AKT regulates NPM dependent ARF localization and p53mut stability in tumors

Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status

Doctor of Philosophy

dissertationCancer is extremely challenging to treat as every patient responds differently to treatments, depending on the specific molecular aberrations and deregulated signaling pathways driving their tumors. To address this heterogeneity and improve patient outcomes, therapies targeting specific pathways have been developed. The use of computational pathway analysis tools and genomic data can help guide the use of targeted therapies by assessing which pathways are deregulated in patient subpopulations and individual tumors. However, most pathway analysis tools do not account for complex interactions inherent to signaling pathways, and are not capable of integrating different types of genomic data (multiomic data). To address these limitations, this dissertation focuses on developing user-friendly multiomic gene set analysis tools, and utilizing bioinformatics tools to measure pathway activation for multiple pathways simultaneously in cancer. Chapter 2 first describes the need for genomics and pathway-based analyses in cancer using the commonly aberrant RAS pathway as an example. Chapter 3 utilizes pathway-based gene expression signatures and the pathway analysis toolkit ASSIGN to interrogate pathways from the growth factor receptor network (GFRN) in breast cancer. Two discrete phenotypes, which correlated with mechanisms of apoptosis and drug response, were characterized from GFRN activity. These phenotypes have the potential to pinpoint more effective breast cancer treatments. Chapter 4 describes the development of Gene Set Omic Analysis (GSOA), a novel gene set analysis tool which uses machine learning to identify pathway differences between two given biologicalconditions from multiomic data. GSOA demonstrated its capacity to identify pathways known to play a role in various cancers, and improves upon other methods because of its ability to decipher complex multigene and multiomic patterns. Chapter 5 describes GSOA-shiny, a novel web application for GSOA, which provides biologists with lack of bioinformatics experience access to multiomic gene set analysis from an easy-to-use interface. Overall, this dissertation presents novel breast cancer phenotypes with clinical implications, provides the research community with gene expression signatures for GFRN components, and presents an innovative method and web application for gene set analysisâ€"all contributing to furthering the field of personalized oncology

Large variety in a panel of human colon cancer organoids in response to EZH2 inhibition

EZH2 inhibitors have gained great interest for their use as anti-cancer therapeutics. However, most research has focused on EZH2 mutant cancers and recently adverse effects of EZH2 inactivation have come to light. To determine whether colorectal cancer cells respond to EZH2 inhibition and to explore which factors influence the degree of response, we treated a panel of 20 organoid lines derived from human colon tumors with different concentrations of the EZH2 inhibitor GSK126. The resulting responses were associated with mutation status, gene expression and responses to other drugs. We found that the response to GSK126 treatment greatly varied between organoid lines. Response associated with the mutation status of ATRX and PAX2, and correlated with BIK expression. It also correlated well with response to Nutlin-3a which inhibits MDM2-p53 interaction thereby activating p53 signaling. Sensitivity to EZH2 ablation depended on the presence of wild type p53, as tumor organoids became resistant when p53 was mutated or knocked down. Our exploratory study provides insight into which genetic factors predict sensitivity to EZH2 inhibition. In addition, we show that the response to EZH2 inhibition requires wild type p53. We conclude that a subset of colorectal cancer patients may benefit from EZH2-targeting therapies

Customizing the therapeutic response of signaling networks to promote antitumor responses by drug combinations

Drug resistance, de novo and acquired, pervades cellular signaling networks (SNs) from one signaling motif to another as a result of cancer progression and/or drug intervention. This resistance is one of the key determinants of efficacy in targeted anti-cancer drug therapy. Although poorly understood, drug resistance is already being addressed in combination therapy by selecting drug targets where SN sensitivity increases due to combination components or as a result of de novo or acquired mutations. Additionally, successive drug combinations have shown low resistance potential. To promote a rational, systematic development of combination therapies, it is necessary to establish the underlying mechanisms that drive the advantages of combination therapies, and design methods to determine drug targets for combination regimens. Based on a joint systems analysis of cellular SN response and its sensitivity to drug action and oncogenic mutations, we describe an in silico method to analyze the targets of drug combinations. Our method explores mechanisms of sensitizing the SN through a combination of two drugs targeting vertical signaling pathways. We propose a paradigm of SN response customization by one drug to both maximize the effect of another drug in combination and promote a robust therapeutic response against oncogenic mutations. The method was applied to customize the response of the ErbB/PI3K/PTEN/AKT pathway by combination of drugs targeting HER2 receptors and proteins in the down-stream pathway. The results of a computational experiment showed that the modification of the SN response from hyperbolic to smooth sigmoid response by manipulation of two drugs in combination leads to greater robustness in therapeutic response against oncogenic mutations determining cancer heterogeneity. The application of this method in drug combination co-development suggests a combined evaluation of inhibition effects together with the capability of drug combinations to suppress resistance mechanisms before they become clinically manifest

