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1
Hewlett, Mark. "The evolution of resistance to multidrug antibiotic therapies." Thesis, University of Exeter, 2015. http://hdl.handle.net/10871/21596.
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The purpose of this thesis is to explore the interaction between antibiotics at sub-lethal doses, and E.coli. Initially we focussed on pairwise antibiotic interaction, and the potential to exploit these interactions to minimise antibiotic resistance. In testing the hypothesis that antagonism will slow adaptation by reducing selection for resistance we determined that there are conditions in which this fails to be the case. We furthermore caution against treating drug interactions as anything other than a dynamic property of the bacteria-drug interaction, by showing that the relationship between two drugs may be both synergistic and antagonistic depending on a variety of factors. Whilst exploring the adaptive response to drug combinations we discovered a highly unusual effect of Doxycycline to act as a growth stimulant to E.coli AG100. Chapter 3 and 4 are devoted to determining the nature and mechanism of this stimulation, and analysing any potential genomic changes using whole genome re-sequencing. Having shown that dose response is not always a monotone function of increasing drug dose, in chapter 5 we also look at the dose response in a diffusive context, using a custom built imaging system to show the common non-monotonicity of disk diffusion type assays, that manifest themselves as bullseye patterns of growth. We use a mathematical model to explore the ecological and adaptive reasons for such patterns. Finally in chapter 6 we look at the coevolutionary history of phage and E.coli REL606 strains, by determining trade-offs caused by lambda phage and the sole carbon source maltotriose both utilising the same porin (lamB) for cell entry.
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Guix, Arnau Marta 1974. "Mechanisms of acquired resistance to anti-EGFR therapies in squamous cell carcinoma." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/565440.
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Targeted therapies against the Epidermal Growth Factor Receptor (EGFR) are useful to treat many human cancers such as non-small cell lung cancer, colorectal cancer and head and neck cancer. However, the efficacy of such treatments is always compromised by resistance. This doctoral thesis has focused in the mechanisms of acquired resistance to targeted therapies against the EGFR (such as the small tyrosine kinase inhibitors gefitinib and erlotinib, or the monoclonal antibody cetuximab) in squamous cell carcinomas. In the first part of the thesis, preclinical studies with cellular and xenograft models were developed to elucidate the molecular mechanisms of resistance; the second part of the thesis was performed in tumor samples from patients with advanced squamous cell carcinomas of the head and neck. The main finding from the preclinical analysis was that the activation of the insulin-like growth factor receptor 1 system, mainly through downregulation of insulin-like growth factor binding proteins, is responsible for the acquired resistance to anti-EGFR therapies. However, these results could not be validated in a small sample set of advanced head and neck cancer patients.Els tractaments dirigits contra el receptor del factor de creixement epidèrmic (EGFR) són útils en diversos càncers en l’home, com el càncer de pulmó de cèl·lula no petita, el càncer colorrectal o els tumors de cap i coll. Però l’eficàcia d’aquests tractaments sempre està limitada per l’aparició de resistències. Aquesta tesi doctoral s’ha centrat en investigar els mecanismes de resistència adquirida a tractaments dirigits contra l’EGFR (com els inhibidors tirosina quinasa gefitinib i erlotinib o l’anticòs monoclonal cetuximab) en carcinomes escamosos. En la primera part de la tesi s’han desenvolupat estudis preclínics amb models cel·lulars i xenoinjerts per desxifrar els mecanismes moleculars de resistència; la segona part de la tesi ha inclòs estudis en mostres de carcinomes escatosos de cap i coll de pacients amb tumors avançats. La troballa principal dels estudis preclínics ha estat que l’activació del sistema del receptor del factor de creixement semblant a la insulina, principalment a través de la disminució dels nivells de les proteïnes d’unió als factors de creixement semblants a la insulina, és la responsable de l’aparició de resistència adquirida als tractaments anti-EGFR. Posteriorment, però, aquests resultats no han estat validats en una petita cohort de pacients amb tumors avançats de cap i coll.
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McGivern, Niamh. "Activation of MAPK signalling results in resistance to therapies for ovarian cancer." Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.695671.
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Ovarian cancer is the fifth most common cancer affecting women in the UK, and is the most lethal of all gynaecological cancers. Treatment primarily consists of debulking surgery followed by a platinum and taxane based chemotherapy, and despite initial high levels of response, the majority of women will relapse with platinum resistant disease, resulting in a poor prognosis. Few targeted therapies have entered the clinic for the treatment of ovarian cancer, however there is strong preclinical data to suggest there is a therapeutic window to target SRC tyrosine kinase in this disease. The SAPPROC trial was a phase 11 trial investigating the addition of the SRC inhibitor saracatinib (AZD0530) to weekly paclitaxel in platinum resistant ovarian cancer, however, this study showed no benefit to survival from this combination. The work presented in this thesis has investigated potential mechanisms of resistance to SRC inhibitors in ovarian cancer, and by using two complementary screening approaches, highlighted a role for activated MEK signalling in driving this resistance. Further work demonstrated that a SRC and MEK are parallel pathways, and inhibition of one leads to activation of the other. This has important therapeutic implications, and a combination of SRC and MEK inhibitors was shown to synergistically inhibit the growth of ovarian cancer cells, and so this combination may be logical in the clinic. Importantly, the SAPPROC trial was performed in platinum resistant ovarian cancer patients, and it has previously been reported that platinum resistant cells exhibit activated MAPK signalling. A novel platinum resistant ovarian cancer cell line was generated in vitro, and demonstrated activated MAPK, as well as resistance to SRC inhibition. This suggests that SAPPROC may have failed to show any benefit of SRC inhibition in platinum resistant patients has their tumours already exhibit activated MEK, and therefore did not respond to SRC inhibition. This highlights the need to further understand mechanisms of drug resistance, the the importance of this in the design of clinical trials.
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Sun, Xiaowen. "An integrin-based mechanism for sensitizing melanomas to therapies." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/6506.
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Metastatic melanoma is unusually lethal with a ten year survival rate of less than 10%. Conventional DNA-damaging agents produce little improvement in patient survival. Vemurafenib (Zelboraf), a targeted therapeutic that inhibits the oncogenic BRAF demonstrates significant survival benefit. Unfortunately, it is now evident that there is both intrinsic and acquired resistance. Consequently, new strategies for sensitizing melanomas to vemurafenib are needed. Melanoma resistance to therapy is fueled in part by the integrins, the major cell surface adhesion receptors which are highly over-expressed in melanoma. Both integrin antagonists and agents that engage defective integrins increase the sensitivity of melanomas to chemotherapy.
Our laboratory has identified a novel peptide, denoted vinculin activating peptide or VAP that targets integrins from within the cell and brings aberrant integrin function intact. VAP sensitizes melanoma to dacarbazine in vitro and in vivo. The effect VAP has on overcoming resistance to targeted therapies like vemurafenib, as well as the mechanism for its effects are not well understood. The goals of this project are to determine if VAP can be employed to improve sensitivity and/or overcome resistance to vemurafenib and to identify the cell surface target of VAP. Our results show that VAP not only improves melanoma sensitivity to vemurafenib but also decreases intrinsic resistance to this promising drug. In addition, we present evidence that β1 and β3 integrins are the target of VAP's effects.
Since peptide-based therapies are not stable in the clinic, we explored another integrin binding partner, kindlin-2. We found that kindlin-2 is over expressed in resistant melanomas. The inhibition of kindlin-2 increases β1 integrin activation and decreases β3 integrin functions. Agents that bring aberrant β1 and β3 integrin function intact can be employed to improve sensitivity and overcome resistance to vemurafenib suggesting that combinatorial therapies that employ vemurafenib and integrin-based agents might be efficacious in combatting resistance in melanoma patients.
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Neto, João Manuel Fernandes. "Improvement of antiangiogenic therapies in colorectal cancer." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15349.
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Mestrado em Biotecnologia – Biotecnologia Industrial e AmbientalAngiogenesis is essential for tumor progression. Antiangiogenic therapies block angiogenesis and cause vessel regression, which leads to an increase of tumor hypoxia. Hypoxia is responsible for many effects in tumor biology, among which, the selection of cells that are more aggressive and more resistant to cancer therapies. In this project we aim to get some molecular insight on the mechanism(s) underlying the resistance to the combination of bevacizumab and cetuximab and to find synthetic lethal interactions with hypoxia. Our results show that: hypoxia induces resistance to EGFR inhibition in WT4 CRC cell; HIF1α is not driving the resistance phenotype; hypoxia activates RAS in WT4 CRC cells; MEK inhibitors increase the sensitivity to EGFR inhibitors in hypoxia and cytokines seem to be involved in the activation of RAS in hypoxia. We also identified four genes as potential candidates to be synthetic lethal with hypoxia. Our findings are of great clinical and biological significance and may lead to better combination therapies, improving current treatments for CRC patients and may also lead to the discovery of biomarkers of response to antiangiogenic therapies.
A angiogénese é essencial à progressão tumoral. As terapias antiangiogénicas bloqueiam a angiogénese e causam regressão dos vasos sanguíneos, o que leva a um aumento da hipóxia nos tumores. A hipóxia é responsável por diversos efeitos na biologia tumoral, entre os quais, a seleção de células cancerígenas mais agressivas e mais resistentes às terapias. Com este projeto pretendemos descobrir o mecanismo molecular envolvido na resistência à combinação de bevacizumab e cetuximab e também encontrar interações de letalidade sintética com hipóxia. Os nossos resultados mostram que: a hipóxia induz resistência à inibição de EGFR em células WT4 de cancro coloretal; o HIF1α não é responsável pelo fenótipo de resistência; a hipóxia ativa RAS em células WT4 de cancro coloretal; os inibidores de MEK aumentam a sensibilidade aos inibidores de EGFR em hipóxia e as citoquinas parecem estar envolvidas na ativação de RAS em hipóxia. Identificámos ainda quatro genes que são potenciais candidatos a terem letalidade sintética com hipóxia. Estes resultados têm uma grande importância clínica e biológica e podem conduzir a melhores terapias combinatórias, contribuindo para melhorar os atuais tratamentos de pacientes com cancro coloretal e podem ainda levar à descoberta de biomarcadores de resposta a terapias antiangiogénicas.
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Phee, Lynette. "Unorthodox antimicrobial combination therapies for the treatment of multi-drug resistant Gram-negative infections." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/44695.
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The rise of antimicrobial resistance (AMR) has culminated in the most pressing problem in modern medicine. The situation is most acute with regards to the management of multi- drug resistant Gram-negative infections (MDRGNB) with common infections increasingly untreatable due to rapidly dwindling therapeutic options. A solution to the problem of AMR is unlikely to be easily found, but revisiting and re-purposing existing antimicrobials is a viable approach in the medium term. This study investigated the use of unorthodox antimicrobial combination therapies for the treatment of MDRGNB, with particular focus on agents of last resort. A systematic review of clinical studies highlighted the potential for polymyxin (colistin) combination therapies (e.g. colistin-rifampicin, colistin-carbapenems), although this could not be supported in a formal meta-analysis. A systematic approach for screening MDRAB for susceptibility to novel colistin combinations using multiple methods was employed and uncovered a number that were more potent than those previously identfied. The most potent combination that was consistently identified was colistin when combined with fusidic acid, despite this drug having no useful activity against MDRGNB on its own. The combination was further evaluated in static time-kill assays against a range of Gram-negative pathogens with defined resistance mechanisms, including to polymyxins and using invertebrate (Galleria mellonella) and murine models of MDRGNB infection. Colistin and fusidic acid combination therapy was subsequently used to successfully treat a case of ventilator-associated pneumonia due to MDR A. baumannii. This work highlights how older drugs can be re-purposed to tackle the problem of AMR using a precision medicine approach. Further studies to elucidate the mechanism of action of the colistin- fusidic acid combination and a formal clinical trial are warranted to investigate the potential utility of this combination in the treatment of MDRGNB with the expressed goal of bridging the current antimicrobial development gap.
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Cerqueira, Vera. "Role of intracellular signalling pathways in conferring resistance to endocrine therapies in breast cancer." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4511.
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Breast cancer is the most prevalent form of cancer in women and accounts for 519,000 annual deaths (WHO Statistics). It has long been established that oestrogen (E2) stimulates tumour growth of oestrogen receptor (ER) positive breast cancer and is involved in the pathogenesis of the disease. Consequently, therapeutic approaches targeting the ER were developed. The use of endocrine therapy is an integral component in treating breast cancer however resistance to such drugs is a major limitation. Unfortunately, even initially responding tumours eventually develop resistance - acquired resistance. The aim of this study was to determine which intracellular pathways may be important in conferring acquired endocrine resistance. In order to do so, a three-stage MCF-7 cell model emulating the clinical development of acquired endocrine was used. MCF-7/LCC1 (LCC1) and MCF-7/LCC9 (LCC9) cells lines were derived from the oestrogen dependent and antioestrogen sensitive MCF-7 cell line. LCC1 cells remain responsive to endocrine therapies but their growth is not dependent on oestrogenic stimulus. LCC9 cells, on the other hand are fully resistant to endocrine therapies and completely oestrogen independent. A number of different cell membrane receptors and intracellular pathways have been implicated in endocrine resistance including HER receptor family, PI3K/Akt & MEK/ERK pathways. These pathways are of particular interest since they are able to activate ER in the absence of oestrogenic stimulus. It is likely that several pathways may be important in conferring resistance to endocrine therapies therefore the experiments in this study focussed on the transcriptional regulation of HER receptors, the activation of the Akt pathway and its implication to basic cellular processes. Following E2 treatment (48h), HER2/3/4 mRNA and protein levels were reduced in MCF- 7 and LCC1 but not in the endocrine-resistant LCC9 cell line as measured by QRT-PCR and Western blotting. The anti-estrogen fulvestrant (ICI 182,780) reversed the E2 modulation. A previous study has shown that ER and the HER2 promoter compete for limiting amounts of SRC-1 in oestrogen-responsive ZR-75-1 cells, causing HER2 repression after E2 stimulation (Newman et al.,Oncogene, 19, 490-7, 2000). ER RNAi abolished E2 repression of HER2 in MCF-7 and LCC1 cells. Furthermore, LCC9 cells have reduced SRC-1 recruitment to ER (assessed by ChIP) allowing SRC-1 to bind to the HER2 promoter. SRC-1 RNAi reduced HER2 transcription in MCF7 cells in a manner similar to E2 whilst it did not restore E2 repression in LCC9 suggesting that the latter cells have alternative mechanisms regulating HER2 transcription. RNAis against the other two p160 co-activators TIF2 and AIB1 did not restore E2 mediated HER2 repression in LCC9 cells. The importance of redundancy between p160 co-activators was also determined by performing double knockouts. SRC-1/TIF2 and TIF2/AIB1 double siRNAs had little effect on HER2 mRNA levels however SRC-1/AIB1 siRNA restored oestrogen mediated downregulation of HER2 transcription in LCC9 cells. This data indicates that SRC-1 and AIB1 co-activators play a role in the transcriptional regulation of HER receptor particularly in MCF-7 and LCC1 cells. The regulation of this transcriptional mechanism is altered in resistant LCC9 cells but, as evidenced by the double knockouts, p160 coactivators are still able to affect HER expression in these cells. This mechanism was further studied in primary breast cancer tumour material. The importance of the Akt pathway in this cell line model was also investigated as phospho-Akt levels are elevated in LCC1 and LCC9 cells. This in turn was shown to activate mTOR and ER (Ser167 residue phosphorylation) thereby contributing to increased growth and ligand independent activation of the oestrogen receptor respectively. Activation of PI3K and PTEN is unchanged in LCC1 and LCC9 cells suggesting that these proteins are not responsible for elevated Akt phosphorylation. In contrast, these cells do express higher levels of phospho-IGFR due to the high availability of receptor ligands (IGFI & IGFII). This is likely to be, at least partially, responsible for the elevated Akt activation. Moreover, the role of Akt isoforms was also determined as they are known to have different functions. The levels of Akt 2 phosphorylation are higher in endocrine resistant cell lines in comparison to parental MCF-7 cells. Interestingly, the Akt 3 phosphorylation is present in all cell lines whilst Akt 1 phosphorylation is minimal. Nevertheless, Akt RNAi studies reveal that Akt 1 and 2 siRNA dramatically reduce growth in MCF-7, LCC1 and LCC9 cells. These results suggest that Akt 2 phosphorylation may play a part in conferring endocrine resistance but the other isoforms are also important for normal cellular growth. The cell cycle profiles of LCC1 and LCC9 are very similar to MCF-7. Similarly, migration levels are unchanged in endocrine resistant cell lines. However, in the presence of antioestrogenic drugs, apoptosis in LCC1 and LCC9 cells in reduced in comparison to the parental MCF-7 cell line. Furthermore, LCC1 and LCC9 cells have higher invasion rates. The deregulation of HER receptor expression and elevated Akt activation may together confer survival advantage in LCC1 and LCC9 cells whilst also increasing their invading potential.
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Yeoman, Kathryn (Kate) Charlotte. "Working the System: Doing Postmodern Therapies in Aotearoa New Zealand." Thesis, University of Canterbury. Humanities, 2012. http://hdl.handle.net/10092/7274.
