Inhibition of Various Receptor Tyrosine Kinase (rtk) Families

Activation of cell surface growth factor receptor tyrosine kinases (RTKs) results in cellular proliferation, differentiation and survival. Humans have 58 RTKs, categorized into twenty subfamilies based on their structural properties and ligand specificity. One of the most well-studied RTKs, the epidermal growth factor receptor (EGFR), is mutated or overexpressed in many human cancers including non-small cell lung cancer and glioblastoma. Several EGFR inhibitors have been approved by the FDA, and numerous additional inhibitors are being pursued as potential cancer therapeutics. Structural studies of the EGFR tyrosine kinase domain suggest that different inhibitors bind selectively to either the active or the inactive conformation of the kinase. Contrary to this supposition, computational studies of inhibitor-stabilized EGFR conformations have suggested that erlotinib can bind equally well to either the active or inactive forms of the EGFR kinase domain. We tested this hypothesis using a mutated EGFR kinase domain that can adopt either the inactive or active kinase conformation in crystals, and determined a structure with erlotinib bound to the inactive conformation – suggesting that this inhibitor can bind either ‘state’. We also determined the crystal structure of this EGFR kinase variant bound to a novel ErbB2 inhibitor (55A) found in screens designed to select inhibitors that bind the active state. When bound to this compound, the EGFR kinase domain adopted only the active conformation, suggesting that erlotinib and 55A select different kinase ‘states’. Collaborative approaches combining biochemical, structural and cell signaling studies suggest new considerations in predicting modes of inhibitor binding in the development of therapeutic agents for cancer. Several activating mutations in the anaplastic lymphoma kinase (ALK) gene have been implicated in neuroblastoma. The small molecule tyrosine kinase inhibitor crizotinib, which inhibits ALK and Met, was recently approved by the FDA for the treatment of non-small cell lung cancer (NSCLC), and is currently in early-phase clinical testing in patients with neuroblastoma (NB). Numerous additional ALK inhibitors are also being pursued as potential therapeutics. However, more and more mutations in the ALK tyrosine kinase domain (TKD) are being found in patients with neuroblastoma. Several of these are associated with primary resistance to currently available ALK inhibitors, and several mutations in oncogenic ALK fusions have been linked to acquired crizotinib resistance. Understanding how different ALK mutations activate its kinase domain, and how they change inhibitor sensitivity, is therefore crucial for further clinical development of ALK-targeted therapeutics. We have studied the biochemical consequences of a wide variety of ALK kinase domain mutations in parallel both with studies of their transforming abilities and computational studies of their structural effects. Our first goal was to use these data to predict the effects of new clinically emerging ALK mutations on ALK’s transforming ability, signaling activity, and drug sensitivity. A second goal was to identify the most potent ALK inhibitor that has inhibitory effects on the widest possible range of ALK mutants using biochemical and cellular assays, with a view to identifying a clinical path forward. Our studies provide valuable mechanistic insight into how ALK is regulated, and also lay important groundwork for guiding more refined targeted therapy in neuroblastoma patients

Belizatinib is a potent inhibitor for non-small cell lung cancers driven by different variants of EML4-ALK fusion proteins carrying L1196M-mutations