Disease modeling on tumor organoids implicates AURKA as a therapeutic target in liver metastatic colorectal cancer

Metastatic spread and resistance to chemotherapy still limit the treatment success of current colorectal cancer therapy, even though multimodal treatment approaches have improved and prolonged patient survival. Here, we used state-of-the-art patient-derived tumor organoids (PDTOs) of liver metastatic colorectal cancer to model the generation of tolerance towards chemotherapy. We achieved this by long-term ex vivo treatment of KRAS wild type PDTOs with a clinically employed first-line therapy consisting of the chemotherapeutic regimen FOLFIRI plus the EGFR-targeting antibody Cetuximab. After up to 9 months of treatment, the PDTOs generated a tolerance towards FOLFIRI/Cetuximab and failed to induce an efficient apoptotic response. This phenotype occurred without the gain of resistance-conferring mutations in clinically relevant genes. Instead, unbiased whole transcriptome sequencing (next generation RNA sequencing) revealed an enrichment in MYC target gene expression in two out of three tolerant PDTO lines. The third PDTO line developed the tolerance towards first-line therapy via a different mechanism, which included upregulation of interferon-α-related gene expression. Intriguingly, all three tolerant PDTO lines were derived from tumors with a genomic amplification of the chromosomal region 20q13.2, which contains the Aurora kinase A (AURKA) locus, and displayed elevated mRNA and protein levels of AURKA compared to normal colonic epithelium. Treatment with the AURKA inhibitor Alisertib, which also represents a strategy to target MYC indirectly in different cancer types, restored an apoptotic response in the three established chemotherapy tolerant PDTO lines. We then introduced a KRASG12D mutation into the FOLFIRI/Cetuximab tolerant PDTO lines via CRISPR/Cas9-mediated genomic engineering and confirmed that this clinically problematic mutation confers resistance towards therapeutic approaches of single or dual targeting of the EGFR-MAPK pathway. Notably, the combination of dual targeting of the EGFR-MAPK pathway with inhibition of AURKA reduced the cell viability of first-line chemotherapy tolerant KRAS mutant PDTOs to a higher extent than each treatment alone. More importantly, the treatment with the AURKA inhibitor restored the apoptotic response and largely diminished the tumor organoid reformation capacity in KRAS mutant PDTOs, sensitized by dual EGFR-MAPK pathway inhibition. This combination treatment strategy was especially effective in the two PDTO lines that had developed increased MYC levels after acquisition of tolerance to first-line therapy. To obtain a deeper insight into AURKA expression levels in metastatic disease, we performed immunohistochemical staining of AURKA of CRCs of a matched patient cohort. Here, we observed that AURKA expression was slightly increased in non-metastatic colorectal cancers compared to liver or lung metastatic colorectal cancers. Moreover, the AURKA expression was positively correlated with the abundance of nuclear beta-catenin, which is a marker of aggressive disease and poor overall patient survival. In conclusion, this Ph.D. thesis provides evidence for the potential of patient-derived tumor organoids for the ex vivo modeling of colorectal cancer therapy tolerance, mutational disease progression, and the evaluation of drug combinations to overcome treatment resistance in a preclinical setting.Metastasierung und Resistenzen gegenüber Chemotherapie limitieren die Behandlungsmöglichkeiten des kolorektalen Karzinoms, obwohl multimodale Behandlungsansätze das Überleben der Patienten verbessert und verlängert haben. Wir haben Patienten-abgeleitete Tumor-Organoide (englisch: patient-derived tumor organoids, PDTOs) des lebermetastasierten kolorektalen Karzinoms angewandt, um die Entwicklung einer Toleranz gegenüber Chemotherapie nachzubilden. Das erreichten wir, indem wir die KRAS-wildtyp PDTOs ex vivo einer Langzeitbehandlung mit einer klinisch üblichen Erstlinientherapie unterzogen, die aus der Chemotherapie-Doublette FOLFIRI und dem EGFR-spezifischen Antikörper Cetuximab besteht. Nach einer bis zu neun Monate andauernden Behandlung entwickelten die PDTOs eine Toleranz gegenüber FOLFIRI/Cetuximab und reagierten nicht mehr mit Apoptose auf diese Behandlung. Dieser Phänotyp trat unabhängig von Resistenz-vermittelnden Mutationen in klinisch relevanten Genen auf. Stattdessen offenbarte die Sequenzierung des gesamten Transkriptoms (RNA-Sequenzierung der nächsten Generation) eine Induktion der Expression von MYC-Zielgenen in zwei von drei toleranten PDTO-Linien. Die dritte PDTO-Linie entwickelte eine Toleranz gegenüber der Erstlinientherapie durch andere Mechanismen, welche die Hochregulierung von Interferon-α-Zielgenen beinhalteten. Interessanterweise stammten alle drei PDTO-Linien von Tumoren ab, die genomische Amplifikation des chromosomalen Bereichs 20q13.2 zeigten, welcher den Aurora Kinase A (AURKA) Locus enthält. Weiterhin zeigten diese Ursprungstumore erhöhte AURKA mRNA- und Proteinmengen im Vergleich zu normalem Kolonepithel. Eine Behandlung mit dem AURKA-Inhibitor Alisertib stellt eine Strategie zur indirekten MYC-Inhibition in verschiedenen Tumorarten dar und induzierte Apoptose in den drei hier etablierten chemotoleranten PDTO-Linien. Anschließend führten wir mittels CRISPR/Cas9-vermittelter Genommanipulation eine KRASG12D-Mutation in die FOLFIRI/Cetuximab-toleranten PDTO-Linien ein. Wir bestätigten, dass diese klinisch problematische Mutation eine Resistenz gegenüber Ansätzen der einfachen oder dualen Inhibition des EGFR-MAPK-Signalwegs vermittelt. Interessanterweise reduzierte die Kombination aus dualer Inhibition des EGFR-MAPK-Signalweges und AURKA-Hemmer die Zellviabilität der Erstlinientherapie-toleranten KRAS-mutierten PDTOs in größerem Ausmaß als die jeweiligen Einzelbehandlungen. Zudem führte die Behandlung mit dem AURKA-Inhibitor von KRAS-mutierten PDTOs, die durch die Inhibierung des EGFR-MAPK-Signalweges sensibilisiert wurden, zu einer Wiederherstellung der Apoptosereaktion und reduzierte zum Großteil die Reetablierungskapazität der Organoide nach Behandlung. Diese Strategie der Kombinationsbehandlung war besonders erfolgreich in den beiden PDTO-Linien, die erhöhte MYC-Level nach der Ausbildung der Toleranz gegenüber der Erstlinientherapie gezeigt hatten. Um einen tieferen Einblick in das AURKA-Expressionslevel in metastasierten Fällen zu erhalten, führten wir einen immunhistochemischen Nachweis von AURKA in einer gepaarten Kohorte von Patienten mit kolorektalen Krebserkrankung durch. Dabei beobachteten wir, dass die AURKA-Expression in nicht-metastasierten kolorektalen Karzinomen im Vergleich zu exklusiv lebermetastasierten oder lungenmetastasierten kolorektalen Karzinomen leicht erhöht war. Weiterhin korrelierte die AURKA-Expression positiv mit der Abundanz von nukleärem Beta-Catenin, welches ein bekannter Marker für eine aggressive Erkrankung und eine schlechte Überlebensrate der Patienten ist. Letztendlich betont diese Ph.D.-Arbeit das Potential von PDTOs als ex vivo Modell für die Therapietoleranz des kolorektalen Karzinoms, für das mutationsbasierte Fortschreiten der Tumorkrankheit sowie für die Evaluierung solcher Wirkstoffkombinationen zu dienen, die in der Lage sind, Behandlungsresistenzen in einem präklinischen Setting zu überwinden