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This thesis documents a qualitative research study of twenty postmodern therapy practitioners in Aotearoa New Zealand, focusing on their experiences in the wider field of therapy. The participants were aligned in their subscribing to postmodern critiques of therapy as a instrument of power, and in their interest in, and use of, therapy techniques and approaches that have grown out of those critiques – including narrative therapy, critical psychology, “Just Therapy”, and feminist poststructuralist therapy approaches. I argue that these practitioners represent a social movement within the field of therapy. The thesis examines the nature of the wider therapy field in Aotearoa New Zealand, analysing the perspectives of the participants. I demonstrate how this field has become increasingly dominated by the twin forces of neoliberalism and bio-science, making postmodern therapy work difficult, particularly within public sector services. In the final substantive part of the thesis, I critically examine and appraise the strategies used by participants to negotiate and resist these forces. This discussion is divided into two main chapters, dealing first with the participants who have difficulty in engaging in official politics and who consequently attempt to operate “under the radar” of management surveillance: these participants are characterised as “battlers”, “burn-outs” and “blow-outs”. Then, I turn my attention to the second group of participants – “infiltrators”, “outsiders” and “accepters” – who strategically utilise symbolic capital to pose resistance, or simply leave the public system. I also consider the professed abilities of this second group to cultivate a postmodern sensibility and to tolerate contradiction and compromise. I conclude this investigation of the possibilites for resistance to neoliberal and bio-scientific discourses by recommending greater strengthening of this local postmodern therapy movement.
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Söderhäll, Thomas. "Antibiotic combination therapies against carbapenamse producing Klebsiella pneumoniae." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-452424.
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The treatment options for multidrug resistant bacteria are dwindling and it is an important issue of research in medicine to solve. One of the more problematic bacterial species is Klebsiella pneumoniae, it can cause infections with high morbidity that are difficult to treat. Common antibiotics for treatment of these infections are carbapenems but K. pneumoniae can produce enzymes called carbapenemases that can hydrolyze carbapenems and most other beta-lactam antibiotics. In this study carbapenemase genes were introduced chromosomally to a previously susceptible K. pneumoniae strain using λ-Red recombineering. Further constructs were made with non-functional porins to examine how they affect combination treatment with carbapenems. Antibiotic combination therapy was evaluated against constructed carbapenemase- (KPC-2, NDM-1 and OXA-48) producing K. pneumoniae strains. Screening was done using time-lapse microscopy (oCelloScope), and combinations with better effect than treatment with a single antibiotic were chosen for time-kill assays. The results shows that a triple combination of colistin, meropenem and the beta-lactamase inhibitor avibactam gives an improved effect, up to twice the effect compared to monotherapy and up to 1.8 times increased effect compared to double combination. The synergistic effect was greater when adding colistin to treat the strains with non-functional porins, indicating that colistin can increase the permeability for other antibiotics into the cell. This is an interesting finding that need to be researched further.
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Salazar, Marcela d'Alincourt. "Genomic Effects of Hormonal Adjuvant Therapies that Could Support the Emergence of Drug Resistance in Breast Cancer." University of Toledo Health Science Campus / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=mco1280929084.
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Costanzini, Anna <1990>. "Bioenergetics of cancer cells in anoxia and role of the miRNAs in melanoma resistance to targeted therapies." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amsdottorato.unibo.it/8810/1/Costanzini_Anna_tesi.pdf.
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Tumours are characterized by a metabolic rewiring that helps transformed cells to survive in harsh conditions.
The endogenous inhibitor of the ATP-synthase IF1 is overexpressed in several tumours and it has been proposed to drive metabolic adaptation. In ischemic normal-cells, IF1 acts limiting the ATP consumption by the reverse activity of the ATP-synthase, activated by ΔΨm collapse. Conversely, IF1 role in cancer cells is still unclear. It has been proposed that IF1 favours cancer survival by preventing energy dissipation in low oxygen availability, a frequent condition in solid tumours. Our previous data proved that in cancer cells hypoxia does not abolish ΔΨm, avoiding the ATP-synthase reversal and IF1 activation. In this study, we investigated the bioenergetics of cancer cells in conditions mimicking anoxia to evaluate the possible role of IF1. Data obtained indicate that also in cancer cells the ΔΨm collapse induces the ATP-synthase reversal and its inhibition by IF1. Moreover, we demonstrated that upon uncoupling conditions, IF1 favours cancer cells growth preserving ATP levels and energy charge. We also showed that in these conditions IF1 favours the mitochondrial mass renewal, a mechanism we proposed driving apoptosis-resistance.
Cancer adaptability is also associated with the onset of therapy resistance, the major challenge for melanoma treatment. Recent studies demonstrated that miRNAs dysregulation drive melanoma progression and drug-resistance by regulating tumour-suppressor and oncogenes. In this context, we attempted to identify and characterize miRNAs driving resistance to vemurafenib in patient-derived metastatic melanoma cells BRAFV600E-mutated. Our results highlighted that several oncogenic pathways are altered in resistant cells, indicating the complexity of both drug-resistance phenomena and miRNAs action. Profiling analysis identified a group of dysregulated miRNAs conserved in vemurafenib-resistance cells from distinct patients, suggesting that they ubiquitously drive drug-resistance. Functional studies performed with a first miRNA confirmed its pivotal role in resistance towards vemurafenib.
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Liu, Ta-Ming. "Kinase Targeting Therapies in Chronic Lymphocytic Leukemia: Mechanisms of Acquired Ibrutinib Resistance and the Pre-Clinical Development of OSU-T315." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1404917090.
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Recondo, Gonzalo. "Resistance Mechanisms to ALK Tyrosine Kinase Inhibitors (TKIs) in NSCLC." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS248/document.
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Les analyses moléculaires et la classification des adénocarcinomes bronchiques ont conduit au développement de thérapies ciblées sélectives visant à améliorer le contrôle de la maladie et la survie des patients. ALK (anaplastic lymphoma kinase) est un récepteur tyrosine kinase de la famille des récepteurs de l'insuline. Des réarrangements chromosomiques impliquant le domaine kinase d’ALK sont présents dans environ 3 à 6% des patients atteints d'un adénocarcinome bronchique. La protéine de fusion provoque une activation du domaine kinase de manière constitutive et indépendante du ligand. Lorlatinib est un inhibiteur d’ALK de troisième génération avec une efficacité et une sélectivité optimale, ainsi qu’une pénétration élevée vers le système nerveux central. Lorlatinib peut vaincre la résistance induite par plus de 16 mutations secondaires dans le domaine kinase d’ALK acquises lors de la progression aux ALK TKI de première et deuxième générations. Le traitement par lorlatinib est donc efficace chez les patients préalablement traités par un ALK TKI de première ou deuxième génération, et est actuellement approuvé pour cette indication. Le spectre complet de mécanismes de résistance au lorlatinib chez les patients reste à élucider. Il a récemment été rapporté que l'acquisition séquentielle de deux mutations ou plus dans le domaine kinase, également appelées mutations composées, est responsable de la progression de la maladie chez environ 35% des patients traités par le lorlatinib, principalement en altérant sa liaison au domaine kinase d’ALK. Cependant, l’effet de ces mutations sur la sensibilité aux différents inhibiteurs d’ALK peut varier, et les autres mécanismes de résistance survenant chez la plupart des patients restent inconnus. Mon travail de thèse avait pour but d’explorer la résistance au lorlatinib chez des patients atteints d'un cancer du poumon ALK réarrangé par la mise en œuvre de biopsies spatiales et temporelles et le développement de modèles dérivés de patients. Dans le cadre de l’étude institutionnelle MATCH-R (NCT02517892), nous avons effectué un séquençage à haut débit de l’exome, de l’ARN et ciblé, ainsi qu’un séquençage des ctDNA afin d’identifier les mécanismes de résistance. Nous avons établi des lignées cellulaires dérivées de patients et caractérisé de nouveaux mécanismes de résistance et identifiés de nouvelles stratégies thérapeutiques in vitro et in vivo. Nous avons identifié trois mécanismes de résistance chez quatre patients avec des biopsies appariées. Nous avons étudié l'induction de la transition épithélio-mésenchymateuse (EMT) par l'activation de SRC dans une lignée cellulaire, dérivée d’un patient, exposée au lorlatinib. Les cellules mésenchymateuses étaient sensibles à l’inhibition combinée de SRC et d'ALK, montrant que même en présence d'un phénotype agressif, des stratégies de combinaison peuvent surmonter la résistance aux ALK TKI. Nous avons identifié deux nouvelles mutations composées du domaine kinase d’ALK, F1174L / G1202R, C1156Y / G1269A survenues chez deux patients traités par le lorlatinib. Nous avons développé des modèles de cellules Ba / F3 exprimant les mutations simples et composées pour étudier leur effet sur la résistance au lorlatinib. Enfin, nous avons caractérisé un nouveau mécanisme de résistance provoqué par la perte de fonction de NF2 au moment de la progression du lorlatinib par l’utilisation de PDX et de lignées cellulaires dérivées de patients, et par CRISPR / CAS9 knock-out de NF2. Nous avons constaté que l'activation de mTOR par la perte de fonction de NF2 provoquait la résistance au lorlatinib et qu'elle pouvait être surmontée par le traitement avec des inhibiteurs de mTOR.Cette étude montre que les mécanismes de résistance au lorlatinib sont plus divers et complexes que prévu. Nos résultats démontrent également comment les études longitudinales de la dynamique tumorale permettent de déchiffrer la résistance aux TKI et d'identifier des stratégies thérapeutiquesThe molecular study and classification of lung adenocarcinomas has led to the development of selective targeted therapies aiming to improve disease control and survival in patients. The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor from the insulin tyrosine kinase receptor family, with a physiologic role in neural development. Gene rearrangements involving the ALK kinase domain occur in ~3-6% of patients with lung adenocarcinoma. The fusion protein dimerizes leading to transactivation of the ALK kinase domain in a ligand-independent and constitutive manner. Lorlatinib is a third generation ALK inhibitor with high potency and selectivity for this kinase in vitro and in vivo, and elevated penetrance in the central nervous system. Lorlatinib can overcome resistance mediated by over 16 secondary kinase domain mutations occurring in 13 residues upon progression to first - and second - generation ALK TKI. In addition, treatment with lorlatinib is effective for patients who have been previously treated with a first and a second generation or a second generation ALK TKI upfront and is currently approved for this indication. The full spectrum of biological mechanisms driving lorlatinib resistance in patients remains to be elucidated. It has been recently reported that the sequential acquisition of two or more mutations in the kinase domain, also referred as compound mutations, is responsible for disease progression in about 35% of patients treated with lorlatinib, mainly by impairing its binding to the ALK kinase domain. However, the effect of these compound mutations on the sensitivity to the repertoire of ALK inhibitors can vary, and other resistance mechanisms occurring in most patients are unknown. My PhD thesis aimed at exploring resistance to lorlatinib in patients with ALK-rearranged lung cancer through spatial and temporal tumor biopsies and development of patient-derived models. Within the institutional MATCH-R study (NCT02517892), we performed high-throughput whole exome, RNA and targeted next-generation sequencing, together with plasma sequencing to identify putative genomic and bypass mechanisms of resistance. We developed patient-derived cell lines and characterized novel mechanisms of resistance and personalized treatment strategies in vitro and in vivo. We characterized three mechanisms of resistance in four patients with paired biopsies. We studied the induction of epithelial-mesenchymal transition (EMT) by SRC activation in a patient-derived cell line exposed to lorlatinib. Mesenchymal cells were sensitive to combined SRC and ALK co-inhibition, showing that even in the presence of an aggressive and challenging phenotype, combination strategies can overcome ALK resistance. We identified two novel ALK kinase domain compound mutations, F1174L/G1202R, C1156Y/G1269A, occurring in two patients treated with lorlatinib. We developed Ba/F3 cell models harboring single and compound mutations to study the differential effect of these mutations on lorlatinib resistance. Finally, we characterized a novel mechanism of resistance caused by NF2 loss of function at the time of lorlatinib progression through the development of patients derived PDX and cell lines, and in vitro validation of NF2 knock-out with CRISPR/CAS9 gene editing. Downstream activation of mTOR was found to drive lorlatinib resistance by NF2 loss of function and was overcome by providing treatment with mTOR inhibitors.This study shows that mechanisms of resistance to lorlatinib are more diverse and complex than anticipated. Our findings also emphasize how longitudinal studies of tumor dynamics allow deciphering TKI resistance and identifying reversing strategies
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Tousignant, Kaylyn Davis. "Investigation of metabolic rewiring in prostate cancer cells during the adaptive response to androgen-targeted therapies." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/180822/1/Kaylyn_Tousignant_Thesis.pdf.
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The development of therapy resistance is a major obstacle in the successful treatment of advanced prostate cancer. This thesis investigated mechanisms that help drive therapy resistance and discovered that prostate cancer cells can utilise different metabolic pathways in order to become resistant to current therapies. This project also explores new therapeutic strategies to use in combination with current treatments to help fight disease progression and improve outcomes for men with prostate cancer.
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Esteve, Arenys Anna. "Innovative targeted therapies for chemorefractory B-cell non-Hodgkin lymphomas." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/565937.
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Lymphomas are a heterogeneous group of tumors characterized by the proliferation of lymphocytes predominantly in lymphoid structures but also in extranodal tissues. More than 90% of patients are afflicted by lymphomas of B-cell origin.
The current World Health Organization (WHO) classification of hematopoietic and lymphoid tumors categorizes B-cell neoplasms in more than 40 distinct disease entities, according to a combination of the morphology, immunophenotype, genetic, molecular and clinical features. Each entity has its own clinical course and requires specific treatments.
The characterization of activated signaling pathways involved in survival and proliferation, together with the development of a wide pharmacological armamentarium against cancer, have facilitated the bench-to-bedside translation of new targeted therapies in B-cell non-Hodgkin lymphomas (B-NHL). These novel therapies include two of the most relevant drugs lately approved: the anti-apoptotic agent venetoclax and the BTK inhibitor ibrutinib.
One major hurdle to their successful application is the rise of drug resistance. Resistance to therapy is observed in many cases of B-cell malignancies. This phenomenon significantly limits the utility of the current therapeutic strategies, and remains a substantial challenge for the clinical management of patients with advanced cancers. Resistance comes in two flavors: intrinsic resistance (also known as innate or de novo resistance) and acquired resistance, resulting from the clonal evolution of resistant variants.
With this concept in mind, in this thesis we have explored new approaches to overcome the development of drug resistance.
Venetoclax (ABT-199) is a first-in-class BH3 mimetic, FDA-approved for use in patients with R/R del17p chronic lymphocytic leukemia. In the clinical setting, it has demonstrated high response rates and good toxicity profiles in other subtypes of relapsed/refractory non-Hodgkin lymphoma.
We proposed a model of double hit lymphoma (DHL) resistance to ABT-199 in which the capacity of CPI203 to regulate the transcriptome of the cells could help to circumvent this problem. In ABT-199 sensitive cells, the BH3 mimetic acts by displacing BIM from BCL-2 complexes, allowing the de-repression and/or direct activation of BAX and leading to an activation of mitochondrial outer membrane permeabilization. In DHL cells, a compensatory upregulation of BFL-1 would bind and inactivate the pool of BIM proteins released from BCL-2 by ABT-199, avoiding MOMP and preserving cell survival. CPI203 primes cells to death by decreasing BFL-1 and
increasing BIM protein levels, and its combination with ABT-199 allows to tip the balance between pro- and anti-apoptotic signaling toward induction of cell death. This concept provides a new insight in the proposed mechanisms of resistance to BH3- mimetics in NHL cell lines, where MCL-1 and BCL-XL have been proposed as the major determinants of drug sensitivity and acquired resistance.
On the other hand, ibrutinib is a BTK inhibitor approved for first-line therapy in patients with chronic lymphocytic leukemia, as well as for the treatment of some relapsed/refractory B-NHL. Despite its high level of clinical activity, acquiring of mutations or re-wiring of the BCR pathway to retain the downstream signaling appears to be a common mechanism of resistance.
The inhibition of more than one BCR kinase might be useful in B-NHL cases that are resistant to the sole inhibition of BTK. We describe the compound IQS019 as a new BCR kinase inhibitor able to counteract both constitutive and ligand-dependent activation of the BCR pathway. Its capacity to inhibit the three upstream BCR kinases, BTK, SYK and LYN, confer an advantage over the inhibition of BTK alone by ibrutinib, in in vitro and in vivo models of B-NHL, being of special importance for the treatment of those patients with non-canonical NF-κB activation, who are low responders to ibrutinib.
Therefore, the development of innovative therapeutic approaches that permit to overcome drug resistance opens a window to important therapeutic advances in the treatment of B-NHL.Las neoplasias linfoides de célula B constituyen un grupo heterogéneo de tumores caracterizados por la proliferación de linfocitos B. Cada entidad clínica posee unas características particulares y requiere de un tratamiento específico. A pesar de los importantes avances terapéuticos, la supervivencia a largo plazo sigue siendo baja y precisa de un desarrollo constante de nuevas aproximaciones terapéuticas. Uno de los mayores problemas asociados a la respuesta a fármacos son las resistencias. En muchos casos estas resistencias se deben a cambios en proteínas diana o a la modulación compensatoria de otras proteínas o vías de señalización. El conocimiento de estos cambios será de gran importancia para poder encontrar aproximaciones terapéuticas que permitan eliminar estas resistencias. El linfoma doble-hit es un linfoma agresivo caracterizado por su baja respuesta a la quimioterapia estándar. Entre los múltiples agentes terapéuticos específicos actualmente en desarrollo encontramos el inhibidor de BCL-2, venetoclax. El venetoclax ha demostrado ser efectivo en varios subtipos de linfoma pero su uso conlleva el problema de la aparición de resistencias. Varios estudios han destacado el papel de proteínas de la familia BCL-2 en este proceso. Nuestros resultados indican que la regulación positiva de BFL-1 es uno de los factores clave en el desarrollo de resistencias al fármaco. Su regulación mediante el CPI203, un modulador epigenético, resulta en una sensibilización al venetoclax, tanto in vitro como in vivo. Por otro lado, la señalización de los receptores de células B (BCR) contribuye a la patogénesis de las neoplasias malignas de células B y ha surgido como una nueva diana terapéutica en varios tipos de linfoma. Así, los inhibidores de quinasas de la vía del BCR constituyen una estrategia terapéutica prometedora. Dentro de este grupo de fármacos destaca el inhibidor de Btk ibrutinib, que ha conseguido esperanzadoras tasas de respuesta pero que también se ve afectado por la aparición de resistencias. Nuestro trabajo muestra que el compuesto IQS019, inhibidor de varias quinasas de la vía del BCR (Btk, Syk y Lyn), posee un potente efecto antitumoral y permite escapar a las resistencias observadas al ibrutinib. Así, supone un buen tratamiento para varios subtipos de linfomas de células B, incluyendo aquellos poco sensibles a los inhibidores de quinasa de BCR actuales.
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Seeber, Tonia Olivia [Verfasser]. "Primary Resistance to Immune Checkpoint Inhibitors in Patients with Metastatic Melanoma : Prognosis, Subsequent Therapies and Survival / Tonia Olivia Seeber." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/123272582X/34.
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Hu, Qiuhua. "Investigating prostate tumour vasculature and oxygenation status in response to androgen-targeted therapies using photoacoustic-ultrasound imaging." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/228679/8/Qiuhua_Hu_Thesis.pdf.
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This project provides a holistic view of changes in the prostate cancer microenvironment in response to androgen targeted therapies. Oxygen saturation and total haemoglobin were monitored using the ultrasound and photoacoustic imaging capabilities of the VEVO LAZR system (FUJIFILM Visual Sonics Inc) and compared with measuring hypoxic and vascular markers using conventional protein and gene expression techniques. Understanding the effects of castration and enzalutamide on the vasculature and oxygenation status of prostate cancer subcutaneous xenografts has the potential to reveal novel mechanisms of therapy resistance and may improve the prediction of patient therapy responses.
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Fernández, Noguera Patricia. "Fibroblast role in the acquisition and maintenance of breast cancer resistance to anti-HER2 therapies. Identification of novel compensatory tyrosine kinase receptors." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/396186.
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HER2 positivity defines a molecular subtype of breast cancer with an aggressive biological behaviour and poor prognosis. Its pharmacological targeting with the monoclonal antibody Trastuzumab, and later on with the small tyrosine kinase inhibitor Lapatinib, changed the prognosis for HER2-positive breast cancer patients. However, despite the positive results from many trials, resistance to anti-HER2 agents occurs in both metastatic and adjuvant settings, in part, because HER2 represent just a part of a more complex biological network, that when deregulated, plays a central role in sustaining the aggressive phenotype of tumour cells.
Moreover, the importance of the tumour microenvironment in drug resistance has been recognized during the last years, and it is now widely accepted that growth, survival and metastasis (as a consequence of treatment failure), are regulated by stromal-cancer cell interactions.
Then, it is likely that the tumour stroma, and cancer associated fibroblasts in particular, influence also the therapeutic outcome. Therefore, our hypothesis is that during the acquisition of drug resistance cancer and stromal cells co-evolve, and that the tumour cells can induce changes in the structure and composition of the microenvironment to support their own growth and progression or dissemination.
Our research has defined a coevolution process between breast cancer cells and fibroblasts where fibroblasts and BC cells crosstalk drives the drug resistant phenotype. We have identified RET and FGFR2 pathways as novel mechanisms of HER2 activation and we have demonstrated the importance of the interaction between these pathways in vitro and in vivo, in promoting breast cancer progression and resistance to Trastuzumab and Lapatinib.
Furthermore, we propose that inhibition of RET and FGFR2 pathways might become a promising strategy after Trastuzumab and Lapatinib failure in patients with HER2-positive breast cancer.El càncer de mama és una malaltia molt heterogènia que inclou diferents subtipus moleculars. El subtipus HER2+ suposa aproximadament el 15-20% dels diferents casos diagnosticats, i es caracteritza per presentar amplificació gènica en la regió 17q 12, que resulta en la sobrexpressió del receptor amb activitat tirosina cinasa HER2. Aquest subtipus presenta una pitjor evolució de la malaltia, estant associat a una major propensió de metàstasis a sistema nerviós, donant lloc a un fenotip més agressiu i de pitjor prognosi. En els darrers anys la evolució clínica d'aquests pacients s'ha vist millorada gràcies al disseny de teràpies dirigides, que han permès el desenvolupament de nous fàrmacs com l'anticòs monoclonal Trastuzumab o l'inhibidor dual dels dominis tirosina quinasa de EGFR i HER2 Lapatinib. Tot i els resultats clínics inicials i l'avantatge que el Lapatinib va suposar pel tractament de pacients amb càncer de mama, alguns pacients no responen de inici al tractament, i de la mateixa manera que amb el Trastuzumab, pacients inicialment receptius desenvolupen resistència durant les primeres fases del tractament. La interacció entre el tumor i un microambient favorable és essencial perquè les cèl.lules tumorals puguin progressar, i durant els darrers anys el paper del microambient en l'establiment de resistències has estat àmpliament estudiat i acceptat. D'aquesta manera, és plausible, que l'estroma tumoral i en concret els fibroblasts influenciïn el pronòstic dels pacients. La nostre hipòtesi es basa en la assumpció que els diferents tractaments terapèutics podrien afavorir la selecció de cèl.lules tumorals amb propietats úniques amb una millor adaptació al microambient, i que aquestes mateixes cèl.lules podrien interaccionar de forma diferent amb el seu entorn, produint canvis en el propi microambient per tal d'afavorir el seu creixement, progressió i disseminació. Els nostres resultats defineixen una coevolució entre les cèl.lules de càncer de mama i els fibroblasts. Hem identificat la via de RET i FGFR2 com un nou mecanisme d'activació de HER2, demostrant també la importància d'aquestes vies en l'establiment del fenotip resistent, així com en la progressió tumoral in vitro i in vivo. També proposem la inhibició de les vies de senyalització de RET i FGFR2 com a estratègia de rescat per aquells pacients amb càncer de mama HER2+ que han generat resistència a Trastuzumab i Lapatinib i que avui en dia presenten poques alternatives terapèutiques.
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Clem, Angela. "Bacteriophage for the elimination of methicillin-resistant staphylococcus aureus (MRSA) colonization and infection." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001568.
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Grockowiak, Élodie. "Role of the Bone Morphogenetic Proteins pathway in tyrosine kinase inhibitors resistance in Chronic Myeloid Leukemia." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1253.
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La leucémie Myéloïde Chronique est un néoplasme myéloprolifératif causé par l'expression de la kinase oncogène BCR-ABL. Les Inhibiteurs de Tyrosine Kinase (ITK) spécifiques de BCR-ABL ont révolutionné la prise en charge de la maladie. Les ITK ne sont cependant pas curatifs ; en effet, certaines cellules souches leucémiques (CSL) sont résistantes aux ITK, et persistent dans la moelle osseuse des patients même en rémission prolongée. Ces CSL sont probablement responsables de la rechute chez 60% de ces patients après arrêt des ITK. 30% des patients développent une résistance aux ITK via des mécanismes inconnus. Dans un contexte sain, les Bone Morphogenetic Proteins (BMP) régulent différentes propriétés des cellules souches hématopoïétiques. Nous avons mis en évidence que les patients atteints de LMC présentent une altération de la voie BMP avant leurs mises sous traitement, avec une hausse de l'expression du récepteur dans les cellules leucémiques immatures, amplifiée par de forts taux de BMP2/4 produits par le microenvironnement des CSL, la niche. Ici, nous démontrons que ces altérations sont maintenues chez les patients sous traitement, et sont activement impliquées dans la résistance aux ITK. Les patients résistants présentent une surexpression de BMPR1b dans les CSL et un maintien de forts taux de BMP produits à la fois par les cellules leucémiques mais aussi par les cellules stromales. Les BMP permettent la survie des CSL via l'expression du récepteur BMPR1b et induisent l'expression de TWIST-1, un facteur de transcription précédemment identifié par l'équipe comme induisant la résistanceChronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm caused by the expression of the oncogenic protein kinase, BCR-ABL. The Tyrosine Kinase Inhibitors (TKI) specifics of BCR-ABL kinase dramatically changed the outcome of CML, turning a life-threatening disease into a chronic illness. However, TKI are not yet curative since most CML patients still retain progenitors and leukemic stem cells (LSC) in bone marrow permanently. Thus, approximately 60% of patients that achieve Complete Molecular Remission =2 years relapse following TKI withdraw. Moreover, some patients develop true resistance to TKI, with ~30% due to unknown mechanisms. In chronic phase CML (CP-CML), LSC survive, sustain interactions with their niche where resistance mechanisms can occur, responsible for disease persistence and relapse following treatment cessation. In normal bone marrow, Bone Morphogenetic Proteins (BMP) pathway regulate the fate and proliferation of normal hematopoietic stem cells, as well as interactions with their niche. The deregulations of this pathway drive early steps of CML development. In newly diagnosed CP-CML patients, high concentration of BMP2/4 in the leukemic niche allows LSC maintenance and sustains a permanent pool of leukemic progenitors expressing elevated levels of BMPR1b receptor. Here, we report that alterations of the BMP pathway persist in TKI-CML resistant patients. As compared to patients in Complete Cytogenetic Remission (CCyR), cells isolated from TKI-resistant patients display a high level of BMPR1b expression in immature cells and high levels of BMP2/4 in bone marrow, provided by the niche and by the leukemic immature cells themselves. BMP allow leukemic stem cells resistance to treatments through binding to BMPR1b. Interestingly, BMP2/4-treated cells overexpressed TWIST-1, a transcription factor that we previously identified as a predictive factor of CML resistance
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Savic, Sinisa. "The role of dysregulated unfolded protein response and resistance to apoptosis in the pathogenesis of rheumatoid arthritis and non- response to anti-TNF therapies." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534440.
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Nguyen, Tran Dang. "The effects of different deployment strategies of artemisinin combination therapies on slowing down the spread of antimalarial drug resistance : investigation with individual-based simulations." Thesis, Open University, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.700132.
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Despite the success of recent global malaria control efforts, which. have halved global malaria mortality since 2000, malaria is still one of the world's most deadly diseases causing an estimated half a million deaths, mostly among African children, and around a quarter of billion clinical episodes every year as reported in 2014 1. Drug resistance is one of the most important challenges to malaria elimination. To contain drug resistance, many efforts have been put forth including improvement of surveillance systems and mass treatment in order. to stop or slow down the transmission of the resistant strain. To find out whether a population-level treatment strategy can have any benefit in containing drug resistance, mathematical models are an appropriate approach to this problem and individual-based models allow us to have a better understanding of the effect of individual heterogeneities on the outcome. The first part of the thesis is about building and validating an individual based microsimulation. The model is implemented as an individual-based discrete-time event simulation model in C++. The behaviors and the state changes of human individuals are determined by relevant events and mathematical formulas. This integrated model combines components that reproduce the most important features of malaria transmission and epidemiology: the infectiousness of human populations; clinical model of acute illness; heterogeneities in individuals' age, biting-rate level, drug absorption, drug action, multiple parasite populations, and human immunity. To validate this individual-based model, two types of validation have been done. The model's parameters were obtained from field or clinical data were used directly in the model. For those parameters that cannot be obtained directly from literature review, sensitivity analysis has been done to find how variation in parameter values affects certain key features of malaria epidemiology .
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Pieper, Natalia [Verfasser], and Annette [Akademischer Betreuer] Paschen. "Impact of IFN-γ resistance & MAPK inhibition on the immune surveillance of malignant melanoma - relevance for immune-based therapies / Natalia Pieper ; Betreuer: Annette Paschen." Duisburg, 2019. http://d-nb.info/1201274095/34.
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Schneider, Matthias [Verfasser]. "A paired comparison between glioblastoma cancer stem cells and differentiated cells in view of proliferation, resistance to conventional therapies and tumour-initiating capabilities / Matthias Schneider." Ulm : Universität Ulm, 2016. http://d-nb.info/112168341X/34.
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Diazzi, Serena. "Le cluster pro-fibrotique miR-143/145 favorise la plasticité phénotypique associée à la résistance des mélanomes aux thérapies ciblées." Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://theses.univ-cotedazur.fr/2021COAZ6006.
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Le mélanome est le cancer de la peau le plus agressif de par sa grande plasticité phénotypique, son potentiel métastatique et sa résistance aux traitements. Malgré la percée des thérapies ciblant la voie oncogénique MAP kinase, la résistance du mélanome a ces traitements demeure un obstacle majeur qui limite le bénéfice pour les patients porteurs de la mutation BRAFV600E. Les cellules de mélanome peuvent transiter vers un état de type mésenchymateux dédifférencie en fonction des pressions du microenvironnement et des traitements. Cette plasticité cellulaire phénotypique adaptative a été décrite comme un facteur essentiel de résistance aux thérapies ciblées. Mon équipe de recherche travaille sur ce type de résistance non-génétique définie comme ≪ mésenchymateuse ≫, dans lequel les cellules tumorales présentent un comportement invasif et acquièrent des caractéristiques observées typiquement dans les fibroses telles que la capacité à accumuler et à remodeler la matrice extracellulaire et activer les voies de mécanotransduction. Dans ce contexte, mon projet a consiste à caractériser un cluster compose de deux ≪ FibromiRs ≫, microARN impliques dans les mécanismes de fibrogènes et qui sont fortement exprimes dans les mélanomes résistants. Mes résultats obtenus à l’aide d’approches in vitro et in vivo démontrent le rôle du locus miR-143/-145 dans la régulation de la résistance non-génétique en raison de sa capacite à remodeler la matrice et façonner une niche de protection et de tolérance pour la tumeur face aux inhibiteurs de la voie MAP kinase. MiR-143 et miR-145 contribuent également au passage d’un phénotype cellulaire différentié prolifératif a un phénotype mésenchymal plus invasif et résistant. Au niveau moléculaire, j’ai identifié parmi les nombreuses cibles potentielles du cluster, la FSCN1 comme un gène clé cible de miR-143 et -145. Ces travaux ont permis de dévoiler le rôle du cluster miR143/-145 dans le comportement agressif des cellules de mélanome dédifférenciées résistantes et de proposer miR-143 et miR-145 comme nouvelles cibles thérapeutiques pour vaincre la résistance mésenchymateuse et mieux combattre la maladie métastatique réfractaireBecause of its intrinsic plasticity and resistance to treatment, melanoma is one of the most aggressive cancers. Due to the MAPK pathway hyperactivation, targeted therapies counteracting this signaling cascade are efficient in most patients harboring BRAFV600E metastatic melanoma. However, innate and acquired resistances constitute major therapeutic challenges. Acquired resistance to MAPK-targeted therapies arises from de novo genetic lesions and non-genetic events such as transcriptional reprogramming and epigenetic changes. Upon MAPK inhibitors exposure, melanoma cells assume functionally different phenotypic states defined by master transcription factors differential activity and fixed by epigenetic events. Among them, the emergence of a poorly differentiated cell state is strongly associated with resistance acquisition and tumor recurrence. Our team has previously shown that melanoma cells switching to a dedifferentiated phenotype in response to MAPK-targeted therapies display features of cancer-associated fibroblasts (CAFs) like extracellular matrix (ECM) remodeling and markers observed in fibrotic diseases, allowing them to generate a drug tolerant microenvironment.This fibrotic state is characterized in vitro and in vivo by increased deposition and altered ECM organization associated with a mechanophenotype regulated by the mechanotransducers YAP and MRTFA. However, post-transcriptional signaling networks that underpin this mesenchymal-like phenotype are still unknown and effective therapeutic treatments to overcome MAPK-targeted therapy resistance are missing. Given the tumorigenic role of ECM in cancer progression and resistance, therapies aimed at “normalizing” the tumorigenic ECM represent promising strategies to overcome non-genetic resistance to MAPK inhibitors. Based on the role of miRNAs in post-transcriptional regulation, I focused on the characterization of a pool of miRNAs, defined as “FibromiRs,” which have been shown to participate in the onset and progression of fibrotic diseases. Their crucial role in the fibrogenic process and the possibility to therapeutically manipulate them make them promising druggable targets to prevent the onset of resistance to MAPK-targeted therapies in melanoma. Starting from a screening designed to compare the expression of “FibromiRs” in MAPK inhibitors resistant mesenchymal melanoma cells compared to therapy-naive parental cells, we have identified the profibrotic miR-143/145 cluster as overexpressed in mesenchymal resistant cells. We then explored the profibrotic function of miR-143/145 cluster in the mesenchymal-like resistant cell state and melanoma phenotypic plasticity. First, we analysed the regulation of miR-143 and miR-145 in melanoma, identifying a negative regulation of the MAPK pathway on its expression and the involvement of signaling pathways typical of the mesenchymal resistant state, such as TGFβ and PDGF signaling, in the activation of their expression. Next, we investigated the function of the cluster in the context of adaptive and acquired resistance, showing its contribution in ECM reprogramming, activation of mechanotransduction pathways, and in driving the switch from a differentiated proliferative phenotype to a dedifferentiated invasive one with decreased sensitivity to MAPK inhibition. We characterized its mechanism of action, identifying FSCN1 as a key target gene of both mature miR-143 and miR-145 in the acquisition of the mesenchymal invasive phenotype. Finally, we tested the cluster as a potential therapeutic target in vitro and in vivo through antisense oligonucleotide-mediated inhibition of its expression or pharmacological modulation combined with MAPK inhibitors administration. Overall, this work highlights the importance of a FibromiR cluster in the acquisition of a dedifferentiated phenotype resistant to MAPK-targeted therapies and proposes new therapeutic strategies based on the inhibition of FibromiRs to overcome such resistance mechanism
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GUARNACCIA, LAURA. "DEVELOPMENT AND VALIDATION OF TARGET THERAPIES FOR PATIENTS WITH BRAIN CANCER, THROUGH THE MODULATION OF ANGIOGENESIS, INVASIVENESS, AND PHARMACOLOGICAL SENSITIVITY/RESISTANCE, IN THE ERA OF PRECISION MEDICINE." Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/888881.
