Brain metastases (BMs) present a formidable obstacle across various primary cancer types, notably small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), melanomas, and breast cancers. In this investigation, we aim to evaluate the potential of genotype-guided targeted therapy while addressing the challenges of co-existing genomic alterations frequently encountered in BMs. This research explores the efficacy of adagrasib (MRTX849), a KRAS G12C inhibitor, and abemaciclib, a CDK 4/6 inhibitor, both individually and in combination against BMs originating from NSCLC cell lines harboring KRAS G12C and CDKN2A mutations. Utilizing a diverse array of methodologies encompassing cell viability assays, cell death assays, western blot analyses, and in vivo xenograft models, we elucidate both the therapeutic potential and underlying mechanisms. Distinct responses to adagrasib and abemaciclib monotherapies were observed across two different cell lines, underscoring the necessity for tailored treatment strategies. While adagrasib exhibited variable efficacy, abemaciclib consistently inhibited CDK 4/6 activity. Notably, the combination therapy demonstrated synergistic effects, suggesting a promising approach for enhanced therapeutic outcomes. Our findings from both in vitro assays and western blot analyses corroborate targeted pathway inhibition, although the observed pathway reactivation underscores the importance of optimizing dosing strategies. In vivo studies further support our in vitro findings, demonstrating efficacy but also raising concerns regarding toxicity with combination therapy. Pharmacokinetic / pharmacodynamic (PK/PD) analyses underscore potential advantages of combination therapy in terms of systemic exposure and brain penetration. Despite histological evidence of therapeutic effects, discrepancies between in vivo and in vitro caspase-dependent apoptosis results highlight the complexity of tumor biology and the challenges of translation. By Focusing on personalized treatment approaches and addressing therapeutic hurdles, this work establishes the foundation for clinical investigation in advancing the management of BMs and improving treatment outcomes in NSCLC patients.

Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l’espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l’oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l’ajout d’un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l’inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l’association de l’inhibiteur de mTOR everolimus et de l’inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l’injection orthotopique d’une lignée bioluminescente de CBP-NPC chez des souris nude a permis d’établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L’association d’un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d’envisager un transfert en clinique. / Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

Tumormodelle, in denen die maligne Transformation durch definierte Onkogene experimentell ausgelöst und unterhalten wird, bieten vielfältige Möglichkeiten, die komplexen Mechanismen der Tumorentstehung und Therapieresistenz zu untersuchen und neue Ansätze für Diagnostik und Therapie auszuarbeiten. KRAS-Onkogen-„getriebene“ Transformationsmodelle spiegeln neben anderen tumorspezifischen Veränderungen insbesondere die charakteristischen Änderungen des Transkriptoms wider. In der vorliegenden Arbeit wird ein Modell für Ovarialtumore auf Grundlage von Rose Zellen („Rat ovarian surface epithelium“) verwendet, um die Rolle von Transkriptionsfaktoren, welche durch die KRAS-vermittelte Signaltransduktion hoch reguliert werden, zu untersuchen. Die KRAS-transfomierten Derivate der normalen Rose Zellen zeigen die typischen Merkmale von ankerunabhängigen und invasiven Tumorzellen. Aufgrund der hohen Komplexität sind die Interaktionen zwischen der zytoplasmatischen Signaltransduktion und dem durch sie regulierten Transkriptionsfaktornetzwerk noch weitgehend unverstanden. Die Transkriptionsfaktoren Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 und RelA wurden systematisch mittels RNA-Interferenz in KRAS-transformierten Rose Zellen transient ausgeschaltet. Danach wurden Proliferation, Morphologie (epithelial-mesenchymale Transition, EMT) und Ankerunabhängigkeit der Zellen bestimmt. Alle untersuchten Transkriptionsfaktoren beeinflussten die KRAS-induzierten morphologischen Veränderungen teilweise, belegt durch die Abnahme der EMT-Merkmale nach siRNA-vermittelter Ausschaltung. Der Knock-down der Transkriptionsfaktoren Otx1, Gfi1 und RelA hemmte die Proliferation, während Fosl1, Hmga2, Klf6 und JunB die generelle Proliferationsfähigkeit nicht beeinflussten, jedoch spezifisch die ankerunabhängige Proliferation blockierten. Diesen Faktoren kommt daher eine spezifische Funktion in der neoplastischen Transformation zu, da die Ankerunabhängigkeit sehr gut mit der Tumorigenität korreliert ist. Um die Beteiligung der Transkriptionsfaktoren an der Deregulation von Zielgenen zu erfassen, wurden Genexpressionsmuster aller Zellen, in denen jeweils ein Faktor durch siRNA ausgeschaltet war, mittels Microarray-Analyse identifiziert. Auf dieser Grundlage wurde ein Netzwerk-Modell der regulatorischen Interaktionen zwischen den Transkriptionsfaktoren berechnet. Die Existenz der beiden funktionellen Gruppen wurde im Modell bestätigt. Darüber hinaus zeigte sich eine gegenseitige Abhängigkeit des transkriptionellen Netzwerks und der zytoplasmatischen Signaltransduktion, gemessen mittels Proteinanalyse der mitogenabhängigen Signalkinasen (MAPK). Diese wird als kompensatorische Regulation interpretiert, welche trotz Pertubation, experimentell durch siRNA, das effiziente Überleben der transformierten Zellen sicherstellt. Die vorliegende Studie schafft somit die Voraussetzung und Motivation, das reduzierte Netzwerk aus sieben Komponenten auf alle differentiell exprimierten Transkriptionsfaktoren zu erweitern. Möglicherweise behindern solche Regulationskreise in der klinischen Situation die effektive Wirkung zielgerichteter Therapien. / Tumor models, in which malignant transformation was experimentally triggered and maintained through defined oncogenes, offer manifold opportunities to determine the complex mechanisms of tumor progression and resistance to therapies, and to develop new strategies for diagnosis and therapy. Particularly, KRAS oncogene driven models of transformation reflect the characteristic alterations of the transcriptome, among other tumor specific changes. In the present work a model for ovarian cancer based on Rose („Rat ovarian surface epithelium“) cells has been used to evaluate the role of transcription factors, which are up-regulated through KRAS dependent signaling. The KRAS transformed derivates of normal ROSE cells exhibit typical characteristics of anchorage-independent and invasive tumor cells. Due to the high complexity of cellular networks, the interactions between cytoplasmic signalling and their regulated transcription factors are not well understood. The transcription factors Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 and RelA were systematically eliminated by transient RNA interference in KRAS transformed ROSE cells. The proliferation, morphology (epithelial-mesenchymal transition, EMT) and anchorage-independence of the cells were determined. All of the selected transcription factors had partial effect on the KRAS induced morphologic changes, documented by reduction of EMT-properties after siRNA treatment. The knock-down of the transcription factors Otx1, Gfi1 and RelA blocked proliferation in general, whereas Fosl1, Hmga2, Klf6 and JunB had no influence on proliferation but specifically blocked the anchorage-independence. Thus, these factors exhibited essential functions in the process of neoplastic transformation, because the anchorage-independence correlates very well with tumorigenicity. In order to elucidate the involvement of the transcription factors in the genetic deregulation of their target genes, microarray based gene expression profiles were determined from all cells in which one factor was eliminated by siRNA. Based on these data, a network model of regulatory interactions among these transcription factors was calculated. The existence of both functional groups was confirmed by the model. Furthermore, an interdependence of the transcriptional networks and cytoplasmatic signaling was observed by protein analysis of the mitogen dependent signal kinases (MAPK). This was interpreted as compensatory regulation, which in spite of experimental perturbation by siRNA, permitted efficient survival of the transformed cells. Thus, the present work provides the basis and motivation to extend the reduced network composed of seven components to all regulated transcription factors. Potentially, such regulatory networks diminish the efficacy of targeted therapies in clinical situations.

Water flow and geochemical processes within the unsaturated zone (UZ) in two distinct types of karst environment were investigated using natural tracers (chemistry, stable isotopes 13 C, 18 O, 2 H, and 3 H, 14 C, 87 Sr/86 Sr). The extent of horizontal flow component and the response of drip water chemistry to recharge events were examined in the Moravian Karst (Czech Republic), while the character of water flow and its chemistry were studied in salt diapirs in southeastern part of the Zagros mountains (Iran). Under the conditions of well-developed epikarst, the horizontal flow component, defined as Hmax/T (Hmax = horizontal migration component, T - thickness of VZ) typically reaches values of 0.1 - 0.6 (Moravian and Slovenian Karst). However, in areas where epikarst was stripped off by glacial or human activity, the proportion of horizontal flow component is far greater (Hmax/T 1.6 - 24). This parameter is vital for the design of water source protection zones above caves. Nitrate mean residence time in 120 m thick VZ of the Moravian karst exceeded 16 years. The VZ above the Ochoz Cave (Moravian Karst) represents a semi-open to open system with respect to soil CO2. Under a high drip rate (high flow), the event water only made 5% of the total. During the year, water degassing and so called prior...

No description available.

Les cancers représentent un problème majeur de santé public d'où la nécessité de rechercher de nouvelles classes de médicaments. Parmi les pistes pour développer de nouveaux traitements, deux ont retenu notre attention et celle de nos collaborateurs : la modulation de l’épissage par des composés comme le NVS-SM2, et l’inhibition de l’oncogène KRAS par des dérivés de produits naturels, les flavaglines. Dans ce contexte, nous avons développé la première synthèse robuste du NVS-SM2, qui peut satisfaire la demande globale de cet agent pour examiner en détail son potentiel thérapeutique dans différents types d’affection. En outre, la stratégie de synthèse rapportée ici pourrait être étendue à de nouveaux analogues de ce composé. D’autre part, nous avons synthétisé de nouvelles flavaglines qui sont en cours d'étude pour leurs effets sur l’inhibition de KRAS. Au cours de cette étude, nous avons découvert de nouvelles réactions, notamment une inversion de configuration d’amines induite par du chlorure de diméthylcarbamoyle. / Cancers represent a major public health problem hence the need to use new classes of medicines. Among the opportunities for developing new treatments, two have caught our attention and that of our collaborators: the modulation of splicing by compounds such as NVS-SM2, and the inhibition of the oncogene KRAS by derivatives of natural products, the flavaglines.In this context, we have developed the first robust synthesis of NVS-SM2, which can satisfy the global demand of this agent to examine in detail its therapeutic potential in different types of disorders. In addition, the synthetic strategy reported here could be extended to new analogues of this compound. Furthermore, we have synthesized new flavaglines that have been examined for their effects on KRAS inhibition. During this study, we discovered new reactions, including a dimethylcarbamoyl chloride-induced amine inversion of configuration.

Lung cancer is the number one cause of cancer related death worldwide, with more than 1.6 million fatalities each year. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers, with KRAS being one of the most prevalent oncogenic driver mutations. Therapeutic approaches for KRAS-mutated NSCLC have been extensively explored due to the US National Cancer Institute RAS Initiative, but methods of directly targeting KRAS or downstream effectors, such as MEK, still have poor results. Previous reports have shown that KRAS-mutated NSCLC activate distinct receptor tyrosine kinases (RTKs) depending on the epithelial or mesenchymal state. Epithelial-to-mesenchymal transition (EMT) is known to play a role in the metastasis and poor prognosis of cancer, and is induced by extracellular matrix (ECM) stiffness. Hallmarks of EMT include loss of E-Cadherin and increase in Vimentin. This research investigates the role of KRAS in EMT transition due to increased ECM stiffness in KRAS mutant NSCLC, and how this affects the efficacy of KRAS and MEK inhibition. To understand how KRAS mutations in NSCLC play a role in this stiffness induced EMT, experiments were performed to detect the gene and protein expression of EMT markers, as well as possible sources of mechanosensing, including primary cilia and receptor tyrosine kinases. We hypothesized that KRAS plays a role in activation of mechanosensors and directly correlates to EMT induced by increased mechanical forces. Results show when KRAS was inhibited and there was increased mechanical forces, either from stretch or substrate stiffness, there was a decreased activation of mechanosensors. KRAS inhibition also prevented the cells from undergoing stiffness-induced EMT. This supports our hypothesis that KRAS plays a key role in ECM stiffness induced EMT. Future studies include examining the mechanism behind this phenomenon and in vivo studies.

No description available.

INTRODUCTION The intention of this study was to formulate guidelines for the implementation of Performance Management System (PMS) regarding line managers (LM) and employees (E) in a level 2 public hospital in the North–West Province. From a preliminary evaluation it was evident that the implementation of performance management as outlined in the NWPG policy No. 13, was not effective. The results of the research was to provide baseline data of the current policy implementation by the line managers and employees; an indication of the ability of the PMS to reach its goal of improved performance between line managers and employees in the long term; and the barriers to the policy implementation discovered over the course of the study. RESEARCH QUESTIONS Based on the statement of the problem, the following research questions were asked: * How is PMS implemented from the perspective of line managers in a level 2 public hospital? * How is PMS implemented from the perspective of employees in a level 2 public hospital? * What guidelines can be formulated for line managers and employees regarding PMS? AIM AND OBJECTIVES OF THE STUDY The principal aim of this study was to formulate guidelines for implementation of PMS by line managers and employees. The objectives below of the research which are derived from the principal aim were: * To describe the implementation of the PMS from the perspective of line managers in a level 2 public hospital. * To describe the implementation of the PMS from the perspective of employees in a level 2 public hospital. * To formulate guidelines for line managers and employees regarding PMS? RESEARCH DESIGN A quantitative, explorative, descriptive, and contextual design was used in this study to reach the overarching aim and respective objectives. RESEARCH METHOD The researcher firstly conducted a literature review to understand implementation of PMS and related constructs. Thereafter the researcher used two similar structured questionnaires for both LM and E to collect data. The questionnaires were developed to measure the perceptions of both LM and E in the implementation of PMS in a level 2 public hospital. The questionnaires were based on the six steps (performance planning, developing performance criteria, performance monitoring, performance review and assessment, annual performance assessment and performance assessment outcomes) of the current PMS policy used in a level 2 public hospital. The study is based on transformational leadership whereby the line managers (LM) as nurse leaders with transformational characteristics are assumed to be empowering the employees (E) and creating enthusiasm for nursing practice. Minor adaptations were made to the questionnaires prior to administration to the nursing personnel in a level 2 public hospital in North West Province. An all inclusive sample was taken, representative of a larger population and this amounted to twenty four line managers (n=24) and fifty five employees (n=55) that participated in the study. RESULTS There were problems identified from both the line managers (LM) and employees (E) in performance planning, developing performance criteria, and monitoring performance, organizing and the process of performance review and assessment and lastly the annual performance assessment. The main two main findings are: * The effect size of all the questions indicates a practically visible and thus significant difference with regard to LM and E perceptions of the implementation of PMS. * There is statistical evidence of improper implementation of PMS from both groups regarding some of the performance implementation items. Based on the empirical evidence and the problems identified from the results, guidelines for the implementation of PMS were formulated. It is suggested that these be used and implemented to streamline the PMS in level 2 public hospitals. Keywords: Key result areas (KRAs), Generic assessment factors (GAFs), reward, work plan, Performance Agreement (PA), Performance Management System (PMS). / Thesis (M.Cur.)--North-West University, Potchefstroom Campus, 2012.

INTRODUCTION The intention of this study was to formulate guidelines for the implementation of Performance Management System (PMS) regarding line managers (LM) and employees (E) in a level 2 public hospital in the North–West Province. From a preliminary evaluation it was evident that the implementation of performance management as outlined in the NWPG policy No. 13, was not effective. The results of the research was to provide baseline data of the current policy implementation by the line managers and employees; an indication of the ability of the PMS to reach its goal of improved performance between line managers and employees in the long term; and the barriers to the policy implementation discovered over the course of the study. RESEARCH QUESTIONS Based on the statement of the problem, the following research questions were asked: * How is PMS implemented from the perspective of line managers in a level 2 public hospital? * How is PMS implemented from the perspective of employees in a level 2 public hospital? * What guidelines can be formulated for line managers and employees regarding PMS? AIM AND OBJECTIVES OF THE STUDY The principal aim of this study was to formulate guidelines for implementation of PMS by line managers and employees. The objectives below of the research which are derived from the principal aim were: * To describe the implementation of the PMS from the perspective of line managers in a level 2 public hospital. * To describe the implementation of the PMS from the perspective of employees in a level 2 public hospital. * To formulate guidelines for line managers and employees regarding PMS? RESEARCH DESIGN A quantitative, explorative, descriptive, and contextual design was used in this study to reach the overarching aim and respective objectives. RESEARCH METHOD The researcher firstly conducted a literature review to understand implementation of PMS and related constructs. Thereafter the researcher used two similar structured questionnaires for both LM and E to collect data. The questionnaires were developed to measure the perceptions of both LM and E in the implementation of PMS in a level 2 public hospital. The questionnaires were based on the six steps (performance planning, developing performance criteria, performance monitoring, performance review and assessment, annual performance assessment and performance assessment outcomes) of the current PMS policy used in a level 2 public hospital. The study is based on transformational leadership whereby the line managers (LM) as nurse leaders with transformational characteristics are assumed to be empowering the employees (E) and creating enthusiasm for nursing practice. Minor adaptations were made to the questionnaires prior to administration to the nursing personnel in a level 2 public hospital in North West Province. An all inclusive sample was taken, representative of a larger population and this amounted to twenty four line managers (n=24) and fifty five employees (n=55) that participated in the study. RESULTS There were problems identified from both the line managers (LM) and employees (E) in performance planning, developing performance criteria, and monitoring performance, organizing and the process of performance review and assessment and lastly the annual performance assessment. The main two main findings are: * The effect size of all the questions indicates a practically visible and thus significant difference with regard to LM and E perceptions of the implementation of PMS. * There is statistical evidence of improper implementation of PMS from both groups regarding some of the performance implementation items. Based on the empirical evidence and the problems identified from the results, guidelines for the implementation of PMS were formulated. It is suggested that these be used and implemented to streamline the PMS in level 2 public hospitals. Keywords: Key result areas (KRAs), Generic assessment factors (GAFs), reward, work plan, Performance Agreement (PA), Performance Management System (PMS). / Thesis (M.Cur.)--North-West University, Potchefstroom Campus, 2012.