Les syndromes myélodysplasiques (SMD) sont un ensemble d’hémopathies clonales de la cellule souche. Ils touchent les sujets âgés et se caractérisent par une hématopoïèse inefficace, une différenciation anormale et une transformation fréquente en leucémie aiguë myéloblastique (LAM). La prise en charge thérapeutique a considérablement évolué ces dix dernières années, principalement avec l’arrivée de la 5-azacytidine (Aza) dans les SMD de haut risque. Malheureusement, il existe fréquemment un échec ou une perte de réponse rapide au traitement responsable d’une survie médiane globale de seulement quelques mois. La compréhension des mécanismes d’action des agents hypométhylants, la mise en évidence des facteurs biologiques impliqués dans la résistance à l’Aza ou encore l’identification de nouvelles associations de molécules constitue donc un enjeu majeur. Plusieurs équipes, dont la nôtre, ont démontré que l’erlotinib (Erlo) (inhibiteur de l’activité kinase de l’EGFR (Epidermal Growth Factor Receptor)) possède des effets antinéoplasiques dans les SMD/LAM. Compte tenu de sa toxicité modérée, cet inhibiteur de tyrosine kinase est actuellement en essai clinique en France et aux États-Unis dans les SMD en échec d’Aza. Dans ce travail, nous avons tenté de comprendre les mécanismes d’action impliqués dans l’activité de l’Aza et de l’Erlo seuls ou en association. Nous avons observé que l’Aza et la décitabine (un autre agent hypométhylant) induisent la déphosphorylation et la translocation dans le noyau du facteur de transcription FOXO3A où il réactive l’expression de gènes cibles tels que les facteurs pro apoptotiques PUMA et BIM. Cet effet observé rapidement, suggère un effet « off target » non lié à une reprogrammation épigénétique. La phosphorylation constitutive de FOXO3A étant considérée comme un facteur de mauvais pronostic dans les LAM, cette observation soulève l’intérêt potentiel des agents hypométhylants dans cette pathologie. Nous avons également identifié deux nouvelles cibles de l’Erlo : les SRC-kinases et la voie mTOR/p70S6K dont l’inhibition par des inhibiteurs biochimiques induit un arrêt du cycle cellulaire en G0/G1 sans apoptose ni différenciation confirmant l’hypothèse d’une action « multikinase » de l’Erlo. Par ailleurs, nous avons mis en évidence une activité synergique sur l’apoptose de l’association Aza et Erlo sur des lignées cellulaires de SMD/LAM et sur des cellules de patients. Cet effet n’a pas été retrouvé avec la décitabine ni les autres inhibiteurs de tyrosine kinase testés. La potentialisation de l’apoptose semble liée à plusieurs mécanismes associant l’augmentation de la concentration intracellulaire d’Aza via l’inhibition des transporteurs ABC, un arrêt de la prolifération, une activation des voies apoptotiques caspases-dépendantes et indépendantes et une activation des dommages à l’ADN. En conclusion, ce travail a permis l’identification de nouvelles cibles de l’Erlo et de l’AZA et a révélé un effet synergique entre ces deux molécules. Ces résultats précliniques encourageants suggèrent que cette association pourrait apporter un potentiel bénéfice chez les patients atteints de SMD/LAM, notamment ceux devenus réfractaires à l’Aza. / Myelodysplasic syndromes (MDS) constitute a diverse group of malignant clonal disorders that typically occur in elderly people. MDS are characterized by ineffective hematopoiesis, refractory cytopenias, morphologic dysplasia and increased potential to transform into acute myeloid leukemia (AML). Treatment of MDS has progressed considerably in recent years with the emergence of new approval agents such as azacytidine (aza)(a hypomethylating agent (HMA)) in higher-risk MDS. However, there are still a significant proportion of patients who do not respond to therapy with aza. Therefore, understanding the mechanisms of action of HMAs, identifying predictive factors for aza resistance and combining HMAs with other active compounds in MDS represent a challenging area to improve MDS/AML treatment. Previous works showed that erlotinib (an inhibitor of the epidermal growth factor receptor (EGFR)) exhibits antineoplastic effects in MDS/AML. Due to its limited toxicity profile, this tyrosine kinase inhibitor is currently being evaluated after failure of aza in two