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Brain tumors represent a group of heterogeneous neoplasms which, despite originating in the same
anatomical region, differ in morphology, etiology, molecular biology, and especially in clinical
behavior. It has been estimated that brain tumors affect about 200,000 people worldwide every year,
representing approximately 2% of cancer deaths. Among all primary human brain tumors,
glioblastoma (GBM) is the most malignant and frequent (~70%), with a median survival of about 14
months and a 5-year survival rate at 5%, thus representing an extreme therapeutic challenge. GBM is
characterized by sustained proliferation and survival, immune system escape, intense angiogenesis,
invasion, cell infiltration, rapid progression, resistance to radio- and chemotherapies, with high
frequency of relapse. In cancer pathogenesis, particularly in high grade tumors as GBM, aberrant
neo-angiogenesis is a vital process for the mass growth: it is driven by neoplastic cells in order to
respond to the tumoral hypoxic environment in the necrotic core, which increases the demand for
oxygen and nutrients by neoplastic cells, and is, therefore, essential to carry out the metabolic
functions on which their survival is based. Several observations led to the knowledge that tumoral
neo-angiogenesis gives rise to ultra-structurally abnormal vessels, larger than their normal
counterparts, dilated, convoluted, irregularly branched and exceptionally permeable due to the
presence of fenestrations and the lack of a complete basal membrane. Due to these destructive
features, despite aggressive therapeutic treatment, consisting in surgical resection followed by
chemotherapy with temozolomide (TMZ), most patients experience tumor recurrence in less than one
year, suggesting the urgent need to implement clinical practice with novel prognostic and therapeutic
strategies. To this aim, this research project has been focused on: i) aberrant angiogenesis mediated
by endothelial cells (ECs), which promote tumor infiltration into surrounding tissues, with consequent
compromission of cognitive skills, and ii) genetic instability, which characterize GBM with a high
intra- and intertumoral heterogeneity. In particular, the underlying hypothesis is that a specific
molecular signature characterizes “resistant” and “sensitive” GBM, and it may be responsible for
patient overall survival. Indeed, although GBMs from short- and long-term survivors (STS and LTS)
are histologically the same, their biological and molecular characteristics are remarkably different,
suggesting that factors that contribute to patients’ longevity are important for precise diagnosis and
correct clinical management of the disease. The genetic profiling obtained by array CGH on STS and
LTS revealed a high number of copy number variation (CNVs) across chromosomes 1 to 22, among
which several novel potential prognostic and predictive biomarkers have been described and
discussed. In particular, aCGH highlighted the presence of an altered chromosomic pattern relative
to calpain family genes. Calpains are a conserved family of cysteine proteinases that catalyze the
controlled proteolysis of many specific substrates. Calpain activity is implicated in several
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fundamental physiological processes, including cytoskeletal remodeling, cellular signalling,
apoptosis and cell survival. Alterations of the calpain activity balance has been observed in numerous
cancer types, as they can reduce apoptosis, increase cell proliferation and stimulate cell migration and
invasiveness. The characterization of calpain expression in primary GBM endothelial cells (GECs)
showed an upregulation of calpains and a positive correlation between expression level and patient
survival. The blockade of calpain activation with natural and synthetic inhibitors resulted in the
inhibition of GEC viability and proliferation, as well as in the promotion of caspase-induced cell
apoptosis. Further, calpain inhibition led to the arrest of tumor angiogenesis and migration in vitro.
Finally, the molecular investigation revealed a downregulation of proliferative signalling as MAPK
and anti-apoptotic regulators as Bcl-2, as well as an upregulation of proapoptotic mediators, as
Caspases and Bax-family. From this evidence, calpains may be considered as novel potential
therapeutic targets to treat cancer and to limit its progression.
Overall, the discovery of novel potential prognostic and predictive biomarkers, by the “omics”
approach, will allow to optimize patient’s management, as early prediction of those patients who are
likely to be STS or LTS, and a full understanding of their clinical course can thus assist clinicians in
providing tailored treatments and support patients and their families. However, beyond prognosis,
what will really impact clinical management of GBM is if genomic analyses can lead to customized
treatment and ultimately improved survival, following the concept of precision medicine.
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Geneste, Aline. "Tissu adipeux et résistance tumorale aux thérapies ciblées." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1115/document.
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Les thérapies ciblées telles que les inhibiteurs de la tyrosine kinase ont permis d'améliorer le traitement du cancer du sein en ciblant HER2. Cependant, il a été observé que le lapatinib était moins efficace chez les patients obèses ou en surpoids que chez les patients de poids normal.Nous avons d'abord reproduit l'effet de résistance des cellules de cancer du sein au lapatinib en présence de tissu adipeux tel qu’il a été observé pour d’autres thérapies. Les cellules tumorales qui surexpriment HER2 étaient partiellement résistantes au lapatinib mais également à d'autres inhibiteurs de la tyrosine kinase lorsqu'elles étaient en contact avec le milieu conditionné d’adipocytes. En implantant du tissu adipeux humain et des tumeurs humaines chez la souris, nous avons pu étudier la résistance du cancer du sein au lapatinib in vivo.Pour comprendre le mécanisme de cette résistance, nous avons exposé les adipocytes à plusieurs modulateurs du métabolisme. La cytotoxicité cellulaire induite par le lapatinib était plus faible pour les cellules tumorales exposées à un milieu conditionné d'adipocytes préalablement incubés avec les alpha-bloquants qu'à un milieu conditionné à partir d'adipocytes seuls. De la même manière, cette toxicité était inférieure pour les agonistes des récepteurs alpha adrénergiques, pour les bêtabloquants et pour les inhibiteurs de lipolyse. Au contraire, la cytotoxicité a été augmentée pour les cellules tumorales en contact avec le milieu conditionné d’adipocytes exposés aux agonistes du récepteur bêta-adrénergiques. Au niveau des cellules cancéreuses, l'arrêt du cycle cellulaire induit par le lapatinib était réduit pour les cellules tumorales exposées au milieu conditionné d’adipocytes en ce qui concerne le pourcentage de cellules dans la phase G0/G1. Ceci s’est vérifié en étudiant l'expression des gènes codant pour plusieurs protéines impliquées dans la progression du cycle cellulaireTargeted therapies as tyrosine kinase inhibitors permitted an improvement of breast cancer therapies by targeting HER2. However, resistance has been observed in obèse patients for lapatinib treatment.We reproduced the effect of resistance of breast cancer cells to laaptinib in presence of adipose tissue as observed for other therapies. Tumor cells overexpressing HER2 was partly resistant to lapatinib but also for other tyrosine kinase inhibitors when in contact with adipocyte-conditioned medium. By impnating human adipose tissue nad human tumors in mice, we were able to study rhe resistance of breast tumor cells in vivo.In order to elucidate the mechanism of such resistance, we exposed the adipocytes to several metabolism modulators. The lapatinib-induced cell cytotoxicity was lower for the tumor cells exposed to the conditioned medium from adipocytes earlier exposed to alpha blockers than to the conditioned medium from adipocytes alone. In the same manner, the toxicity was lower for the agonists of alpha-adrenergic receptors , for beta-blockers and for the lipolysis inhibitors. At the opposite, the cytotoxicity was enhanced for tumor cells in contact with the conditioned medium of adipocytes exposed to the agonists of beta adrenergic receptors.At the tumor cell level, the laaptinib-induced cell cycle arrest was reduced for the tumor cells exposed to the conditioned medium regarding the G0/G1 phase. That was verified by the study of the expression of genes involved in the cell cycle progression
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Ben, Jouira Rania. "Implication de la matrice extracellulaire tumorale dans la transition phénotypique et la résistance aux thérapies ciblées du mélanome." Thesis, Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4140.
Full textAbstract:
Le mélanome cutané est le cancer de la peau le plus agressif de par sa grande plasticité phénotypique, son fort caractère métastatique et sa résistance aux traitements. L’émergence d’inhibiteurs ciblant la forme mutée de la kinase BRAF (BRAF V600E) a produit des réponses thérapeutiques spectaculaires, malheureusement suivies par l’apparition rapide de résistances secondaires très agressives. La compréhension des mécanismes cellulaires et moléculaires impliqués dans ces résistances constitue donc un prérequis indispensable à l’amélioration de ces thérapies ciblées. A côté des altérations intrinsèques au mélanome, les interactions entre les cellules malignes et leur microenvironnement favorisent la survie tumorale et contribuent à la résistance aux thérapies. En particulier, la matrice extracellulaire (MEC), qui constitue un réseau dynamique de macromolécules de composition et de propriétés physico-chimiques variables, influence l’architecture des tissus tumoraux, l’invasion et la réponse aux traitements. De façon importante, l’acquisition par les cellules de mélanome d’un phénotype mésenchymateux invasif a été décrite comme un mécanisme d’échappement aux thérapies ciblant la mutation oncogénique de BRAF. Dans ce contexte, l’objectif de mon travail de thèse a été de préciser le rôle de cette signature phénotypique sur les propriétés bio-mécaniques des cellules de mélanome et la réponse aux thérapies ciblées. Dans la première partie de ma thèse, j’ai observé que les cellules résistantes présentant un phénotype invasif mésenchymateux produisent, assemblent et remodèlent une matrice ayant des propriétés mécaniques et biochimiques proches de myofibroblastes. Ce phénotype est associé à une activation de la voie YAP/TAZ et une mécano-sensibilité amplifiée. La caractérisation par spectrométrie de masse du matrisome des cellules résistantes a révélé la présence abondante de protéines matricielles comme la Fibronectine, le Collagène 1(I) et la THBS1 mais également de protéines de réticulation du collagène comme LOXL2 et TGM2. Nos données montrent aussi que ces modifications sont conférées de novo par un traitement aux inhibiteurs de BRAF ou de MEK dans des cellules de mélanome mutées BRAF in vitro, et que chez la souris le traitement au Vémurafénib de cellules de mélanome xénogreffées induit l’assemblage de fibres de collagène associé à une rigidification tumorale. Finalement, j’ai pu montrer que la matrice produite par les cellules mésenchymales résistantes protège les cellules de mélanome naïves des effets anti-prolifératifs liées à l’inhibition de BRAF ou MEK. Dans une deuxième partie de ma thèse, je me suis intéressée à la protéine de réticulation du collagène de la famille des lysyl oxidases (LOX), LOXL2 exprimée par les cellules mésenchymales résistantes. Nos analyses bioinformatiques et biochimiques montrent que l'expression de cette enzyme est fortement associée à la signature invasive MITFlow AXLhigh des mélanomes. En utilisant des approches d'interférence à ARN, j'ai aussi montré que la suppression de LOXL2 dans les cellules de mélanome invasif diminue la migration cellulaire et augmente la prolifération cellulaire in vitro et in vivo, suggérant un rôle de LOXL2 dans la transition phénotypique du mélanome. Dans l’ensemble, mes travaux de thèse révèlent un rôle paradoxal de l’inhibition de la voie MAPK qui induit des changements du phénotype tumoral associés à la production autonome par la cellule maligne d’une MEC pathologique capable d'altérer le comportement cellulaire et la réponse au traitement. Cet environnement matriciel 'sanctuaire', associée à une intense hétérogénéité tumorale, pourrait jouer un rôle majeur dans le développement et l'émergence des résistances thérapeutiques du mélanome. Ces résultats permettent une meilleure compréhension du rôle de la MEC du mélanome et devraient proposer de nouvelles pistes pour améliorer les traitementsCutaneous melanoma remains one of the most challenging and difficult cancers to treat because of its high plasticity, metastatic potential and resistance to treatment. New therapies targeting oncogenic BRAFV600E mutation have shown remarkable clinical efficacy. However, drug resistance invariably develops. Thus, the need for improving existing therapies remains critical. Recent studies have indicated that tumor resistance arises from the tumor microenvironment in which the extracellular matrix (ECM) is a determinant factor. Here, we found that BRAF inhibitor (BRAFi)-resistant melanoma cells, but not BRAFi-sensitive cells display an increased mechanosensitivity associated with a capacity to produce and remodel a 3D ECM displaying increased levels of matrix proteins such as fibronectin (FN) and collagen fibers. Interestingly, our results show that this 3D ECM is able to protect therapy-sensitive cells from the anti-proliferative effects of MAPKi. In addition, short exposures of naive melanoma cells to MAPKi augment matrix proteins production and assembly in vitro and in vivo. This 3D ECM also promotes drug tolerance within BRAFi sensitive cells. In conclusion, our results suggest that a subset of resistance to MAPK targeted therapies is associated with the production by melanoma cells of a pathological fibrotic matrisome that may affect cell behavior and therapeutic response
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Ben, Jouira Rania. "Implication de la matrice extracellulaire tumorale dans la transition phénotypique et la résistance aux thérapies ciblées du mélanome." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4140.
Full textAbstract:
Le mélanome cutané est le cancer de la peau le plus agressif de par sa grande plasticité phénotypique, son fort caractère métastatique et sa résistance aux traitements. L’émergence d’inhibiteurs ciblant la forme mutée de la kinase BRAF (BRAF V600E) a produit des réponses thérapeutiques spectaculaires, malheureusement suivies par l’apparition rapide de résistances secondaires très agressives. La compréhension des mécanismes cellulaires et moléculaires impliqués dans ces résistances constitue donc un prérequis indispensable à l’amélioration de ces thérapies ciblées. A côté des altérations intrinsèques au mélanome, les interactions entre les cellules malignes et leur microenvironnement favorisent la survie tumorale et contribuent à la résistance aux thérapies. En particulier, la matrice extracellulaire (MEC), qui constitue un réseau dynamique de macromolécules de composition et de propriétés physico-chimiques variables, influence l’architecture des tissus tumoraux, l’invasion et la réponse aux traitements. De façon importante, l’acquisition par les cellules de mélanome d’un phénotype mésenchymateux invasif a été décrite comme un mécanisme d’échappement aux thérapies ciblant la mutation oncogénique de BRAF. Dans ce contexte, l’objectif de mon travail de thèse a été de préciser le rôle de cette signature phénotypique sur les propriétés bio-mécaniques des cellules de mélanome et la réponse aux thérapies ciblées. Dans la première partie de ma thèse, j’ai observé que les cellules résistantes présentant un phénotype invasif mésenchymateux produisent, assemblent et remodèlent une matrice ayant des propriétés mécaniques et biochimiques proches de myofibroblastes. Ce phénotype est associé à une activation de la voie YAP/TAZ et une mécano-sensibilité amplifiée. La caractérisation par spectrométrie de masse du matrisome des cellules résistantes a révélé la présence abondante de protéines matricielles comme la Fibronectine, le Collagène 1(I) et la THBS1 mais également de protéines de réticulation du collagène comme LOXL2 et TGM2. Nos données montrent aussi que ces modifications sont conférées de novo par un traitement aux inhibiteurs de BRAF ou de MEK dans des cellules de mélanome mutées BRAF in vitro, et que chez la souris le traitement au Vémurafénib de cellules de mélanome xénogreffées induit l’assemblage de fibres de collagène associé à une rigidification tumorale. Finalement, j’ai pu montrer que la matrice produite par les cellules mésenchymales résistantes protège les cellules de mélanome naïves des effets anti-prolifératifs liées à l’inhibition de BRAF ou MEK. Dans une deuxième partie de ma thèse, je me suis intéressée à la protéine de réticulation du collagène de la famille des lysyl oxidases (LOX), LOXL2 exprimée par les cellules mésenchymales résistantes. Nos analyses bioinformatiques et biochimiques montrent que l'expression de cette enzyme est fortement associée à la signature invasive MITFlow AXLhigh des mélanomes. En utilisant des approches d'interférence à ARN, j'ai aussi montré que la suppression de LOXL2 dans les cellules de mélanome invasif diminue la migration cellulaire et augmente la prolifération cellulaire in vitro et in vivo, suggérant un rôle de LOXL2 dans la transition phénotypique du mélanome. Dans l’ensemble, mes travaux de thèse révèlent un rôle paradoxal de l’inhibition de la voie MAPK qui induit des changements du phénotype tumoral associés à la production autonome par la cellule maligne d’une MEC pathologique capable d'altérer le comportement cellulaire et la réponse au traitement. Cet environnement matriciel 'sanctuaire', associée à une intense hétérogénéité tumorale, pourrait jouer un rôle majeur dans le développement et l'émergence des résistances thérapeutiques du mélanome. Ces résultats permettent une meilleure compréhension du rôle de la MEC du mélanome et devraient proposer de nouvelles pistes pour améliorer les traitementsCutaneous melanoma remains one of the most challenging and difficult cancers to treat because of its high plasticity, metastatic potential and resistance to treatment. New therapies targeting oncogenic BRAFV600E mutation have shown remarkable clinical efficacy. However, drug resistance invariably develops. Thus, the need for improving existing therapies remains critical. Recent studies have indicated that tumor resistance arises from the tumor microenvironment in which the extracellular matrix (ECM) is a determinant factor. Here, we found that BRAF inhibitor (BRAFi)-resistant melanoma cells, but not BRAFi-sensitive cells display an increased mechanosensitivity associated with a capacity to produce and remodel a 3D ECM displaying increased levels of matrix proteins such as fibronectin (FN) and collagen fibers. Interestingly, our results show that this 3D ECM is able to protect therapy-sensitive cells from the anti-proliferative effects of MAPKi. In addition, short exposures of naive melanoma cells to MAPKi augment matrix proteins production and assembly in vitro and in vivo. This 3D ECM also promotes drug tolerance within BRAFi sensitive cells. In conclusion, our results suggest that a subset of resistance to MAPK targeted therapies is associated with the production by melanoma cells of a pathological fibrotic matrisome that may affect cell behavior and therapeutic response
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Beganton, Benoît. "Caractérisation des réseaux d’interactions protéiques associés aux mutations oncogéniques principales retrouvées dans le cancer du poumon non à petites cellules." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT157/document.