Structural fusions of EML4 with ALK kinase might lead to signaling abnormalities that drive NSCLC. Although approved specific ALK inhibitors have led to excellent initial responses in ALK positive NSCLC patients, acquired resistance to these inhibitors due to the occurrence of mutations is a major clinical challenge. Studies have shown that different mutations are related with unique and specific responses to certain inhibitors, and that the sensitivity of EML4-ALK fusion variants to ALK specific inhibitors varies. To investigate the response spectrum of combinations of each resistance mutation and EML4-ALK fusion gene variant (V) to different ALK specific inhibitors, individual cellular Ba/F3 models were constructed and used for ALK-TKIs screening in this thesis. Based on cell viability assays, the results gained here suggested that brigatinib might be priority recommended for G1269A and L1152R mutations, lorlatinib for C1156Y and I1171T mutations and belizatinib for G1269A and L1196M mutation. Moreover, a tendency was observed that V2 was most sensitive to ALK-TKIs, V1 and V3b had intermediate sensitivity and V3a was the least sensitive. But there were situations where drug sensitivity was not in line with the expected additive effects of mutation- and variant- dependent sensitivities, which indicated the necessity of taking both variants and mutations into consideration. Notably, belizatinib, a drug in development, was approximately twenty times as potent as lorlatinib for ALK-L1196M in all types of fusion variants. The promising efficacy of belizatinib against L1196M mutation of EML4-ALK were also proven by molecular dynamics simulations. In conclusion, the results of this study provided sensitivity spectra with clinical confirmed EML4-ALK mutation-fusion combinations to nine ALK-TKIs, offered a possible optimal sequence of ALK-TKIs for treating ALK-positive patients developing resistance mutations during therapy and suggested belizatinib as a promising possible targeted inhibitor for the L1196M mutation of EML4-ALK in NSCLC

Mechanistic Insight into RET Kinase Inhibitors Targeting the DFG-out Conformation in RETrearranged Cancer