clinical trials. In this project, we aimed at understanding the molecular mechanisms involved in the activity of aza and erlo alone or in combination. We observed that aza and decitabine (another HMA related to aza) induces dephosphorylation and translocation to nucleus of the transcriptional regulator FOXO3A promoting the upregulation of the pro-apoptotic factors PUMA and BIM. This effect could be an “off target” effect and could contribute the bebenfical role of HMA in AML as constitutive phosphorylation of FOXO3A has been shown to be an adverse prognostic factor. We discovered new target for erlo, Src-kinase kinases and mTOR that are implicated in the cell-cycle arrest but not in the induction of apoptosis or differentiation confirming the “multikinase” activity of erlo. We found that the combination of aza and erlo demonstrated synergistic induction of apoptosis in MDS/AML cell lines and in some patient cells. This effect was not observed with decitabine or other tyrosine kinase inhibitors frequently used in onco-hematology. We demonstrated that potentiation of cell death is associated with different mechanisms such as intracellular accumulation of aza (via inhibition of ABC transporters), cell cycle arrest with inhibition of leukemic cells growth, caspase-dependent and -independent induction of apoptosis and DNA damage level. In conclusion, this work identified new targets of aza and erlo and revealed a synergistic induction of apoptosis upon co-treatment suggesting that this drug combination might be promising for SMD/AML treatment SMD/AML, especially the resistant patients.

Objetivos: Desenvolver e caracterizar nanocápsulas poliméricas contendo erlotinib, bem como avaliar sua atividade antitumoral in vitro em células de adenocarcinoma de pulmão humano. Metodologia: As nanocápsulas contendo erlotinib (0,5 mg.mL-1) foram obtidas pelo método de nanoprecipitação utilizando poli(ε-caprolactona) e óleo de copaíba como parede polimérica e núcleo oleoso, respectivamente. Os parâmetros físico-químicos avaliados foram: diâmetro médio e distribuição de tamanho, índice de polidispersão, potencial zeta, concentração de partículas e pH. Para determinação do teor e eficiência de encapsulação do erlotinib, utilizou-se metodologia validada por CLAE-UV. O estudo de liberação in vitro, utilizando sacos de diálise, foi realizado para obter o perfil de liberação do fármaco a partir das nanocápsulas. As nanocápsulas contendo erlotinib foram avaliadas quanto ao seu potencial de inibir o crescimento, induzir a apoptose, interferir com o ciclo celular e sobrevivência clonogênica de células de adenocarcinoma de pulmão, linhagem A549. Resultados: As nanocápsulas apresentaram diâmetro médio de 171 ± 2 (PDI < 0, 10), potencial zeta de −8,17 ± 2.26 mV, número de partículas por mL de 6,97 ± 0,22 × 1013 e pH de 6,24 ± 0,02. O teor e a eficiência de encapsulação foram próximos de 100%. Com exceção do pH, todos parâmetros mantiveram-se iguais após 30 dias de armazenamento em temperatura ambiente. Observou-se uma liberação controlada do fármaco devido à nanoencapsulação. Os ensaios de citotoxicidade demonstraram que as nanocápsulas contendo erlotinib apresentaram maior atividade antitumoral quando comparado com o fármaco livre. Também foi demonstrado indução de apoptose, pela análise de ciclo celular e marcação por Anexina-V conjugada ao 7-AAD. No ensaio clonogênico, as nanocápsulas contendo erlotinib reduziram 100% o número de colônias formadas. Conclusões: Foram obtidas nanocápsulas com propriedades nanotécnologicas adequadas e capazes de controlar a liberação do erlotinib. Os estudos in vitro na linhagem celular A549 demonstraram aumento no efeito antitumoral e foi demonstrado que o encapsulamento do fármaco é imprescindível para essa melhor atividade. / Purpose: To develop and characterise erlotinib-loaded polymeric nanocapsules and to evaluate its in vitro antitumor activity in human lung adenocarcinoma cells. Methodology: The erlotinib-loaded nanocapsules (0.5 mg.mL-1) were obtained by nanoprecipitation method using poly (ε-caprolactone) and copaiba oil as the polymeric wall and oily core, respectively. The physicochemical parameters evaluated were: mean diameter and size distribution, polydispersity index, zeta potential, particle