Full textAbstract:
Le cancer du poumon est la première cause de mortalité liée au cancer en France et dans le monde. Il s’agit d’un cancer de mauvais pronostique, diagnostiqué généralement aux stades tardifs III ou IV et avec une survie à 5 ans faible, respectivement de 6 et 1%. Ce cancer comprend différents types histologiques, parmi lesquels les adénocarcinomes sont les plus fréquents et comptent pour 40% des diagnostics. L’analyse génétique par séquençage des tumeurs des patients en stade IV permet de mettre en évidence des mutations récurrentes, et généralement mutuellement exclusives, au niveau des gènes KRAS, EGFR, BRAF et ALK. L’identification de ces mutations permet dans le cadre de tests compagnons de décider de l’éligibilité du patient aux thérapies ciblées lorsqu’elles sont disponibles.Bien que ces thérapies ciblées représentent une véritable révolution dans la prise en charge des patients, une majorité de patients ne peuvent bénéficier de ces traitements car ils ne présentent pas au niveau tumoral de mutation activatrices actionnables (absence de mutation EGFR, BRAF et ALK, présence d’une mutation KRAS…). De plus, dans l’éventualité où ils peuvent être traités par une molécule orale, le bénéfice observé reste malheureusement de courte durée et tous finiront à terme par échapper au traitement, c’est le cas par exemple lors de mutations de l’EGFR.Ainsi, afin de mieux comprendre quels sont les mécanismes moléculaires associés aux phénomènes de résistances thérapeutiques observés en clinique, et dans l’objectif de proposer de nouvelles voies thérapeutiques pour les patients non éligibles aux thérapies ciblées, j’ai étudié, au niveau protéique, l’impact des mutations sur les réseaux d’interactions protéiques en utilisant la technique du BioID (proximity-dependent biotinylation identification). Plus particulièrement, je me suis intéressé aux mutations activatrices de l’EGFR, KRAS, BRAF et ALK. Les protéines de la famille RAS (HRAS, NRAS, KRAS) étant mutées dans plus de 30% des cancers, je me suis également intéressé aux réseaux d’interactions protéiques portés par ces trois isoformes afin de mettre en évidence des interactions protéiques spécifiques à l’isoforme KRAS.Au cours de ma thèse, j’ai montré que les réseaux d’interaction caractérisés par le BioID sont bien plus denses que ceux obtenus par la technique plus conventionnelle de purification par affinité, et qu’ils permettent de mettre en évidence des interacteurs spécifiquement dérégulés par l’apparition de mutations activatrices. Par ailleurs les modèles cellulaires HEK293 (cellules humaines embryonnaires de rein) et BEAS2B (cellules pulmonaires d’origine non-cancéreuses) ont montré un fort recouvrement des interacteurs identifiés, ce qui suggère que la stratégie employée est pertinente pour identifier des interacteurs spécifiques de mutations. Ce travail de thèse a permis d’identifier une série de plusieurs interacteurs spécifiques des formes mutante de KRAS, d’EGFR, de BRAF, de NRAS et de la fusion EML4-ALK. Treize interacteurs spécifiques de la forme mutée de KRAS ont été validés fonctionnellement dans des modèles cellulaires de cancers du poumon. Enfin, en utilisant les données de BioID, un modèle préliminaire de résistance de l’EGFR aux thérapies ciblées a pu être élaboré, lequel fait apparaitre les protéines CBL et IGF2R comme des protéines potentiellement impliquées dans l’acquisition des résistances aux thérapies ciblées.Ce travail de thèse propose ainsi de nouvelles perspectives quant à la détermination des mécanismes de résistance spécifiques aux thérapies ciblées et à l’identification de nouvelles cibles thérapeutiques chez les patients présentent des mutations activatricesLung cancer is the leading cause of cancer-related death in France and in the world. It is a cancer of poor prognosis, diagnosed at the late stage III or IV, with a 5-years survival of 6% and 1%, respectively. This cancer encompass several histological types, and among them adenocarcinoma account for 40% of the diagnosis. Genetic sequencing of stage IV tumors highlights redundant mutations, and generally exclusives from each other’s, of KRAS, EGFR, BRAF and ALK genes. The identification of these mutations enable, within companion test, to make eligible patients for targeted therapies when molecules are available.Even though these targeted therapies represent a true revolution in patient’s care, the majority of them cannot benefit from these treatments because their tumors do not harbor activating mutations that are targetable (e.g. absence of EGFR, BRAF and ALK mutation, presence of KRAS mutation). Additionally, when they can be treated using an oral molecule, the benefit observed is unfortunately poor in terms of period of time, and all the patients will escape from the treatment. This is for example the case with EGFR mutations.To better understand the molecular mechanisms associated with the resistance events observed in the clinic, and to propose new therapeutics for patient not-eligible for targeted therapies, I studied at the proteome level, the impact these mutations on protein networks, using the BioID technology (proximity-dependent biotinylation identification). More particularly, I have been interested in the activating mutation of EGFR, KRAS, BRAF and ALK. Considering that proteins from the RAS family (HRAS, NRAS and KRAS) are mutated in around 30% of cancers, I have been also interested in the protein network of these proteins to highlight interaction specific to the KRAS isoform.During my thesis, I showed that the protein networks characterized using BioID are much more dense compared to those identified with the more conventional technic of AP-MS (Affinity-purification and mass spectrometry), and that they enable to identify interactors specifically deregulated upon activating mutation. Additionally, the HEK293 cell model (Human Embryonic Kidney) and BEAS2B cell model (non-cancerous lung cell line) showed a high overlapping degree of the interactors identified, suggesting that the strategy used is relevant to identify interactors specific to mutations. This thesis enabled to identify several interactors specific to the mutant KRAS, EGFR, BRAF, NRAS and EML4-ALk fusion. Thirteen interactors specific to the mutated-KRAS have been functionally validated in lung-cancer cell lines models. Finally, using BioID data I have been able to propose a model of EGFR resistance to targeted therapies. This model shows that CBL and IGH2R might be the EGFR partners responsible for therapeutic escape.Altogether, this thesis propose new perspective to determine resistance mechanisms and to identify new therapeutic targets for KRAS-mutated patients
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Frentzel, Julie. "Rôle et régulation de l'autophagie dans les lymphomes anaplasiques à grandes cellules ALK positifs." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30180/document.
Full textAbstract:
L'oncogène ALK (Anaplastic Lymphoma Kinase) est une tyrosine kinase constitutivement active, impliquée dans divers cancers, tels que les lymphomes anaplasiques à grandes cellules (LAGC), ou certains carcinomes bronchiques. Ces tumeurs sont traitées par chimiothérapie, ce qui n'est pas un traitement optimal (30% de rechutes, abaissement de la qualité de vie). Dans ce contexte, de nombreux inhibiteurs spécifiques de la tyrosine kinase ALK tels que le Crizotinib ont été développés et ont prouvé leur efficacité à la fois dans des modèles in vitro, in vivo ainsi que chez les patients. Néanmoins, le succès de cette thérapie ciblée est limité par l'apparition de résistances. Il est donc essentiel de mettre au jour de nouvelles stratégies thérapeutiques permettant de contrecarrer ces résistances. Récemment, l'autophagie, un processus catabolique intracellulaire de dégradation lysosomale, a été proposée comme nouvelle cible thérapeutique dans le traitement des cancers résistants aux inhibiteurs de tyrosine kinase. Le premier objectif de mon projet de thèse a été de caractériser ce processus autophagique dans les LAGC ALK+, en réponse à différents traitements. Nous avons montré que (1) l'autophagie était activée dans des lignées de LAGC en réponse à l'inhibition de ALK, (2) que cette autophagie jouait un rôle cytoprotecteur dans ce modèle et (3) que les traitements par chimiothérapies de ces cellules n'induisaient pas de réponse autophagique. Dans un deuxième temps, nous nous sommes intéressés à la régulation potentielle de l'autophagie par les microARNs. Nous avons montré que plusieurs microARNs, dont le miR-7, étaient sous-exprimés en réponse au traitement par le Crizotinib et que la réexpression ectopique de ce miR-7 permettait une potentialisation de l'effet du Crizotinib, par l'induction d'autophagie cytotoxique dans notre modèle. Nous avançons également l'hypothèse que le " switch " autophagique de la cytoprotection à la cytotoxicité, que nous observons pourrait s'expliquer par la régulation de l'expression de plusieurs protéines cibles de miR-7 telles que Bcl-2 ou c-Raf. Ainsi, l'ensemble de nos résultats nous permettent de mieux comprendre le rôle et la régulation de l'autophagie induite en réponse à l'inhibition de ALK dans les LAGC ALK+, et pourrait à terme contribuer à l'amélioration des thérapies actuelles de divers cancers dépendants de l'oncogène ALKThe ALK oncogene (Anaplastic Lymphoma Kinase) is a constitutively activated tyrosine kinase implied in various cancers including Anaplastic Large Cell Lymphomas (ALCL), or some lung adenocarcinomas. The current operative treatment is standard chemotherapy, which is not optimal (30% of relapses, low quality of life). In this context, new specific ALK inhibitors such as Crizotinib have been developed, and have showed their efficiency in vitro, in vivo and in patients. However, the emergence of resistant mutations has been described. Thus, the identification of alternative therapies targeting new pathways appears as mandatory to counteract those resistances. In this context, autophagy, an intracellular catabolic lysosomal process, has been described as a new therapeutic target in the treatment of cancers resistant to tyrosine kinase inhibitors. The first aim of my project was to characterize the autophagic process in ALK+ ALCL, upon different treatments. We showed that (1) autophagy was activated in ALK+ ALCL cell lines in response to ALK inhibition (2) that this autophagy played a cytoprotective role in our model and (3) that treatment with chemotherapies did not trigger an autophagic response. In a second part of the project, we focused on the potential regulation of autophagy by microRNAs. We showed that several microRNAs including miR-7 were down-regulated upon Crizotinib treatment and that ectopic re-expression of this miR-7 potentiates the effects of Crizotinib by induction of cytotoxic autophagy in our model. We hypothesized that this switch in the role of autophagy from cytoprotection to cytotoxicity observed in our model, could be explained by the regulation of several protein targets of miR-7 such as Bcl-2 or c-Raf. Altogether, these results enable a better understanding of the role and regulation of autophagy induced upon ALK inhibition in ALCL, and could in the longer term, contribute to improvement of current therapies of cancers involving the ALK oncogene
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Silva, Ana Cristina Marques da. "Microbiota e colorectal cancer: a preliminary study in Portuguese patients." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/21959.
Full textAbstract:
Mestrado em MicrobiologiaThe colorectal cancer (CRC) is the third deadly cancer in the world, and in the last years its incidence rate has been increasing in Portugal. Given the relevance of the symbiosis between the intestinal microbiota and the host for body homeostasis, many studies have been focusing on the analysis of the microbiota associated with health and disease scenarios, namely with CRC. It is increasingly more important to know the microbial community associated with CRC, once it can be exploited as a tool for different clinical applications against CRC. As such, the present study intended to perform a preliminary characterization of the non-cultivable and cultivable bacterial community isolated from tumoral (TT) and adjacent healthy (TN) mucosa tissues of patients with CRC. Additionally, it was tested the antimicrobial potential and antibiotic resistance of cultivable bacterial isolates in order to verify how they behave under stressful conditions (i.e., presence of pathogens and antibiotics). It is also presented a short review on the applications of microorganisms or their abilities to fight CRC. In a general view it was observed some difference between the diversity of bacterial community from TN and TT samples, according to the DGGE profiles. Identical genera of bacteria were identified in TN and TT samples (e.g., Escherichia, Klebsiella, Pseudomonas), although some were only found in TN (e.g., Citrobacter) or TT (e.g., Enterococcus). Some bacterial isolates showed antimicrobial activity against Gram-positive and Gramnegative pathogens, and all of them were resistant to at least three different antibiotics. These responses help to understand the behavior of gut bacteria under infectious aggressions, which often occur in CRCaffected patients. On the other hand, given the relevance of gut microbiota on CRC development, the biotechnological abilities of bacteria have been explored as complementary or adjuvant therapeutics for controlling CRC. They mainly involve microbiota modulation through the consumption of pro- and prebiotics, and fecal microbiome transplantation, bacteriophage therapy, but also other groundbreaking strategies targeting CRISPR, essential and resistant bacterial genes, and quorum sensing systems.
O cancro colorectal (CCR) é o terceiro tipo de cancro fatal no mundo e, nos últimos anos, Portugal tem vindo a assistir a um aumento da sua incidência. Dada a relevância da simbiose entre a microflora intestinal e o hospedeiro para a manutenção da homeostase do organismo, vários estudos têm-se focado na análise do microbiota associado a situações de saúde e doença, nomeadamente ao CCR. Cada vez mais é relevante conhecer a comunidade microbiana intestinal associada a CCR, pois pode constituir uma ferramenta para diferentes aplicações clínicas no âmbito desta patologia. Portanto, o presente estudo pretendeu realizar uma caracterização preliminar da comunidade bacteriana não cultivável e cultivável, extraída a partir de tecidos tumorais (TT) e tecidos adjacentes saudáveis (TN) da mucosa intestinal de pacientes portugueses que apresentem CCR. Adicionalmente testou-se o potencial antimicrobiano e a resistência a antibióticos das estirpes bacterianas isoladas no sentido de verificar como se comportam em situações de stresse (presença de bactérias patogênicas e antibióticos). Por fim, é apresentada uma revisão sumária acerca das aplicações de microrganismos como estratégias terapêuticas complementares para o combate do CCR. De um modo geral, observou-se alguma diferença na diversidade da comunidade bacteriana entre TN e TT de cada paciente, conforme os perfis genéticos obtidos por DGGE. No que concerne as bactérias isoladas foram identificados alguns géneros semelhantes em TN e TT (e.g., Escherichia, Klebsiella, Pseudomonas), muito embora, outros tivessem sido registados apenas em TN (e.g., Citrobacter) ou TT (e.g., Enterococcus). Alguns dos isolados bacterianos revelaram resistência a bactérias Gram-positivas e Gram-negativas, apresentando todos eles resistência a, pelo menos, três antibióticos diferentes. Estas respostas auxiliam na compreensão da resposta do microbiota a agressões infeciosas em situações de CCR. Por outro lado, e tendo em conta a relevância do microbiota na evolução da doença, as potencialidades biotecnológicas das bactérias têm vindo a ser exploradas para terapias complementares ou adjuvantes no combate ao CCR. Estas envolvem a modelação do microbiota através de pro- e prebióticos, transplante de microbioma fecal e terapia bacteriofágica, para além de outras estratégias inovadoras basedas em sistemas CRISPR, genes bacterianos essenciais e de resistência, e sistemas de comunicação entre bactérias.
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Balachandran, Banujan. "Investigating novel therapies for breast cancers resistant to Trastuzumab." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117212.
Full textAbstract:
Approximately 20% of all metastatic breast cancers overexpress the HER-2 receptor, a cell surface-bound receptor tyrosine kinase upstream of crucial proliferation and cell survival pathways. Trastuzumab, a humanized monoclonal antibody binding to the extracellular domain of HER-2, has proven to be a beneficial treatment for patients diagnosed as HER-2+ but continues to have limitations. With low response rates for patients when administered alone and for patients having received prior chemotherapy, the true potential of trastuzumab seems to arrive only through combination therapies. However, the majority of advanced HER-2 positive breast cancer patients still develop resistance to the therapy by the end of the first year or present de novo resistance. For this reason, it is important to continue to investigate therapies to give in combination with trastuzumab to improve progression-free survival and overall survival in this clinical setting. Two Phase II Astra Zeneca compounds: AZD0530, a dual Src and Abl kinase inhibitor and AZD8931, a pan-erbb tyrosine kinase inhibitor abrogating EGFR-, HER-2-, and HER-3-mediated signaling were explored in this context using established trastuzumab-naïve and trastuzumab-resistant cell lines. AZD0530 was not effective when administered alone and any combinations with trastuzumab showed positive responses only in those models in which some form of response to AZD0530 alone had been seen. Clinically-relevant responses to AZD8931 were seen in all cell lines tested and for this reason a head-to-head comparison was carried out with lapatinib, the FDA-approved therapy for patients progressing on trastuzumab. AZD8931 alone and in combination with trastuzumab worked effectively to stop proliferation and induce cell death in both trastuzumab-naïve and trastuzumab-resistant cell lines at clinically relevant doses. AZD8931 had similar activity to lapatinib in the SKBR3-based ER-negative cell lines, but was less active in the BT474-based ER-positive cell lines. From this study, it appears AZD8931 is a therapeutic candidate for overcoming trastuzumab resistance but further investigation is required, before translation into the clinical setting, including the use of animal models of trastuzumab-resistance, specifically in the ER-negative subtype.20% des cancers du sein sur-expriment le récepteur membranaire, HER-2 ayant une forte activité enzymatique tyrosine kinase et qui est impliqué dans l'activation de plusieurs voies de signalisation comme la prolifération et la survie cellulaire. Trastuzumab, un anticorps monoclonal spécifique pour la portion extracellulaire de HER-2 et a été démontré efficace dans le traitement des cancers du sein positifs pour HER-2, mais son efficacité cliniques a des limites. En effet, avec un faible taux d'efficacité lorsqu'administré seul chez les patients ayant été préalablement traités ou non avec la chimiothérapie, le vrai potentiel de Trastuzumab ne semble se dévoiler qu'en combinaison avec d'autres thérapies. Effectivement, la majorité des patients atteints d'un cancer du sein avancé et sur-exprimant HER-2 deviennent insensibles au Trastuzumab avant la fin de leur première année de traitement ou présentent une résistance intrinsèque. Pour cette raison, il est important de poursuivre la recherche pour permettre la découverte de nouvelles thérapies à administrer en combinaison avec Trastuzumab dans le but de prévenir la progression de la maladie et d'améliorer la survie des patients. Deux médicaments développés par AstraZeneca font présentement l'objet d'essai cliniques de phase II : AZD0530, un double inhibiteur des kinases Src et Abl ainsi que AZD8931, un inhibiteur de tyrosine kinase bloquant la signalisation via les récepteurs EGFR, HER2 et HER3. Dans la présente étude, l'efficacité de AZD0530 et AZD8932 a été analysée dans des lignées cellulaires n'ayant jamais été exposées au Trastuzumab ainsi que des lignées résistantes au trastuzumab. Non-efficace lorsqu'administré en monothérapie, AZD0530 s'est montré actif en combinaison avec Trastuzumab, mais seulement à des concentrations où AZD0530 utilisé seul avait un effet positif. Quant à AZD8931, son efficacité a pu être observée à des concentrations pertinentes en clinique dans toutes les lignées cellulaires testées. Conséquemment, une étude comparative entre AZD8931 et Lapatinib a été menée, lapatinib étant le traitement approuvé par la FDA pour les patients dont la maladie progresse sous Trastuzumab. Les résultats démontrent que AZD8931, en monothérapie ou en combinaison avec Trastuzumab limite efficacement la prolifération et induit la mort cellulaire dans chacune des lignées évaluées, résistante ou non au Trastuzumab, à des doses cliniquement pertinentes. Cette étude nous permet aussi de montrer que AZD8931 a une activité similaire à celle de Lapatinib dans la lignée cellulaire SKBR3 qui ne sur-exprime pas le récepteur à l'estrogène (ER), mais qu'il était toutefois moins efficace dans la lignée positive pour ER, BT474. Cette étude nous permet donc de conclure que AZD8931 pourrait être un important candidat thérapeutique pour contrer la résistance au Trastuzumab. D'autres analyses sont toutefois requises, tel que l'utilisation d'un modèle vivant de résistance au Trastuzumab dans un contexte de lignées négatives pour le ER, avant de confirmer le potentiel de AZD8931 comme agent de seconde ligne et d'appliquer ces résultats en clinique.
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Cuomo, Alessandro. ""TREATMENT-RESISTANT DEPRESSION" AND USE OF INTRAVENOUS KETAMINE AND INTRANASAL ES-KETAMINE." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1148507.
Full textAbstract:
Treatment-Resistant Depression (TRD) is defined as major depression characterized by the unsatisfactory response of two or more antidepressant therapies (adequate for dosage and duration), associated with a high incidence of comorbidities, with more marked impairment of functioning socio-occupational and greater risks of relapse, recurrence and suicidality. The high incidence of TRD has advanced scientific research towards the study of molecules with antidepressant action and with different mechanisms of action than those based on the monoaminergic theory. Ketamine is one of these molecules. A non-competitive antagonist of the glutamate N-methyl-D-aspartate (NMDA) receptor, ketamine is mainly used in clinical practice as an anaesthetic and analgesic agent. It has recently been shown that it exerts an antidepressant activity with few and transient side effects at sub anaesthetic doses. Esketamine is the S-enantiomer of ketamine and has recently been FDA approved in the United States for treating depression that has failed to respond to trials of two or more antidepressants. Although the complete molecular mechanism of ketamine is very complex and still partly unknown, it appears that NMDA blockade induces a modulation of several synaptogenic signalling pathways, such as brain-derived neurotrophic factor (BDNF), improving synaptic plasticity of the prefrontal cortex and hippocampus. The design of the observational and retrospective study object of this thesis is evaluating the clinical response following repeated administration of intravenous ketamine (off-label use) and intranasal esketamine in a sample of 16 patients referring to the Psychiatric Clinic of the Siena University Hospital suffering from TRD. The efficacy assessment was performed by administering the Montgomery-Asberg Depression Rating Scale (MADRS) by an expert clinician before and during each entire treatment cycle of ketamine or esketamine intake.
The data appeared to be significant in terms of benefit deriver from intravenous ketamine or intranasal S-ketamine therapy. In particular on the core symptoms of depression, such as sadness, apathy, inability to concentrate and fatigue, as shown by a reduction in the total score MADRS at last dose> 27% of baseline score. The most significant effects were achieved in the first 4 weeks of administration. The study limit is the small number of samples, which are still being implemented.
To date, ketamine and S-ketamine are proven to be one of the few innovative drug therapies effective and safe for the treatment of TRD.
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Shreim, Amani. "Cibler le splicéosome : une nouvelle stratégie thérapeutique pour contrecarrer la résistance thérapeutique dans les cancers du poumon ?" Electronic Thesis or Diss., Université Grenoble Alpes, 2024. http://www.theses.fr/2024GRALV023.
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Le cancer du poumon est un problème de santé publique majeur et la première cause de décès par cancer dans le monde. Chez les patients porteurs de carcinomes pulmonaires non à petites cellules (CBnPC), la chimiothérapie à base de sels de platine demeure l’une des pierres angulaires du traitement. Même chez les 30% de patients répondeurs, une résistance survient en quelques mois, rendant impérative la recherche d'alternatives thérapeutiques. L’épissage des ARNs participe au contrôle de l’expression des gènes et à la diversité protéique et met en jeu une machinerie multi-protéique, connue sous le nom de splicéosome. Cette dernière décennie, une dérégulation massive de l’expression de certains composants du splicéosome a été observée dans les cancers, conduisant au développement d’inhibiteurs pharmacologiques le ciblant, tels que le SPHINX31 qui inhibe SRPK1, une kinase impliquée dans la régulation de l'épissage par la phosphorylation de divers facteurs d’épissage riches en sérine/arginine (SR). Bien qu’apparaissant comme des médicaments anticancéreux prometteurs, leurs effets sur les cellules cancéreuses demeurent largement méconnus. Dans cette étude, nous montrons que le SPHINX31 inhibe la voie de signalisation ATR, la principale voie impliquée dans la gestion du stress réplicatif, notamment dans des cellules de CBnPCs présentant une résistance acquise aux sels de platine. Cela conduit à une inhibition de la croissance cellulaire, à une augmentation de l'instabilité génomique et à la mort cellulaire. Au niveau moléculaire, nous montrons que SRPK1, la cible du SPHINX31, est recrutée au niveau des fourches de réplication bloquées en cas de stress réplicatif, qu'elle co-immunoprécipite avec le complexe ATR/ATRIP/TOPBP1, et qu'elle est nécessaire au recrutement de TOPBP1/ATRIP à la chromatine et à la formation de foyers nucléaires de TOPBP1. Ces effets de SRPK1 pourraient participer à l’activation complète d’ATR. Nous montrons aussi que SRPK1 interagit directement avec TOPBP1 et que les domaines BRCT-7 et -8 de TOPBP1 sont nécessaires pour cette interaction. Parallèlement, des données de RNA-Seq nous ont permis de montrer que le SPHINX31 et la kinase SRPK1 régulent l'épissage de WIZ, en faveur de variants d'épissage impliqués dans l'activation d'ATR, identifiant ainsi des fonctions dépendantes et indépendantes de l'épissage de SRPK1 par lesquelles cette protéine contrôle la voie de signalisation ATR. Enfin, nos résultats montrent que les effets cytotoxiques du SPHINX31 sont atténués par l'activation de la kinase DNA-PKcs, une voie de secours activée en réponse à l’inhibition d’ATR, et identifient un effet cytotoxique synergique de la combinaison du SPHINX31 avec un inhibiteur de DNA-PKcs in vitro. Dans l’ensemble, ces résultats identifient un nouveau rôle biologique de la kinase SRPK1 dans la gestion du stress réplicatif de l'ADN et le contrôle de la stabilité génomique des cellules cancéreuses pulmonaires. Ils suggèrent aussi fortement que l'utilisation d'inhibiteurs de SRPK1 en combinaison avec des inhibiteurs de DNA-PKcs pourrait induire la mort de cellules tumorales résistantes aux sels de platine dans le cancer du poumonLung cancer is a major public health problem and the leading cause of cancer-related deaths worldwide. In patients with non-small cell lung carcinoma (NSCLC), platinum-based chemotherapy remains a cornerstone of treatment. Even among the 30% of patients who initially respond, resistance typically develops within a few months, making the search for alternative therapies imperative. RNA splicing plays a key role in gene expression control and protein diversity, involving a multi-protein machinery known as the spliceosome. Over the past decade, significant deregulation of the expression of certain spliceosome components has been observed in cancers, leading to the development of pharmacological inhibitors targeting the spliceosome, such as SPHINX31, which inhibits SRPK1, a kinase involved in the regulation of splicing by phosphorylating various serine/arginine-rich (SR) splicing factors. Although these inhibitors show promise as anticancer drugs, their effects on cancer cells remain largely unknown. In this study, we show that SPHINX31 inhibits ATR signaling, the main pathway involved in managing replicative stress, especially in NSCLC cells with acquired resistance to platinum salts. This leads to inhibited cell growth, increased genomic instability, and cell death. At the molecular level, we demonstrate that SRPK1, the target of SPHINX31, is recruited to stalled replication forks during replicative stress, co-immunoprecipitates with the ATR/ATRIP/TOPBP1 complex, and is necessary for the recruitment of TOPBP1/ATRIP to the chromatin as well as the formation of TOPBP1 nuclear foci. All these events contribute to the full activation of ATR. Further examining this interaction, we found that SRPK1 directly interacts with TOPBP1, and that the BRCT-7 and -8 domains of TOPBP1 are necessary for this interaction. Concurrently, RNA-seq data showed that SPHINX31 and SRPK1 regulate the splicing of WIZ, favoring splice variants involved in ATR activation. These results thereby identify both splicing-dependent and -independent functions of SRPK1 in controlling the ATR signaling pathway. Finally, our results indicate that the cytotoxic effects of SPHINX31 are mitigated by the activation of the DNA-PKcs kinase pathway, a backup response mechanism activated in response to ATR inhibition, and identify a synergistic cytotoxic effect of combining SPHINX31 with a DNA-PKcs inhibitor in vitro. Overall, these findings identify a new biological role of SRPK1 kinase in managing DNA replicative stress and controlling genomic stability in lung cancer cells. They also strongly suggest that using SRPK1 inhibitors in combination with DNA-PKcs inhibitors could induce the death of tumor cells resistant to platinum salts in lung cancer
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Blandin, Anne-Florence. "Rôle de l'intégrine α5β1 dans la biologie du glioblastome et dans la résistance aux thérapies anti-EGFR." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ066/document.
Full textAbstract:
Le glioblastome multiforme (GBM) est la tumeur cérébrale primaire la plus fréquente. Une dérégulation des voies de signalisation de l’EGFR et un fort potentiel invasif sont les caractéristiques principales du GBM. Malheureusement, les essais cliniques impliquant des thérapies anti-EGFR dans le traitement des GBM demeurent inefficaces. Nous avons précédemment montré que le récepteur de la fibronectine, l’intégrine α5β1, est associé avec un mauvais pronostic et une résistance des patients au temodal. Les intégrines peuvent coopérer avec les récepteurs aux facteurs de croissance et ainsi amplifier leur potentiel oncogénique. Ici, nous avons cherché à déterminer le rôle de l’intégrine α5 dans la résistance aux thérapies anti-EGFR. Utilisant la lignée U87 de GBM, on a dans un premier temps confirmé que l’activation de l’intégrine sous l’influence de la fibronectine, potentialisait la signalisation de l’EGFR. La perte d’expression d’α5 sensibilise les cellules U87 aux anti-EGFR (cetuximab, gefitinib) dans des essais de clonogénicité en soft agar. L’expression d’ α5 favorise la résistance aux 2 drogues lors de la migration cellulaire. Pour aller plus loin, nous avons développé un nouveau test basé sur la quantification de l’évasion cellulaire à partir d’une sphère tumorale. La perte d’ α5 augmente la sensibilité des cellules U87 à 2 TKI réversibles spécifiques de l’EGFR, gefitinib et erlotinib, mais n’a pas d’effet sur l’efficacité du lapatinib, un TKI irréversible ciblant EGFR, ErbB2, ErbB3 et ErbB4. Grâce à la microscopie confocale, nous avons montré l’effet important du gefitinib sur l’endocytose de l’intégrine et de l’EGFR. Ces résultats suggèrent que l’expression d’ α5 favorise la résistance aux TKI par l’activation des voies de signalisation des récepteurs ErbB ou en contrôlant le trafic membranaire de l’EGFR. On a aussi montré que pour favoriser l’adhésion cellulaire, l’intégrine α5 stimulait la fibrillogénèse. Dans les cellules migrant à distance de la sphère, l’intégrine α5 est strictement engagée dans des adhésions cellule-substrat contenant la protéine FAK activée. Nos résultats soulignent le rôle central du couple fibronectine/ intégrine α5 dans l’invasivité du GBM et la résistance aux thérapies anti-EGFRGlioblastoma multiforme (GBM) is the most common primary brain tumor. Alteration of the EGFR pathway and high invasive potential are hallmarks of GBM. Unfortunately, trials using anti-EGFR therapies for the treatment of GBM reveal limited efficacy. We previously showed that overexpression of the fibronectin receptor, α5β1 integrin, is associated with a poor prognosis for patients and is responsible for chemoresistance to temodal. Integrins can cross-talk with growth factor receptors and amplified their oncogenic activity. Here, we sought to determine the potential role of α5 integrin in resistance to anti-EGFR therapy. Using U87 GBM cell line, we first confirmed that fibronectin-mediated integrin activation potentiated EGFR signaling. Loss of α5 integrin expression sensitized U87 cells to anti-EGFR drugs (cetuximab, gefitinib) in soft agar clonogenic assay. α5 expression can trigger resistance to both drugs on cell migration. To go further, we developed a new assay based on the quantification of cell evasion from tumor spheroids. α5 depletion increased U87 cell sensitivity to gefitinib and erlotinib, 2 EGFR-selective reversible TKI, but had not effect on lapatinib efficacy, an irreversible TKI that target EGFR, ErbB2, ErbB3 and ErbB4. Confocal microscopy revealed a strong impact of gefitinib on EGFR and integrin endocytosis. These results suggested that α5 expression may trigger resistance to TKI either by activating ErbB pathways or by controlling EGFR membrane trafficking. We also showed that to promote cell adhesion, α5 integrin stimulated fibronectin fibrillogenesis. As cells moved away from the spheroids, α5 became strictly engaged in cell-substratum adhesion sites where it recruited activated FAK. Our work highlights the pivotal role of fibronectin/α5β1 integrin in invasivity of GBM and resistance to anti-EGFR drugs
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Pohorecka, Magdalena. "Rôle de c-Jun dans la réponse aux inhibiteurs de la voie des MAPK dans les mélanomes." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30373.