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Markers and mechanisms of resistance to Toceranib phosphate (Palladia®) in canine cutaneous mast cell tumor

2014 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

How cell death shapes cancer

Apoptosis has been established as a mechanism of anti-cancer defense. Members of the BCL-2 family are critical mediators of apoptotic cell death in health and disease, often found to be deregulated in cancer and believed to lead to the survival of malignant clones. However, over the years, a number of studies pointed out that a model in which cell death resistance unambiguously acts as a barrier against malignant disease might be too simple. This is based on paradoxical observations made in tumor patients as well as mouse models indicating that apoptosis can indeed drive tumor formation, at least under certain circumstances. One possible explanation for this phenomenon is that apoptosis can promote proliferation critically needed to compensate for cell loss, for example, upon therapy, and to restore tissue homeostasis. However, this, at the same time, can promote tumor development by allowing expansion of selected clones. Usually, tissue resident stem/progenitor cells are a major source for repopulation, some of them potentially carrying (age-, injury- or therapy-induced) genetic aberrations deleterious for the host. Thereby, apoptosis might drive genomic instability by facilitating the emergence of pathologic clones during phases of proliferation and subsequent replication stress-associated DNA damage. Tumorigenesis initiated by repeated cell attrition and repopulation, as confirmed in different genetic models, has parallels in human cancers, exemplified in therapy-induced secondary malignancies and myelodysplastic syndromes in patients with congenital bone marrow failure syndromes. Here, we aim to review evidence in support of the oncogenic role of stress-induced apoptosis

THE PHARMACOGENOMICS OF EGFR-DEPENDENT NSCLC: PREDICTING AND ENHANCING RESPONSE TO TARGETED EGFR THERAPY

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses