épidermique (EGFR) contrôlant la prolifération, la migration et l'invasion cellulaire. Cependant, le mécanisme par lequel l’EGFR active la GTPase ainsi que le rôle de cette dernière dans la régulation de la fonction du récepteur demeure inconnue. Dans cette thèse, nous avions comme objectifs de définir le mécanisme d'activation de ARF1 dans les cellules de cancer du sein hautement invasif et démontrer que l’activation de cette isoforme de ARF joue un rôle essentiel dans la résistance de ces cellules aux inhibiteurs de l'EGFR. Nos études démontrent que les protéines d’adaptatrices Grb2 et p66Shc jouent un rôle important dans l'activation de ARF1. Alors que Grb2 favorise le recrutement d’ARF1 à l'EGFR ainsi que l'activation de cette petite GTPase, p66Shc inhibe le recrutement du complexe Grb2-ARF1 au récepteur et donc contribue à limiter l’activation d’ARF1. De plus, nous démontrons que ARF1 favorise la résistance aux inhibiteurs des tyrosines kinases dans les cellules de cancer du sein hautement invasif. En effet, une diminution de l’expression de ARF1 a augmenté la sensibilité descellules aux inhibiteurs de l'EGFR. Nous montrons également que de hauts niveaux de ARF1 contribuent à la résistance des cellules à ces médicaments en améliorant la survie et les signaux prolifératifs à travers ERK1/2, Src et AKT, tout en bloquant les voies apoptotiques (p38MAPK et JNK). Enfin, nous mettons en évidence le rôle de la protéine ARF1 dans l’apoptose en réponse aux traitements des inhibiteurs de l’EGFR. Nos résultats indiquent que la dépletion d’ARF1 promeut la mort cellulaire induite par gefitinib, en augmentant l'expression de facteurs pro-apoptotiques (p66shc, Bax), en altérant le potentiel de la membrane mitochondriale et la libération du cytochrome C. Ensemble, nos résultats délimitent un nouveau mécanisme d'activation de ARF1 dans les cellules du cancer du sein hautement invasif et impliquent l’activité d’ARF1 comme un médiateur important de la résistance aux inhibiteurs EGFR. / The small GTPase ADP-ribosylation factor-1 (ARF1) has been well described for its role in regulating transport within the Golgi. Recently, in breast cancer cells, we have characterized ARF1 as important mediator of epidermal growth factor receptor (EGFR) signals leading to cell proliferation, migration and invasion. However, the mechanisms regulating ARF1 activity downstream of the EGFR had yet to be defined. Here, we aim to characterize these mechanisms of ARF1 activation in invasive breast cancer cells and demonstrate that activated ARF1 plays an essential role in mediating the resistance of breast cancer cells to EGFR tyrosine kinase inhibitors. We show that the adaptor proteins Grb2 and p66Shc regulate EGF-dependent ARF1 activation. While Grb2 was shown to be essential in the recruitment of ARF1 to the EGFR as well as the activation of this small GTPase, p66Shc blocked the recruitment of this Grb2-ARF1 complex to the receptor and thus suppressed EGF-induced ARF1 activation. Additionally, we demonstrate that ARF1 promotes EGFR tyrosine kinase inhibitor resistance in invasive breast cancer cells. Indeed, the depletion of ARF1 was associated with an increased sensitivity to EGFR inhibition. We show that ARF1 promotes resistance by enhancing survival and proliferative signals through Erk1/2, Src and AKT, while blocking the apoptotic p38MAPK and JNK pathways. Furthermore, ARF1 was shown to stabilize EGFR dynamics (Expression, activation, dimerization and down-regulation) in response to treatment with EGFR inhibitors. Finally, we highlight the role of ARF1 in mediating mitochondrial-dependent apoptosis in response to EGFR tyrosine kinase inhibitor treatment. The depletion of ARF1 was shown to promote gefitinib-induced cell death as measured by increase expression of pro-apoptotic factors(p66Shc, Bax), altered mitochondrial membrane potential and cytochrome C release. Together, our results delineate a novel mechanism of ARF1 activation in breast cancer cells and implicate ARF1 activity as an important mediator of EGFR inhibitor resistance further supporting the importance of targeting this GTPase in breast cancer patients.

L’étude des mécanismes aboutissant à la tumorigénèse a permis de révéler, dans beaucoup de cancers, une amplification ou une mutation de divers oncogènes, avec pour conséquence des capacités de prolifération et de survie accrues pour la cellule tumorale. L’identification des protéines kinases comme étant des éléments centraux de ces processus en ont fait des cibles thérapeutiques prometteuses. Plusieurs inhibiteurs ciblant de façon plus ou moins spécifique les tyrosines kinases oncogéniques ont ainsi été développés. Parmi eux, l’imatinib mesylate (Gleevec®, Novartis) a constitué la première chimiothérapie ciblée. Il correspond aujourd’hui au traitement de première intention contre la LMC. Cependant, malgré sa très grande efficacité, il est apparu que certains mécanismes de résistances pouvaient être mis en place pour diminuer son effet pro-apoptotique. Le travail de cette thèse a consisté à mieux comprendre les mécanismes d’apoptose induits par les inhibiteurs de tyrosine kinases (ITK), et à rechercher quelles voies alternatives de survie devront être bloquées pour leur assurer une meilleure efficacité. Trois modèles ont été utilisés : la leucémie myéloïde chronique (LMC), les leucémies aiguës myéloïdes (LAM) et les glioblastomes (GBM). La LMC a été utilisé comme modèle et la démarche utilisé pour essayer d’augmenter l’efficacité des ITK, a été transposée aux modèles des LAM et des GBM. L’ensemble des résultats obtenus a démontré qu’une meilleure compréhension de la réponse apoptotique et des mécanismes de résistance permettait l’identification de nouvelles cibles thérapeutiques. Nous avons pu observer que, tout en favorisant la diminution des doses de molécules administrées, l’inhibition simultanée de plusieurs cibles apportait plusieurs bénéfices. Elle permet d’augmenter l’action pro-apoptotique des ITK, de contrer certains mécanismes de résistances, d’atteindre la cellule souche cancéreuse résistance et par conséquent de cibler simultanément des populations a plusieurs de stades de différenciation. / Protein kinases have been identified as playing fundamental roles in cancer development, suggesting that they could represent a promising therapeutic target. Several kinase inhibitors have been developed and the most successful of them, by far, is Gleevec® (imatinib, STI57; Novartis), a BCR-ABL inhibitor. It is currently used as the treatment of reference for chronic myeloid leukemia. However, despite a huge efficiency, some resistance mechanisms could be used to decrease its pro-apopototic effect. The global aim of my PhD was to understand the apoptotic mechanisms induced by tyrosine kinase inhibitors (TKI) to identify new potential therapeutic targets. I work on three different tumors: Chronic Myeloid Leukemia (CML), Acute Myeloid Leukemia (AML) and Glioblastomas (GBM). CML has been used as a model and the approach followed to increase TKI efficiency has been transposed to AML and GBM models. Altogether, our results showed that a better understanding of apoptotic response and resistance mechanisms could lead to the identification of new therapeutic targets. We observed that combination therapy brings several benefits. It allows to increase the TKI-induced apoptotic response, to counter some resistance mechanism, to reach the resistant cancer stem cells, and thus, to target simultaneously several populations in the tumour.