concentration and pH. An HPLC-UV validated method was used to determine the drug content and encapsulation efficiency. The in vitro release study using dialysis bags was performed to obtain the drug release profile from nanocapsules. The erlotinib-loaded nanocapsules were evaluated regarding their potential to inhibit the growth, induce apoptosis, interfere with the cell cycle and clonogenic survival of lung adenocarcinoma cell (A549). Results: The nanocapsule formulation presented z-average diameter of 171 ± 2 (PDI <0.10), zeta potential value of -8.17 ± 2.26 mV, number of particles per mL of 6.97 ± 0.22 × 1013, and pH value of 6.24 ± 0.02. The drug content and the encapsulation efficiency were nearly 100%. Except for the pH value, all these parameters remained the same after 30 days of storage. A controlled release of the drug was observed due to nanoencapsulation. The cytotoxicity assays demonstrated that the erlotinib-loaded nanocapsules showed higher antitumor activity compared to free drug. Induction of apoptosis was demonstrated by cell cycle analysis and Annexin-V/7AAD staining. In the clonogenic assay, erlotinib-loaded nanocapsules reduced 100% the number of colonies formed. Conclusions: Nanocapsules with appropriate nanotechnological properties and capable of controlling the erlotinib release were obtained. The in vitro studies in the A549 cell line showed an increase in antitumor effect and was demonstrated that the drug encapsulation is essential for this better activity.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.

Objetivos: Desenvolver e caracterizar nanocápsulas poliméricas contendo erlotinib, bem como avaliar sua atividade antitumoral in vitro em células de adenocarcinoma de pulmão humano. Metodologia: As nanocápsulas contendo erlotinib (0,5 mg.mL-1) foram obtidas pelo método de nanoprecipitação utilizando poli(ε-caprolactona) e óleo de copaíba como parede polimérica e núcleo oleoso, respectivamente. Os parâmetros físico-químicos avaliados foram: diâmetro médio e distribuição de tamanho, índice de polidispersão, potencial zeta, concentração de partículas e pH. Para determinação do teor e eficiência de encapsulação do erlotinib, utilizou-se metodologia validada por CLAE-UV. O estudo de liberação in vitro, utilizando sacos de diálise, foi realizado para obter o perfil de liberação do fármaco a partir das nanocápsulas. As nanocápsulas contendo erlotinib foram avaliadas quanto ao seu potencial de inibir o crescimento, induzir a apoptose, interferir com o ciclo celular e sobrevivência clonogênica de células de adenocarcinoma de pulmão, linhagem A549. Resultados: As nanocápsulas apresentaram diâmetro médio de 171 ± 2 (PDI < 0, 10), potencial zeta de −8,17 ± 2.26 mV, número de partículas por mL de 6,97 ± 0,22 × 1013 e pH de 6,24 ± 0,02. O teor e a eficiência de encapsulação foram próximos de 100%. Com exceção do pH, todos parâmetros mantiveram-se iguais após 30 dias de armazenamento em temperatura ambiente. Observou-se uma liberação controlada do fármaco devido à nanoencapsulação. Os ensaios de citotoxicidade demonstraram que as nanocápsulas contendo erlotinib apresentaram maior atividade antitumoral quando comparado com o fármaco livre. Também foi demonstrado indução de apoptose, pela análise de ciclo celular e marcação por Anexina-V conjugada ao 7-AAD. No ensaio clonogênico, as nanocápsulas contendo erlotinib reduziram 100% o número de colônias formadas. Conclusões: Foram obtidas nanocápsulas com propriedades nanotécnologicas adequadas e capazes de controlar a liberação do erlotinib. Os estudos in vitro na linhagem celular A549 demonstraram aumento no efeito antitumoral e foi demonstrado que o encapsulamento do fármaco é imprescindível para essa melhor atividade. / Purpose: To develop and characterise erlotinib-loaded polymeric nanocapsules and to evaluate its in vitro antitumor activity in human lung adenocarcinoma cells. Methodology: The erlotinib-loaded nanocapsules (0.5 mg.mL-1) were obtained by nanoprecipitation method using poly (ε-caprolactone) and copaiba oil as the polymeric wall and oily core, respectively. The physicochemical parameters evaluated were: mean diameter and size distribution, polydispersity index, zeta potential, particle concentration and pH. An HPLC-UV