Full textAbstract:
Il est clairement admis que la voie des MAPK est essentielle à la mélanomagenèse. Le développement de nouveaux médicaments ciblant cette voie, tels que les inhibiteurs de BRAF et/ou de MEK, a constitué une avancée majeure dans la prise en charge thérapeutique du mélanome. Cependant, les patients rechutent systématiquement sous traitement, ce qui suggère l'émergence de mécanismes de résistances. De nombreuses études montrent que l'expression et l'activation du facteur de transcription c-Jun sont induites après traitement de cellules de mélanome BRAF-mutées par des inhibiteurs de la voie des MAPK (MAPKi). De plus, la déplétion de c-Jun sensibilise ces cellules à ces inhibiteurs en induisant l'apoptose. Nous avons transfecté des lignées cellulaires de mélanome BRAF-mutées par des siARN dirigés contre c-Jun et traité ces cellules avec un inhibiteur de BRAF (PLX4032). Nous avons ensuite analysé l'expression du génome par une étude de transcriptomique afin de déterminer les gènes cibles de c-Jun qui pourraient être impliqués dans la réponse pharmacologique aux MAPKi. Cette étude a révélé que SLIT And NTRK Like Family Member 6 (SLITRK6) est un gène cible de c-Jun qui pourrait être associé à une réponse pharmacologique antitumorale aux MAPKi. En effet, l'ARNm et la protéine SLITRK6 sont induits dans les lignées cellulaires de mélanome BRAF-mutées après traitement par inhibiteur de BRAF seul ou en association avec un inhibiteur de MEK (AZD6244). Nous avons également montré que la combinaison des inhibiteurs de la voie des MAPK avec un anticorps conjugué à un cytotoxique ciblant SLITRK6 augmente la mort des cellules de mélanome BRAF-mutées en induisant de l'apoptose in vitro. Finalement, nos travaux montrent que SLITRK6 pourrait être une nouvelle cible pharmacologique pour le traitement du mélanome métastatique BRAF-muté et/ou être un biomarqueur potentiel de cellules résistantes aux MAPKiIt is clearly recognized that the MAPK pathway is essential for melanomagenesis. The development of new drugs targeting this pathway such as BRAF inhibitors and/or MEK inhibitors has been a major advance in the therapeutic management of melanoma. However, patients still relapse suggesting the emergence of mechanisms of resistance. Many data show that both the expression and activation of the transcription factor c-Jun are induced after treatment of BRAF-mutant cells with MAPK pathway inhibitors (MAPKi). Furthermore, depletion of c-Jun sensitizes cells to these inhibitors triggering apoptosis. We depleted BRAF-mutant melanoma cell lines for c-Jun by siRNA and treated cells with a BRAF inhibitor (PLX4032). Whole genome expression was then analysed by transcriptomic study to determine target genes of c-Jun that could be associated with pharmacological response to MAPKi. This study revealed that SLIT And NTRK Like Family Member 6 (SLITRK6) is a target gene of c-Jun that could be associated with antitumor pharmacological response to MAPKi. Indeed, SLITRK6 mRNA and protein are induced in BRAF-mutant melanoma cell lines after BRAF inhibitor treatment alone or in combination with MEK inhibitor (AZD6244). We also show that the combination of MAPKi with an antibody conjugated with a cytotoxic drug targeting SLITRK6 increases BRAF-mutant melanoma cell death triggering apoptosis in vitro. Finally, our data show that SLITRK6 could be a new pharmacological target for the treatment of BRAF-mutant metastatic melanoma and/or a potential biomarker of resistant cells to MAPKi
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Goodwin, Annabelle Michelle. "An Exploration of Feminist Family Therapists' Resistance to and Collusion with Oppression." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/27701.
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In this study, I explore the ways in which feminist family therapists encourage exploration of, resistance to, and collusion with, oppression. I explore qualitatively the critical dialogues, both inner, and with others, that feminist family therapists employ to address oppressive systems. My research questions are: a. How do family therapists who identify as feminist describe how their feminist identities and ideas about feminism have evolved over time? b. How do feminist family therapists report stories of their own resistance to gender-based oppression? c. How do feminist family therapists report stories of their own collusion with the oppression of others? And d. How do feminist family therapists encourage clients to examine oppression and collusion of oppression of others? I use tape-recorded, one-on-one interviews with a theoretical sample of self-identified feminist participants who have demonstrated rigorous attention to feminist inquiry and practice in the field of family therapy. Consistent with a contemporary grounded theory methodology, generation of theory is based on constructivist methods, which recognize that there are multiple coexisting realities and not one objective truth (Charmaz, 2000). By way of constructivist grounded theory analysis the following four categories emerged: (a) Actions and Strategies of a Feminist Family Therapist, (b) Itâ s a Sensibility: The Development of a Feminist Identity, (c) Recognizing Oppression and Injustice: A Quest for Liberation and (d) Resisting: Exploring Why, How, and at the Risk of Which Consequences.Ph. D.
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Kayyali, Yousef John. "Therapist Personal and Professional Experience as Predictors of Gestalt Therapy Contact Resistances." Thesis, Adler School of Professional Psychology, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10936230.
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Gestalt therapy postulates psychological illness and health are interwoven with how a person gets in touch or interrupts contact with self, others, the environment, and the spiritual field (Brownell, 2018; Corey, 2005; Perls, 1969, 1973, 1942/1993; Perls, Hefferline, & Goodman, 1951/1994; Perls, 1976; Polster & Polster, 1973; Prosnick & Woldt, 2014; Yontef, 1993, 1999). Preliminary research found support for psychological wellness and disturbance relative to Gestalt therapy contact styles between human organism and environment (Byrnes, 1975). The purpose of this study was to investigate four predictor variables of therapist age, professional experience, theoretical orientation, and preferred theoretical orientation as a client vis-à-vis seven criterion variables of Gestalt therapy contact resistances —or interruptions to contact. Essentially, this study hypothesized therapists with more experience (i.e., age, and professional practice) and Gestalt therapy exposure (i.e., practicing, and receiving Gestalt therapy as a client) would obtain lower scores on the seven Gestalt contact resistances: Confluence, Desensitization, Introjection, Projection, Retroflection, Deflection, and Egotism—as gauged by the Gestalt Inventory of Resistance Loadings (GIRL; Woldt & Prosnick, 2014a). Archival datasets comprising 291 mental health trainees, professionals, and affiliates were utilized. Age and Gestalt therapy theoretical orientation were most supported suggesting Gestalt therapy coupled with aging process attenuate Gestalt contact resistances. Professional experience and Gestalt therapy preference as a client produced partial support indicating these two variables also aid in the diminution of Gestalt contact resistances. In sum, 13 out of 28 hypotheses (i.e., 46%; four more neared significance) were statistically significant cementing credence for the utility of Gestalt therapy modi in helping both psychotherapists and clients alike transcend obstacles precluding growth, maturation, actualization, organismic self-regulation, authenticity, and wholeness .
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Gharbi, Leïla. "Portees et limites de la guidance infantile parentale dans une therapie d'enfants marocains : analyse des resistances rencontrees." Toulouse 2, 1987. http://www.theses.fr/1987TOU2A027.
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Le present travail porte sur les possibilites d'utiliser la guidance parentale dans le contexte marocain. Deux axes de reflexion s'en degagent : le premier est consacre a l'approche theorique des differentes relations therapeutiques et de la place qui y occupe la guidance parentale. Son originalite reside dans l'interet porte a la famille en tant que partie prenante dans la prise en charge de l'enfant. La famille, un des lieux de socialisation de l'enfant, occupe une place preponderante dans la guidance parentale. Or cette derniere est nee dans un contexte socio-cu\turel vehiculant un systeme de valeur (famille, personne, representation de la maladie mentale) completement different de celui qui caracterise le contexte marocain en mouvance (acculturation). C'est pour cette raison que ce dernier a fait l'objet d'une analyse afin de degager les possibilites d'utiliser la technique de guidance parentale et de reperer les resistances rencontrees. Le second axe fournit les elements cliniques qui ont permis de repondre a la problematique posee. A partir de l'analyse de vingt observations se degage la possibilite d'utiliser cette technique. Cette derniere a ete legerement remaniee (une plus grande utilisation de la symbolique et de l'expression non-verbale parallelement a une interpretation rationnelle moindre) pour tenir compte de la specificite du contexte marocain. Cette specificite transparait egalement dans les modes d'expression a connotation socio-culturelle (avec une predominance des themes religieux) des resistances rencontrees. Cependant comme toute relation therapeutique, la guidance parentale presente des limites. Outre les limites individuelles propres a chacun, les limites resident au niveau de la croyance dans le lien entre la problematique du parent et celle de l'enfantThe present work relates to the possibilities of using parental guidance in the moroccan context. Two axis of reflexion emerge : the first one is centered on the theoretical approach of different therapeutical relation with the parental guidance involved in it. Its originality lies in the interest focused on the family involved as it is in taking care of the child. The family - one of the socialization places of the child - holds an outstanding rank in the parental guidance. But this guidance has emerged in a social and cultural context that carries a system of values (family, individual representation of the mental illness) completely different of the one caracterizing the moroccan context in constant change (acculturation). It is for this reason that the moroccan context is analyzed, in order to bring out the technique of parental guidance, and to outline the resistance encountered. The second axis offers the clinical elements which have lead to consider as well as to answer the stated problematic. From the analysis of twenty observations, it appears that it is possible, to use this technique. Even though it has been slightly changed (wider use of the symbolic and the non verbal expression as well as a lesser rational interpretation) to take into consideration the specificity of the moroccan context. This specificity appears as well in the ways of expression that have a social and cultural connotation (with a predominance of the religious themes) and in the resistance encountered nevertheless as in any therapeutical relation the parental guidance has its limits. Besides the individual limits specific to everyone, the limits lie also at the level of the belief in the link between the parent problematic and the one of the child
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Bargou, Ralf. "Die Bedeutung von Apoptoseresistenzmechanismen für die Pathogenese und Therapie maligner Lymphome." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2001. http://dx.doi.org/10.18452/13750.
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Apoptoseresistenzmechanismen spielen bei der Pathogenese maligner Lymphome eine zentrale Rolle. So konnte bei Hodgkin/Reed-Sternbergzellen eine Deregulation des Transkriptionsfaktors NF-_B beobachtet werden, die zu verstärkter Apotoseresistenz führt und so zum malignen Wachstum dieser Zellen wahrscheinlich entscheidend beiträgt. Es konnte gezeigt werden, dass die selektive Blockade von NF-_B sowohl zu erhöhter Apoptosesensitivität als auch zur Inhibition der Zellzyklusprogression in kultivierten Hodgkinzellen führt. Der genaue molekulare Mechanismus der NF-_B-Deregulation in Hodgkinzellen ist jedoch noch unklar. Apoptoseresistenzmechanismen sind nicht nur bei der Pathogenese maligner Lymphome, sondern auch bei der Entstehung von Therapieresistenz von Bedeutung. So konnte gezeigt werden, dass die Überexpression proapoptotischer Gene der bcl-2 Familie in resistenten malignen Zellen sowohl die Empfindlichkeit gegenüber Zytostatika als auch gegenüber Antikörperbehandlung wiederherstellen kann. Neben der bcl-2 Familie spielt wahrscheinlich auch das Apo-I/Fas-System eine wichtige Rolle bei der Entstehung von Zytostatikaresistenz und immunologischer Resistenz. Somit stellt neben der Überexpression des P-Glykoproteins (MDR1), das als transmembranes "Pumpenprotein" Zytostatika aus der Tumorzelle heraustransportieren kann, die Deregulation Apoptose-steuernder Gene einen weiteren wichtigen Therapie-Resistenzmechanismus dar. Eine Möglichkeit, intrazelluläre Resistenzmechanismen zu umgehen, stellt die indirekte Induktion von Zelltod mit Hilfe bispezifische Antikörper dar. Durch diese Moleküle kann eine T-Zell-vermittelte Zellyse von Lymphomzellen erreicht werden.Resistance towards apoptosis plays an important role in the pathogenesis of malignant lymphomas. It could be demonstrated that deregulation of the transcription factor NF-kB is a common molecular defect of Hodgkin/Reed-Sternberg cells that leads to enhanced resistance towards apoptosis and therefore probabaly contributes to the malignant growth of these cells. It couldbe shown that blocking of NF-kB leads to increased sensitivity towards apoptosis and decreased cell cycle progression. The precise molecular mechanism that leads deregulation of NF-kB is still unknown. Besides its role in the pathogenesis of malignant lymphoma resistance towards apoptosis plays an important role in the development of drug resistance. It could be shown that overexpression of pro-apoptotic members of the bcl-2 family in resistant tumor cells can restore sensitivity towards both cytotoxic drugs as well as antibody treatment. In addition to the bcl-2 family the Apo-I/Fas system is also involved in the development of drug resistance. Thus, besides overexpression of p-glycoprotein (MDR-1) that might pump cytotoxic drugs out of a malignant cell deregulation of apoptosis regulating genes is another important mechanisms of drug resistance development. One possibility to overcome drug resistance is the induction of cell death via bispecific antibodies. These molecules can induce T cell mediated lysis of lymohoma cells.
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Brodzinski, Annika. "Langzeiteffektivität der Therapie mit Lamivudin bei Patienten mit chronischer Hepatitis-B-Virus-Infektion – Prädiktion des Langzeitansprechens und Spektrum der Resistenzentwicklung." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-165685.
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Lamivudin war der erste Inhibitor der Hepatitis-B-Virus(HBV)-Polymerase, der für die Therapie der chronischen HBV-Infektion in Deutschland zugelassen wurde und seitdem zu einer signifikanten Senkung der HBV-assoziierten Letalität beigetragen hat. Aufgrund des hohen Resistenzrisikos unter Lamivudin wird der Einsatz der Substanz jedoch in den aktuellen HBV-Leitlinien nicht mehr empfohlen. Stattdessen sollen hochpotente Nukleos(t)id-Analoga wie Entecavir oder Tenofovir angewendet werden, die jedoch gerade in den ärmeren Endemiegebieten nur selten verfügbar sind. Zudem gibt es auch in den westlichen Industrieländern Patienten, die seit Jahren resistenzfrei mit Lamivudin behandelt wurden. Ob diese von einer prophylaktischen Therapieumstellung profitieren, ist bislang unklar.
Ziel dieser Studie war es, die Langzeiteffektivität der Lamivudin-Therapie sowie die Häufigkeit und die prädiktiven Faktoren der Lamivudin-Resistenz zu ermitteln. In einer retrospektiven Untersuchung wurden hierzu die Verläufe von 147 HBeAg-positiven und 80 HBeAg-negativen Patienten aus 3 hepatologischen Zentren in Deutschland analysiert. Zur Sicherung der Lamivudin-Resistenz erfolgte eine genetische Resistenztestung. Zusätzlich wurde der klinische Nutzen einer hoch-sensitiven Real-Time-PCR zur Bestimmung der HBV-DNA evaluiert.
Im Langzeitverlauf zeigte sich bei einer bestimmten Subgruppe von Patienten ein gutes virologisches, serologisches und biochemisches Ansprechen. Die vorliegende Studie bestätigte jedoch auch das hohe Resistenzrisiko unter einer Langzeittherapie mit Lamivudin, welches nach 7 Jahren 55% erreichte. Maßgeblicher prädiktiver Faktor für eine spätere Resistenzentwicklung war das virologische Ansprechen im Therapieverlauf. Entgegen den Ergebnissen früherer Studien stellte die Höhe der HBV-DNA zum Zeitpunkt des Therapiebeginns keinen prädiktiven Faktor der Resistenzentwicklung dar, sondern beeinflusste die Wahrscheinlichkeit des virologischen Ansprechens. Der Einsatz hoch-sensitiver Verfahren zur Bestimmung der HBV-DNA zur Therapiekontrolle oder Prädiktion der Lamivudin-Resistenz ist unseren Beobachtungen nach nicht erforderlich. Bei HBeAg-positiven Patienten hatte zudem ein HBeAg-Verlust einen günstigen Einfluss auf die Resistenzwahrscheinlichkeit. Auffällig war außerdem, dass HBeAg-positive Patienten nach einem virologischen Ansprechen nur noch ein sehr geringes Resistenzrisiko aufwiesen, während bei HBeAg-negativen Patienten trotz eines virologischen Ansprechens im gesamten Therapiezeitraum neue Fälle von Lamivudin-Resistenz beobachtet wurden. Auch durch die Kombination verschiedener Merkmale, die die Resistenzwahrscheinlichkeit nachweislich beeinflussten, konnte letztlich keine Subgruppe identifiziert werden, die im Verlauf resistenzfrei blieb oder nur ein sehr geringes Resistenzrisiko aufwies.
Zusätzlich wurden diverse weitere Wirts-, virale und therapieassoziierte Charakteristika untersucht, die jedoch keinen prädiktiven Wert in Hinblick auf eine Resistenzentwicklung aufwiesen. Insbesondere natürlich vorkommende HBV-Mutationen, die mit einer Lamivudin-Resistenz assoziiert sind, wurden bei 9% der unvorbehandelten Patienten nachgewiesen, ohne dass dieser Befund einen signifikanten Einfluss auf das virologische Ansprechen oder die Resistenzwahrscheinlichkeit zeigte.
Zusammenfassend ist festzustellen, dass wir in unserer Studie Parameter definieren konnten, die mit einem Langzeitansprechen assoziiert sind und damit die Grundlage einer individualisierten Therapie darstellen könnten. Die Ergebnisse sind vor allem für Länder mit eingeschränkten Therapiemöglichkeiten von Relevanz. Sofern höher potentere Nukleos(t)idanaloga zur Verfügung stehen, sollte in Anbetracht des hohen Resistenzrisikos allerdings von einer Therapie mit Lamivudin abgesehen werden. Liegt bereits eine langjährige Lamivudin-Therapie ohne Zeichen einer Resistenzentwicklung vor, so erscheint die Fortsetzung jedoch gerechtfertigt. Dennoch sind regelmäßige Kontrollen erforderlich, da Lamivudin-Resistenzen auch nach mehr als 10 Jahren auftreten können.