RET Fusions-Gene können in 1-2% aller Lungen-Adenokarzinom Patienten nachgewiesen werden. Diese genetischen Veränderungen stellen mittels Tyrosin-Kinase Inhibitoren potentiell therapierbare molekulare Zielstrukturen dar (Pao and Hutchinson 2012), jedoch haben klinische Studien im Jahr 2018 für diese Lungenkarzinome bisher noch keine ausreichend erfolgreichen Therapieansätze zeigen können (Drilon et al. 2018). Um systematisch das therapeutische Profil von AD80 und einer Vielzahl weiterer Tyrosinkinase Inhibitoren gegen RET-Fusion getriebene Zellmodelle auswerten zu können, habe ich zunächst Ba/F3 Zellen viral mit KIF5B-RET sowie CCDC6-RET transduziert. Das Wachstum von Ba/F3 Zellen in vitro ist im Normalzustand abhängig von IL-3. Sobald sie mit einem starken Onkogen jedoch transduziert werden, sind sie unabhängig von IL-3 und proliferieren nur noch abhängig von der Aktivität des entsprechenden Onkogens. Durch dieses Modelsystem konnte ich eine große Anzahl verschiedener Inhibitoren gegen RET testen und ihre Potenz untereinander vergleichen. Die selbst etablierten Ba/F3 Zell-Linien zusammen mit der RET-Fusion getriebenen Lungen-Adenokarzinom Zelllinie LC-2/AD bildeten den Ausgangspunkt für mein Projekts. Zusätzlich habe ich ein endogen RET-mutiertes Zell-Model mittels der Genom-Editierungstechnik CRISPR/Cas9 etabliert. Dafür habe ich einen Vektor mit Cas9 mRNA sowie zwei Promotoren für die Expression von spezifischen „single-guided RNAs“ (sgRNA) kloniert und murine Fibroblast Zellen (NIH-3T3) transfiziert. Mittels sgRNAs gegen die jeweiligen spezifischen Introns von RET und KIF5B konnte ich somit in selektionierten NIH-3T3 Zellen KIF5B-RET Translokationen generieren. Indem ich diese neu etablierten Zelllinien zusammen mit einer größeren Anzahl humaner Lungenkrebs Zell-Linien gegen potentielle RET-Inhibitoren getestet habe, konnte ich die klinische Erfahrung in vitro bestätigen, dass die zurzeit gängigen Therapieansätze mit Tyrosinkinase- Inhibitoren wie z.B. Cabozantinib, Alectinib oder Vandetanib wahrscheinlich eine nicht ausreichend hohe therapeutische Potenz besitzen, um eine effektive Wirkung auf RET getriebene Tumore zu entwickeln. Weiterhin deuteten die Daten darauf hin, dass andere Tyrosin-Kinase Inhibitoren, wie z.B. AD80 und Ponatinib, im Vergleich dazu 100 bis 1000-fach potenter sind und spezifisch die RET-Kinase inhibieren. Als nächstes habe ich die Unterschiede in den Zell-Viabilitäts-Assays mit den Veränderungen auf der Protein-Ebene zu verglichen. Die folgenden Western Blot und Phosphoproteom Analysen haben eine entsprechende Reduktion in phospho-RET und den weiteren nachgeschalteten Signal-Molekülen gezeigt. Zusätzlich haben die in vivo Ergebnisse unserer CCDC6-RET PDX-Mausmodelle unsere in vitro Daten mit gutem Tumoransprechen unter Therapie mit AD80 bestätigt. Parallel zu meiner Arbeit für das Projekt haben wir mit anderen Arbeitsgruppen zusammengearbeitet, um ein tieferes Verständnis über die funktionellen Mechanismen hinter der hohen Aktivität von AD80 gegen RET zu erhalten. Mittels computerbasierter Modelle haben wir ableiten können, dass AD80 mit hoher Wahrscheinlichkeit als Typ II Inhibitor die RET-Kinase in der inaktiven „DFG-out“ Konformation bindet, was mit einer erhöhten Kinase- Thermostabilität im Vergleich zu Typ I Inhibitoren als Bindungspartner einhergeht. Dies wiederum ist ein Surrogat-Parameter für eine engere Kinase-Bindung durch die Inhibitoren und könnte eine mögliche Erklärung für die hohe Cytotoxitität in unseren Experimenten sein. Im Folgenden hat sich das Projekt mehr auf die Rolle von Resistenz-Mechanismen in RET mutierten Zelllinien konzentriert. Mittels zielgerichteter Mutagenese (site-directed mutagenesis) habe ich an der sogenannten „Gatekeeper Position“ mutierte Ba/F3 KIFRETV804M und CCDC6-RETV804M etablieren können, um die Wirksamkeit der Inhibitoren dagegen zu testen. Wieder zeigten AD80 und Ponatinib den stärksten inhibitorischen Effekt gegen RET mit nur einer geringen Reduktion der Cytotoxitität in den Zell-Viabilitäts-Assays und RET-Dephosphorylierung im Vergleich zu RETwt. Mittels Sättigungsmutagenese (saturated mutagenesis screening) habe ich versucht, Resistenzmutationen zu finden, die neben der bekannten „Gatekeeper Position“ pV804M zur Resistenz gegen AD80 führen könnten. Es zeigte sich in der Sequenzierung von resistenten Ba/F3CCDC6-RET Zellen die missense Mutation pI788N (c.2363T>A) im Bereich der RET-Kinase Domäne als potentielle sekundäre Resistenzmutation unter Therapie mit AD80. Diesen Resistenz-Effekt durch die neue Mutation konnte ich dann in den folgenden zellulären Modellen mittels Zell-Viabilitäts-Assays und Western Blots bestätigen. Zusätzlich haben Ergebnisse einer sekundär gegen AD80 resistent gewordenen TPC-1 Schilddrüsenkarzinom-Zelllinie sowie RNASequenzierungen von LC-2/AD Zelllinien unter Therapie gegen AD80 ergeben, dass MAPKReaktivierung potentiell eine Rolle als Resistenz-Mechanismus in RET-getriebenen Tumoren besitzen könnte. Um formell die Rolle von MAPK-Reaktivierung in Bezug auf Resistenz-Effekte zu testen, wurden LC-2/AD Zellen von mir lentiviral mit KRASG12V transduziert, was zu einer Überexprimierung von KRAS und zu einer folgenden Resistenz gegen die Behandlung mit AD80 geführt hat. Für Ponatinib gibt es bereits klinische Phase 3 Studien an Patienten mit Chronisch Myeloischer Leukämie, die jedoch trotz gutem initialen Tumor Ansprechen aufgrund von erhöhten Therapie assoziierten Komplikationen beendet werden musste (Lipton et al. 2016). Der RET-Kinase Inhibitor AD80 wäre daher ein geeigneter Kandidat für zukünftige klinische Studien. Bedenkt man den hohen Bedarf an neuen, wirksamen und verträglichen Therapieansätzen im Rahmen der individualisierten Lungenkarzinom-Therapie, bietet unsere Studie eine Vielzahl neuer mechanistischer Einsichten in die aktuelle anti-RET Therapie und trägt zur deren zukünftigen Entwicklung bei

Simulation & Experiment Learning From Kinases In Cancer

The decreasing cost of genome sequencing technology has lead to an explosion of informa- tion about which mutations are frequently observed in cancer, demonstrating an important role in cancer progression for kinase domain mutations. Many therapies have been devel- oped that target mutations in kinase proteins that lead to constitutive activation. However, a growing body of evidence points to the serious dangers of many kinase ATP competitive inhibitors leading to paradoxical activation in non-constitutively active proteins. The large number of observed mutations and the critical need to only treat patients harboring activat- ing mutations with targeted therapies raises the question of how to classify the thousands of mutations that have been observed. We start with an in depth look at the state of knowl- edge of the distribution and effects of kinase mutations. We then report on computational methods to understand and predict the effects of kinase domain mutations. Using molecular dynamics simulations of mutant kinases, we show that there is a switch-like network of la- bile hydrogen bonds that are often perturbed in activating mutations. This is paired with a description of a software platform that has been developed to streamline the execution and analysis of molecular dynamics simulations. We conclude by examining a machine learning method to demonstrate what kinds information derived from protein sequence alone have the most value in distinguishing activating and non-activating mutations

Molecular mechanisms of vascular remodeling in pulmonary arterial hypertension : the implication of tyrosine kinase inhibitors and epigenetic events in the disease

L'hypertension artérielle pulmonaire (HTAP) est une maladie rare caractérisée par une obstruction progressive et un remodelage vasculaire des artères pulmonaires distales, conduisant à une pression artérielle pulmonaire moyenne supérieure à 20mmHg. L'augmentation de la pression aboutit à une dysfonction du ventricule droit et la mort. À l'heure actuelle, il n'existe pas de traitement curatif pour l'HTAP. Il est donc primordial d'identifier de nouvelles cibles thérapeutiques. Comme dans le cancer, les cellules musculaires lisses de l'artère pulmonaire des patients atteints d'HTAP présentent un phénotype hyper-prolifératif et résistant à l'apoptose, entraînant un remodelage vasculaire pulmonaire. Ainsi, plusieurs stratégies thérapeutiques anti cancéreuses pourraient être utiles pour le traitement de l'HTAP. Compte tenu de l'expression altérée des récepteurs tyrosines kinases dans l'HTAP ainsi que dans le cancer, les inhibiteurs de tyrosine-kinases (ITK) ont été mise sur le marché pour le traitement de différents types de cancers et ont été envisagées dans le cadre de l'HTAP. Ainsi, l'ITK, Imatinib, a été capable de régresser l'HTAP induite dans des modèles expérimentaux, tandis que l'administration d'un autre inhibiteur, le Dasatinib, était associée au développement de l'HTAP. Récemment, plusieurs études observationnelles ont démontré le développement de l'HTAP chez des patients atteints d'un cancer du poumon non à petites cellules (CPNPC) présentant des réarrangements de la kinase lymphocytaire anaplasique (ALK) et qui ont reçu des ITK ALK/cMET, notamment Xalkori (R-Crizotinib), Ceritinib, Brigatinib et Lorlatinib. Étant donné que l'hypertension pulmonaire peut être associée à plusieurs maladies, y compris le cancer du poumon, la question demeure de savoir si le développement de l'HTAP chez les patients atteints d'un cancer du poumon recevant l'ITK ALK/c-MET représente un événement indésirable du médicament ou un résultat de la propagation de la maladie. Ainsi, l'objectif principal de chapitre 1 est de déterminer si le R-Crizotinib (connu sous le nom de Xalkori et qui est une première ligne de traitement des patients ayant un CPNPC-ALK positif) exacerbe l'HTAP et/ou prédispose à l'HTAP dans des modèles animaux. In vivo, le traitement par R-Crizotinib a entraîné une élévation marquée de la pression systolique du ventricule droit et de la pression artérielle pulmonaire moyenne associée à une augmentation de l'épaisseur de la paroi médiale des artères pulmonaires distales. De plus, R-Crizotinib, administré avant l'exposition à une faible dose de monocrotaline (MCT), induit une réponse hypertensive pulmonaire exagérée, comme en témoigne une augmentation de la pression systolique du ventricule droit et de la pression artérielle pulmonaire moyenne, de l'épaisseur de la paroi médiale et une diminution du débit cardiaque. In vitro, nous avons démontré que le traitement avec R-Crizotinib réduit la prolifération des cellules endothéliales contrôles de l'artère pulmonaire, effet associé à la formation de cellules multinucléées, ce qui est généralement observée dans les cellules qui meurent d'une une catastrophe mitotique. En conclusion, nous avons démontré que l'agent anticancéreux R-Crizotinib favorise le dysfonctionnement des cellules endothéliales, conduisant à la prédisposition et à l'exacerbation de l'HTAP dans des modèles animaux. Les dernières années de recherche ont approuvé l'importance des marques épigénétiques dans le développement de l'HTAP. En effet, nous nous sommes intéressés, dans le deuxième volet de la thèse, au facteur épigénétique « G9a », qui s'est révélé surexprimé dans différents types de cancers, favorisant la survie et la prolifération des cellules. Compte tenu de l'analogie cancer/HTAP, G9a fut un candidat idéal pour l'étude de son potentiel rôle dans le développement de l'HTAP. Ainsi, l'objectif principal du chapitre 2 est de déterminer si G9a est impliqué dans la progression et la pathogenèse de l'HTAP et de déterminer si son inhibition est bénéfique dans les modèles animaux. Nous avons démontré que G9a est surexprimé dans les artères pulmonaires de patients HTAP et dans les modèles expérimentaux. In vitro, l'inhibition pharmacologique de G9a à l'aide de BIX01294 diminue drastiquement la capacité d'hyper prolifération et la résistance à l'apoptose des cellules musculaires lisses HTAP. Grâce au séquençage d'ARN, nous avons démontré que l'inhibition de G9a s'accompagnait d'une altération du flux d'autophagie et d'une accumulation de lipides. Enfin, le traitement thérapeutique avec BIX01294 a réduit le remodelage vasculaire pulmonaire et la pression artérielle pulmonaire moyenne dans un modèle expérimentale de rat et a également amélioré l'hémodynamique pulmonaire et la fonction ventriculaire droite dans un autre modèle de souris. Ces résultats suggèrent que l'inhibition de G9a pourrait représenter une nouvelle approche thérapeutique dans l'HTAP.Pulmonary arterial hypertension (PAH) is a rare and fatal disease characterized by "a progressive loss and obstructive remodeling of pulmonary arteries (PAs) leading to a mean pulmonary arterial pressure (mPAP) greater than 20mmHg. The persistent elevation of pulmonary pressures leads to right ventricular dysfunction and death. Currently, there is no cure for patients with PAH, which increases the need to develop new and effective therapeutic strategies. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients exhibit a "cancer-like" hyperproliferative and apoptosis-resistant phenotype leading to pulmonary vascular remodeling. Therefore, several anti-cancer therapies could be useful for the treatment of PAH. Given the altered expression of receptor tyrosine kinases and their ligands in PAH as well as in cancer, tyrosine kinase inhibitors (TKIs) have been marketed for the treatment of different types of cancers and have been in the spotlight for anti-PAH drug research. Indeed, the tyrosine kinase inhibitor, Imatinib, was able to regress established PAH in experimental models, while the administration of another inhibitor, Dasatinib, was associated with the development of PAH. Recently, several observational studies have highlighted the development of PAH in patients with non-small cell lung cancer (NSCLC) with anaplastic lymphocyte kinase (ALK) rearrangements who received ALK/cMET TKIs, including Xalkori (R-crizotinib), Ceritinib, Brigatinib, and Lorlatinib. Since pulmonary hypertension can be associated with several diseases, including lung cancer, the question remains whether the development of PAH in lung cancer patients receiving cMET/ALK TKIs represents an adverse drug event or a result of disease spread. Thus, the main objective of Chapter 1 is to determine whether R-Crizotinib (known as Xalkori and which is the first-line treatment for patients with advanced ALK-positive NSCLC) exacerbates PAH and/or predisposes to PAH in experimental animal models. In vivo, R-Crizotinib treatment resulted in a marked elevation of the right ventricular systolic pressure (RVSP) and mPAP which was associated with an increased medial wall thickness of the distal PAs. Additionally, we found that pretreatment of rats with R-Crizotinib, induced an exaggerated pulmonary hypertensive response, as evidenced by the increased RVSP, mPAP, medial wall thickness, and decreased cardiac output. In vitro, we have demonstrated that treatment with R-Crizotinib reduces the proliferation of control pulmonary artery endothelial cells, which was accompanied by the appearance of multinucleated cells, a feature commonly seen in cells dying from mitotic catastrophe. In conclusion, we have demonstrated for the first time that the anticancer agent R-Crizotinib promotes endothelial cell dysfunction, leading to susceptibility and exacerbation of PAH in animal models. Previous studies have demonstrated the importance of epigenetic marks in the development and progression of PAH. Indeed, we were interested in the second part of the thesis, in the epigenetic factor "G9a", which was found to be overexpressed in different types of cancers, promoting cell survival and proliferation. Given the similarities between PAH and cancer, G9a was the ideal candidate to study in PAH. Thus, the main objective of Chapter 2 is to determine if G9a is involved in the progression and pathogenesis of PAH and to determine if its inhibition is beneficial in PAH animal models. We demonstrated that G9a is overexpressed in PAs of PAH patients and in experimental models. In vitro, we found that pharmacological inhibition of G9a using BIX01294 drastically reduces the PAH-PASMC proliferation and survival. Through RNA sequencing analysis, we demonstrated that G9a inhibition is accompanied by an impaired autophagy flux and lipid accumulation. Finally, therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling as well as mPAP in an experimental rat model and also improved pulmonary hemodynamics and right ventricular function in another PAH mouse model. These results suggest that G9a inhibition could represent a new therapeutic approach in PAH

Novel gene fusions identified as new drug targets in paediatric glioma and their pre-clinical characterisation

Gliomas are the most common paediatric brain tumours, accounting for about 50% of all brain tumours in children. They are typically classified by the putative cell type they arise from based on morphology, the location they are found and malignancy grade. The most common glioma found in adults is glioblastoma, which is a WHO grade IV tumour with an average survival of less than 15 months. Extensive work has gone into characterising, modelling and treating this tumour in vivo and in vitro, but it is now clear that glioblastoma in children is biologically very different from that in adults. Paediatric high-grade gliomas do, however, share their aggressiveness with their adult counterparts, with few patients achieving long-term survival - dictating an urgency to find more precise therapies for these patients. Additionally, relatively little work has focused on low-grade gliomas to date. Nevertheless, these tumours also deserve a lot of attention, because low-grade gliomas make up 30-50% of all paediatric brain tumours and more than half of all paediatric gliomas. Although the tumour itself does not necessarily lead to a massively reduced life span and should rather be considered a chronic disease, the impact on the patients’ and families’ lives caused by the therapy load and possible recurrences remains a major clinical burden. Unlike high-grade gliomas, which are very heterogeneous with multiple oncogenic drivers, most low-grade gliomas are driven by alterations in the mitogen-activated protein kinase (MAPK) pathway through different mechanisms. Examples are BRAF alterations, FGFR1 mutations, and NTRK or MYB fusions. After analysing around 200 paediatric glioma samples by whole-genome/whole-exome and RNA sequencing within the ICGC PedBrain Tumour Project and the INFORM personalised medicine study, fusions involving an additional candidate gene (ALK) were found to be of interest in terms of their pattern of occurrence and availability of targeted inhibitors. Clinically these fusions arose in an interesting patient population, affecting infants (<2 years old) with histologically malignant tumours that however showed outcomes more similar to low-grade glioma than glioblastoma. PPP1CB:ALK was the most common gene fusion found in this context, which was then modelled in vivo using two different state-of-the-art methodologies: in utero electroporation and p0 injection using the RCAS-tva system. Through in utero electroporation, a novel mouse model was generated that nicely recapitulates the human tumours. Different ALK-specific inhibitors already used in clinical trials for other tumours, like non-small cell lung cancer or neuroblastoma, were tested on the tumour cells in an in vitro sphere culture setting and in addition also in vivo on allografted tumour cells. The results show a promising effect of the third-line, blood-brain-barrier penetrant ALK inhibitor lorlatinib, with IC50 values below 1.3nM and a significant increase in lifespan with a decrease in tumour signal, respectively. Thus, the project led from the genomic discovery of a novel driving event in paediatric glioma through to its modelling and identification of a promising new option for therapy. Further proof-of-concept application with other oncogenic combinations leads to the conclusion that the mouse model strategy and the methodology behind this can be further applied to test other candidate genes and specific inhibitor therapies. The overall aim is thus to accelerate the approval of targeted drugs by authorities after running a stratified clinical trial on a small infant patient population carrying the gene of interest, to enable patients to get the most potent therapy with the fewest side effects

The Role of NPM-ALK Signaling in Tumor Cell Metabolism.

NPM-ALK is a fusion tyrosine kinase that drives oncogenesis in a subset of anaplastic large cell lymphoma. Based on the role of NPM-ALK in cancer initiation and progression, we pursued a mass spectrometry-based phosphoproteomic approach aimed at the unbiased identification of novel mediators of NPM-ALK signaling. The analysis of cell lines under differing NPM-ALK activation conditions revealed a significant number of proteins that regulate cellular metabolism to be affected by NPM-ALK signaling. We therefore, pursued an untargeted metabolomic screen aimed at the identification of specific metabolites and metabolic pathways that are regulated by NPM-ALK. This analysis revealed significant alterations in pathways implicated in the Warburg effect and biomass production, including glycolysis, the pentose phosphate pathway, pyrimidine metabolism, etc. Metabolic flux analysis revealed an NPM-ALK-driven up-regulation of these pathways and down-regulation of energy production. We hypothesized that PKM2 and GSK3β, both identified in the phosphoproteomic study, mediate the metabolic changes. Biochemical studies revealed that NPM-ALK directly phosphorylated PKM2, decreasing its enzymatic activity and driving a metabolic shift away from energy production and toward biomass production. Chemical activation of PKM2 or expression of a mutant PKM2 resulted in a reversal of this metabolic shift and decreased tumorigenesis. The phosphoproteomic analysis identified another candidate mediator of NPM-ALK signaling, GSK3beta. Through the PI3K/AKT pathway, NPM-ALK regulated pS9-GSK3beta, inhibited its activity and provided proteasomal protection for Mcl-1, CDC25A and GYS. This pathway increased proliferation and survival, while increasing glycogen production as a form of metabolic regulation. Studies described in this dissertation globally characterize the ALK driven phosphoproteome and metabolome while providing mechanistic discoveries of two novel NPM-ALK driven pathways. This work provides significant advances to our understanding of oncogenesis and will lead to advances in targeted therapies for ALK driven neoplasms.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97975/1/scottmcd_1.pd