validated method was used to determine the drug content and encapsulation efficiency. The in vitro release study using dialysis bags was performed to obtain the drug release profile from nanocapsules. The erlotinib-loaded nanocapsules were evaluated regarding their potential to inhibit the growth, induce apoptosis, interfere with the cell cycle and clonogenic survival of lung adenocarcinoma cell (A549). Results: The nanocapsule formulation presented z-average diameter of 171 ± 2 (PDI <0.10), zeta potential value of -8.17 ± 2.26 mV, number of particles per mL of 6.97 ± 0.22 × 1013, and pH value of 6.24 ± 0.02. The drug content and the encapsulation efficiency were nearly 100%. Except for the pH value, all these parameters remained the same after 30 days of storage. A controlled release of the drug was observed due to nanoencapsulation. The cytotoxicity assays demonstrated that the erlotinib-loaded nanocapsules showed higher antitumor activity compared to free drug. Induction of apoptosis was demonstrated by cell cycle analysis and Annexin-V/7AAD staining. In the clonogenic assay, erlotinib-loaded nanocapsules reduced 100% the number of colonies formed. Conclusions: Nanocapsules with appropriate nanotechnological properties and capable of controlling the erlotinib release were obtained. The in vitro studies in the A549 cell line showed an increase in antitumor effect and was demonstrated that the drug encapsulation is essential for this better activity.

Objetivos: Desenvolver e caracterizar nanocápsulas poliméricas contendo erlotinib, bem como avaliar sua atividade antitumoral in vitro em células de adenocarcinoma de pulmão humano. Metodologia: As nanocápsulas contendo erlotinib (0,5 mg.mL-1) foram obtidas pelo método de nanoprecipitação utilizando poli(ε-caprolactona) e óleo de copaíba como parede polimérica e núcleo oleoso, respectivamente. Os parâmetros físico-químicos avaliados foram: diâmetro médio e distribuição de tamanho, índice de polidispersão, potencial zeta, concentração de partículas e pH. Para determinação do teor e eficiência de encapsulação do erlotinib, utilizou-se metodologia validada por CLAE-UV. O estudo de liberação in vitro, utilizando sacos de diálise, foi realizado para obter o perfil de liberação do fármaco a partir das nanocápsulas. As nanocápsulas contendo erlotinib foram avaliadas quanto ao seu potencial de inibir o crescimento, induzir a apoptose, interferir com o ciclo celular e sobrevivência clonogênica de células de adenocarcinoma de pulmão, linhagem A549. Resultados: As nanocápsulas apresentaram diâmetro médio de 171 ± 2 (PDI < 0, 10), potencial zeta de −8,17 ± 2.26 mV, número de partículas por mL de 6,97 ± 0,22 × 1013 e pH de 6,24 ± 0,02. O teor e a eficiência de encapsulação foram próximos de 100%. Com exceção do pH, todos parâmetros mantiveram-se iguais após 30 dias de armazenamento em temperatura ambiente. Observou-se uma liberação controlada do fármaco devido à nanoencapsulação. Os ensaios de citotoxicidade demonstraram que as nanocápsulas contendo erlotinib apresentaram maior atividade antitumoral quando comparado com o fármaco livre. Também foi demonstrado indução de apoptose, pela análise de ciclo celular e marcação por Anexina-V conjugada ao 7-AAD. No ensaio clonogênico, as nanocápsulas contendo erlotinib reduziram 100% o número de colônias formadas. Conclusões: Foram obtidas nanocápsulas com propriedades nanotécnologicas adequadas e capazes de controlar a liberação do erlotinib. Os estudos in vitro na linhagem celular A549 demonstraram aumento no efeito antitumoral e foi demonstrado que o encapsulamento do fármaco é imprescindível para essa melhor atividade. / Purpose: To develop and characterise erlotinib-loaded polymeric nanocapsules and to evaluate its in vitro antitumor activity in human lung adenocarcinoma cells. Methodology: The erlotinib-loaded nanocapsules (0.5 mg.mL-1) were obtained by nanoprecipitation method using poly (ε-caprolactone) and copaiba oil as the polymeric wall and oily core, respectively. The physicochemical parameters evaluated were: mean diameter and size distribution, polydispersity index, zeta potential, particle concentration and pH. An HPLC-UV validated method was used to determine the drug content and encapsulation efficiency. The in