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Giusti, Veronica <1990>. "Preclinical Development of novel therapeutic approaches in models resistant to targeted therapies in HER2-positive mammary breast cancer." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amsdottorato.unibo.it/8849/1/Giusti_Veronica_tesi.pdf.
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HER2 enriched mammary breast cancer represents 15-25% of mammary carcinomas and is associated to increased aggressiveness and worse prognosis. Advent of targeted therapies against HER2 has improved 5-year survival up to 75%, nevertheless receptor discordance, which is observed in 10.8% of metastasis, as well as resistance to targeted therapies render it a still challenging disease.
On the one hand, taking advange of a recently estabilished murine model of spontaneous loss of HER2 expression, we sought to understand the underlying mechanism, to evaluate role of trastuzumab and to identify identify druggable targets in HER2-negative metastasis or relapses of HER2-positive tumors. The study of transcriptome of cell lines with different HER2 expression has permitted us to identify pathways related to the modulation of this more malignant phenotype, which appeared to be promoted by trastuzumab. Some indications emerged for inhibition of PDGFR-B by sunitinib in tumours which have lost HER2 expression.
On the other hand, a recently established collection of patient derived xenografts (PDX) was used to obtain models of progression where to evaluate the effect of neratinib, a pan-HER tyrosine kinase inhibitor. No abrupt loss of HER2 expression was registered in these models. Collectively, our data, obtained in ER-/PR- HER2+ PDX, strongly indicate a great and long-lasting efficacy of neratinib even in trastuzumab-resistance and after progression and call for further evaluation of neratinib in advanced clinical settings. In our PDX diagnosed as luminal B expressing HER2 (score 2+), neratinib alone had no effect but synergized with tamoxifen and their combination tended to confer a little survival benefit in vivo, thus underscoring the possible relevance of dual blockade in tumors expressing both hormone receptors and HER2.
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Aust, Julia Katharina. "Einfluss von Polymorphismen der Methylentetrahydrofolat Reduktase sowie des Multi-Drug-Resistance Proteins auf die Therapie der Rheumatoiden Arthritis mit Methotrexat." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-130248.
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ROUSSEAU, JEANROY CHRISTINE. "Diabete insulino-requerant : etude chez 22 patients des effets d'une insulino-therapie intensive transitoire sur l'insulino-secretion et l'insulino-resistance." Nice, 1990. http://www.theses.fr/1990NICE6509.
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FERRANI, KARIMA. "Etude de la selectivite d'incorporation et de retention de derives d'hematoporphyrine par les cellules normales et tumorales : bases rationnelles pour la therapie photodynamique des cancers." Strasbourg 1, 1995. http://www.theses.fr/1995STR1M405.
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Chhouri, Houssein. "Analysis by DNA barcoding of the heterogeneous response to anticancer drugs by different subpopulations of lung cancer cells." Electronic Thesis or Diss., Normandie, 2022. http://www.theses.fr/2022NORMR037.
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Le cancer est une maladie évolutive, caractérisée par l’existence d’un mélange complexe de plusieurs sous-populations cellulaires qui peuvent évoluer en réponse aux conditions environnementales. Cette hétérogénéité intratumorale a des conséquences extrêmement importantes, non seulement sur la progression tumorale, les métastases, mais aussi sur l’efficacité des traitements. Le cancer bronchique non à petites cellules (CBNPC) avec mutations activatrices de l’Epidermal Growth Factor Receptor (EGFR) est traité avec des inhibiteurs spécifiques de ce récepteur. Malheureusement, après une réponse favorable de plusieurs mois, ces tumeurs presque invariablement développent une résistance à ces médicaments. Au cours des traitements, des sous-populations de cellules résistantes ou tolérantes sont sélectionnées et vont émerger pour conduire à une rechute de la tumeur.Dans cette étude, nous avons utilisé une approche d’étiquetage cellulaire pour marquer plusieurs milliers de populations de cellules PC9 de CBNPC avec des codes-barres génétiques et suivre leur évolution clonale en réponse aux thérapies anticancéreuses. Nos résultats ont révélé que certains clones présentent une réponse spécifique et prédéterminée en fonction du traitement, ce qui indique que le phénotype tolérant ou encore hautement sensible représente une propriété intrinsèque de certaines cellules qui préexistent au sein de la population cellulaire initiale. Nous avons également montré que chaque type de médicament anticancéreux exerce un effet caractéristique sur l'architecture clonale de la population cellulaire, ce qui entraîne un profil de code-barres spécifique qui peut être utilisé comme signature pour comparer différents composés et étudier leurs mécanismes d'action. Nous avons généré une collection de profils de codes-barres à partir de 87 composés connus, agissant sur des processus cellulaires différents, et les avons utilisés pour identifier le mécanisme d'action d’une nouvelle molécule capable d'inhiber spécifiquement la croissance des cellules de CBNPC.Dans la dernière partie de la thèse, grâce à la technologie CRISPR/Cas9, nous avons développé Barcode-Tracker, une nouvelle stratégie pour identifier et isoler des clones d'intérêt à partir d’une population de cellules tumorales, basée sur la reconnaissance d'un code-barres génétique spécifique. Contrairement à d'autres approches, Barcode-Tracker n'utilise pas d’inhibiteur pour sélectioner des cellules, et peut être utilisé pour purifier les clones selon leur capacité intrinsèque à se comporter d'une manière particulière en présence d'un traitement, imitant ainsi la réponse d'une tumeur naïve. Avec une meilleure compréhension de la manière dont le traitement anticancéreux peut affecter l’évolution tumorale, ces études devraient permettre une amélioration des stratégies thérapeutiques pour les patients atteints de CBNPCCancer is a dynamic disease, characterized by the co-existence of multiple subpopulations of tumor cells that can evolve in response to environmental changes. This intratumor heterogeneity has dramatic consequences, not only for cancer progression and metastatic spread, but also for treatment. Specific tyrosine kinase inhibitors are effective against non-small cell lung cancers harboring activating mutations of the epidermal growth factor receptor (EGFR), but the response is not durable, and cure remains elusive because of the inevitable development of acquired resistance. During therapeutic intervention, small subpopulations of resistant or tolerant cells are selected by virtue of their higher fitness, ultimately resulting in tumor relapse.In this study, we used cellular barcoding to label several thousand populations of PC9 NSCLC cells and monitor their clonal dynamics in response to anti-cancer therapies. Our results revealed that some clones display a specific and predetermined response to treatment, indicating that cells that are primed to behave as tolerant or highly sensitive pre-exist in the original mass population. We extended these findings and showed that each type of anti-cancer drugs exerts a characteristic effect on the clonal architecture of the cell population, resulting in a specific barcode pattern that can be used as a signature to compare different compounds and investigate their mechanism of action. We have generated barcode profiles from 87 drugs targeting various pathways and used it to predict the mechanism of action of a new compound that can specifically inhibit NSCLC cell growth.In the last part of the thesis, we took advantage of CRISPR/Cas9 technology to devise Barcode-Tracker, a new strategy to identify and isolate clones of interest from a mass population based on the recognition of a specific genetic barcode. Contrary to other approaches, Barcode-Tracker doesn’t involve drug selection of the cells and it can be used to sort clones according to their intrinsic capacity to behave in a particular manner in the presence of treatment, thus mimicking the response of a therapy-naïve tumor. By providing a better understanding of how treatment can shape clonal evolution, our studies should help to improve therapeutic strategies for NSCLC patients
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Crane, Sarah Becker. "Therapists' descriptions of their beliefs and practices regarding engaging resistant caregivers and adolescents : a project based upon an independent investigation /." View online, 2008. http://hdl.handle.net/10090/5877.
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Walther, Wolfgang. "In vitro- und in vivo Untersuchungen für eine nicht-virale und Therapie-regulierbare Tumorgentherapie." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2004. http://dx.doi.org/10.18452/13932.
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Die Gentherapie hat in den letzten Jahren wesentliche Entwicklungen im Vektordesign, der kontrollierte Expression sowie der Sicherheit ihrer Anwendung durchgemacht. Die Erkenntnis, dass die Tumorgentherapie allein nur in begrenztem Maße zum erhofften therapeutischen Benefit für den Patienten beitragen kann, führte zum Konzept der lokalen Gentherapie als Teil anderer, etablierter Tumortherapien. In diesem Zusammenhang wird die Gentherapie als eine moderne Option zur Steigerung der Effizienz von Chemotherapie, Strahlentherapie oder Hyperthermie verstanden. Zum Erreichen dieses Zieles ist die Etablierung Therapie-regulierbarer Vektorsysteme von besonderer Attraktivität. Im Rahmen der Strategie des lokalen Transfers therapeutischer Gene bietet inzwischen die Anwendung nicht-viraler Transfersysteme, wie z.B. in vivo-Elektrotransfer, Gene-Gun oder Jet-Injection eine klinisch applikable Technologie. Die Etablierung einer effizienten, auf der Jet-Injection basierenden nicht-viralen Transfertechnologie und die Analyse ihres Potentials für eine klinische Anwendung in einem multimodalen Therapiekonzept war ein wesentliches Ziel der Arbeit. Es wurde gezeigt, dass die Jet-Injection in tierexperimentellen Tumormodellen zur effizienten Expression der Transgene führt, dass sowohl Eindringtiefen, als auch Verteilung der Jet-Injection optimal für einen effizienten Gentransfer sind und die Höhe der Genexpression mit etablierten Gentransfer-Technologien, wie z.B. der in vivo-Lipofektion, vergleichbar ist. Basierend auf der Strategie des Einsatzes der Gentherapie in Kombination mit anderen Therapien, bestand ein weiteres Ziel der Arbeit in der Charakterisierung und Anwendung konditioneller Vektorsysteme, mit denen die Expression therapeutischer Gene durch Chemotherapie oder Hyperthermie kontrollierbar ist. Derartige Vektoren, in denen der humane Multidrug Resistenzgen 1- (mdr1) Promotor genutzt wurde, exprimierten vor allem Zytokingene, die die therapeutische Effizienz von Zytostatika oder der Hyperthermie verbessern. Die Zytostatika-und auch Hitze-Induzierbarkeit der mdr1-Promotor gesteuerten Genexpression konnte in verschiedenen Tumormodellen in vitro und in vivo erfolgreich demonstriert werden Diese Untersuchungen zeigten, dass eine Zytostatika-induzierte Gentherapie zu einer besseren Tumortherapie beiträgt. Die Kombinations-Experimente der konditionellen Gentherapie im Kontext einer Hyperthermie geben erste Hinweise, dass auch hier die therapeutische Effektivität in vitro und in vivo gesteigert werden kann. Im Rahmen des Konzepts der kombinierten Gen- und Chemotherapie von Tumoren ist in der Arbeit vor allem auf das chemosensitivierende Potential von Zytokinen gesetzt worden. Besonders für TNF-a, IL-2 sowie IFN-g konnte gezeigt werden, dass diese Zytokine zu einer Modulation der Expression MDR-assoziierter Gene, wie dem mdr1, MVP/LRP und auch MRP1 in der Lage sind und dadurch zur Chemosensitivierung in verschiedenen Tumormodellen führt. Diese Befunde bildeten eine wichtige Rationale für den Einsatz von Zytokingenen im Rahmen der Tumorgentherapie zur Überwindung der MDR. Gentransferexperimente mit TNF-a- und IL-2-exprimierenden Vektoren konnten analog zur Applikation rekombinanter Zytokine die Modulation der Gene mdr1 und MVP/LRP zeigen, die mit der Erhöhung der Sensitivität gegenüber Zytostatika wie Vincristin oder Adriamycin assoziiert ist.Gene therapy has made great achievements in vector design, controlled gene expression and in safety. The fact, that gene therapy as single therapy has only limited potential for the benefit in the therapy for cancer patients, has led to the concept of local gene therapy as part of other, established therapies. In this context, gene therapy serves as a modern option to improve the efficiency of chemotherapy, radiotherapy or hyperthermia. To achieve this goal, the establishment of therapy-regulatable vectors is of particular attractiveness. For the concept of local transfer of therapeutic genes non-viral transfer systems, such as in vivo electrotransfer, gene gun or jet-injection represent clinically applicable transfer technologies. One major issue of this work was the establishment of an efficient, jet-injection based non-viral transfer technology and the analysis of its potential for clinical application in a concept of multimodal therapy. It has been shown in vivo, that efficient transgene expression can be achieved by jet-injection, that penetration and distribution of the transgene are optimal for an efficient gene transfer and that the level of gene expression is comparable to established gene transfer technologies, sch as in vivo lipofection. Based on the strategy of combination of gene therapy with other therapies, another goal of this work aimed at the characterization and utilization of conditional vector systems, by which expression of therapeutic genes is controllable by chemotherapy or hyperthermia. By such vectors, in which the human multidrug resistance gene 1 (mdr1) promoter was employed, cytokine genes were expressed, which are capable to improve the therapeutic efficacy of cytostatic drugs or of hyperthermia. The drug- and heat-inducibility of mdr1 promoter-driven gene expression has successfully been demonstrated in in vitro and n vivo tumor models. The studies have also shown, that drug-induced gene therapy leads to improved tumor treatment. Combination experiments of conditional gene therapy in the context with hyperthermia give first indication of an increased therapeutic efficiency in vitro and in vivo. For the concept of combined gene- and chemotherapy the chemosensitizing potential of cytokines was exploited. It has been shown, particularly for TNF-a, IL-2 and IFN-g, that these cytokines are capable to modulate the expression of MDR-associated genes, such as mdr1, MVP/LRP or MRP1 leading to chemosensitization in different tumor models. These observations represent an important rationale for the use of cytokine genes in gene therapy for MDR-overcoming. Gene transfer experiments with TNF- or IL-2 expressing vectors showed the modulation of mdr1 or MVP/LRP expression, associated with increased sensitivity towards cytostatic drugs, such as vincristine or adriamycin.
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Billaud, Amandine. "Analyse moléculaire, enjeux et limites des thérapies ciblées en oncologie : extension des sensibilités aux anti-PARP dans les cancers ovariens par caractérisation de variants non annotés et nouveaux mécanismes de résistance dans les cancers bronchiques. Caractérisation moléculaire de l’EGFR dans les cancers bronchiques non à petites cellules : étude prospective comparative des technologies NGS et automate Idylla Somatic mRNA analysis of BRCA1 splice variants provides a direct theranostic impact on PARP inhibitors." Thesis, Angers, 2020. http://www.theses.fr/2020ANGE0003.
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Malgré les bénéficies cliniques considérables de la prise en considération du contexte moléculaire tumoral, les thérapies ciblées présentent certaines limitations majeures. La première partie de ces travaux se concentre sur l’étude des inhibiteurs de tyrosine kinase ciblant l’EGFR dans les cancers bronchiques non à petites cellules. L’amélioration des méthodes de détection des biomarqueurs a été complétée par la caractérisation in vitro d’un mécanisme de résistance acquise non précédemment identifié. L’exposition de cellules pulmonaires à un agent mutagène puis une pression de sélection a ainsi mis en évidence la signature TBK1 et l’effet synergique de la co-inhibition s’est révélé intéressant. Le deuxième aspect concernent les inhibiteurs de PARP, incontournables dans la prise en charge des cancers de la sphère gynécologique. Ces thérapies reposant sur le principe de létalité synthétique, la présence de mutations pathogènes de BRCA1/2 est un pré-requis, illustrant la problématique des variants de signification inconnue. Face à la nécessité de leur caractérisation fonctionnelle, une étude transcriptionnelle des variants d’épissage par extraction de l’ARNm depuis les prélèvements FFPE a d’abord été réalisée. Puis, afin d’évaluer tous les variants, l’édition génomique a été développée comparant les efficacités d’édition d’un variant d’intérêt et d’un variant silencieux dans un contexte haploïde où ces gènes sont essentiels. La signification biologique de variants de BRCA1/2, et la généralisation de notre approche à l’ensemble des gènes suppresseurs de tumeur essentiels de nos cellules, peut ainsi être évaluée en 3 semaines, délai compatible avec les impératifs cliniquesDespite significant clinical benefit from the consideration of molecular context, targeted therapies are still challenging. First part of this work focused on tyrosine kinase inhibitors targeting EGFR in non small cell lung cancers. Thus, improvement of biomarkers detection methods was completed by in vitro characterization of an unreported mechanism of acquired resistance. Briefly, pulmonary cells were exposed to a mutagen agent and a selection pressure was applied with EGFR inhibitors allowing the detection of TBK1 signature. Finally, synergic effect of that co-inhibition was highlighted. Now essentials in gynaecological cancers management, PARP inhibitors represent the second part of that work. Those targeted therapies are based on synthetic lethality. Consequently, BRCA1/2 pathogenic mutations are required for their administration, illustrating the issue of variants of uncertain significance. Toward their functional characterization necessity, a transcriptional analysis of splicing variant was first conducted on mRNA extracted from FFPE samples. Then, to evaluate functional signification of all types of variants, genomic edition was developed. Editing efficiencies of the unknown variant and a silent control one were compared in a haploid model where those genes are essentials. Functional signification of BRCA1/2 variants, and thereby mutations from all essential tumor suppressor genes in our model, can be evaluated in three weeks which is compatible with clinical management