vitro release study using dialysis bags was performed to obtain the drug release profile from nanocapsules. The erlotinib-loaded nanocapsules were evaluated regarding their potential to inhibit the growth, induce apoptosis, interfere with the cell cycle and clonogenic survival of lung adenocarcinoma cell (A549). Results: The nanocapsule formulation presented z-average diameter of 171 ± 2 (PDI <0.10), zeta potential value of -8.17 ± 2.26 mV, number of particles per mL of 6.97 ± 0.22 × 1013, and pH value of 6.24 ± 0.02. The drug content and the encapsulation efficiency were nearly 100%. Except for the pH value, all these parameters remained the same after 30 days of storage. A controlled release of the drug was observed due to nanoencapsulation. The cytotoxicity assays demonstrated that the erlotinib-loaded nanocapsules showed higher antitumor activity compared to free drug. Induction of apoptosis was demonstrated by cell cycle analysis and Annexin-V/7AAD staining. In the clonogenic assay, erlotinib-loaded nanocapsules reduced 100% the number of colonies formed. Conclusions: Nanocapsules with appropriate nanotechnological properties and capable of controlling the erlotinib release were obtained. The in vitro studies in the A549 cell line showed an increase in antitumor effect and was demonstrated that the drug encapsulation is essential for this better activity.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.

Cancer cells are believed to exist in a condition of metabolic oxidative stress compared to normal cells because of inherent mitochondrial dysfunction. Cancer cells up regulate antioxidant defense mechanisms to combat the toxic effect of reactive oxygen species (ROS). Many anticancer agents block ROS detoxification mechanisms and utilize oxidative stress to cause cytotoxicity to cancer cells. However, ROS also up-regulate many pro-survival signaling pathways that may mediate resistance to chemotherapy. I hypothesize that ROS induces both cytotoxicity and pro-survival mechanisms in cells treated with chemotherapeutic agents such as the EGFR inhibitor erlotinib. This thesis explores how oxidative stress may induce both pro-survival and pro-death mechanisms in HNSCC cells and how this can be exploited to increase the cytotoxicity of erlotinib. The combined use of buthionine-[S,R]-sulfoximine, an inhibitor of glutathione and auranofin, an inhibitor of thioredoxin metabolism enhanced human head and neck cancer cell killing by a mechanism involving oxidative stress both in vitro and in vivo and sensitized cells to erlotinib in vitro. However, in other studies erlotinib as a single agent induced oxidative stress and this was mediated by NADPH oxidase 4 (NOX4). NOX4 mediated oxidative stress activated a process called autophagy which protected cancer cells from cytotoxic effect of erlotinib and inhibition of autophagy sensitized cells to erlotinib in vitro. These studies show that oxidative stress may have a dual role in cancer chemotherapy. ROS generated from various drug treatments can cause oxidative damage of cells culminating in cell death. However, it may also activate autophagy protecting cells against the stress and leading to decreased efficacy of the treatment. Hence inhibiting autophagy and hydroperoxide metabolism can be effective treatment modalities to enhance the cytotoxicity of erlotinib and achieve maximum therapeutic efficacy.

Im Vergleich zu anderen Krebsentitäten, konnten Patienten mit PDAC bisher kaum von Therapieerfolgen der Präzisionsmedizin profitieren. Um diese Problematik zu adressieren, habe ich eine umfassende molekularbiologische Studie durchgeführt, um prädiktive Biomarker zu identifizieren und die Risikostratifizierung der Patienten zu verfeinern. Mittels gen-spezifischer Sequenzierung und gezielter RNA-Expressionsanalyse wurden 293 R0-resezierte Patienten aus einer multizentrischen Phase-III-Studie untersucht. Ziel der klinischen Studie war der Vergleich von adjuvanter Chemotherapie mit Gemcitabin entweder mit oder ohne Zusatz von Erlotinib. Für meine Arbeit wurden die Patientenproben unter Verwendung einer nicht-negativen Matrixfaktorisierung (NMF) basierend auf ihren Einzelnukleotidvarianten (SNV) und ihren Kopienzahlveränderungen (CNA) gruppiert und auf klinische und molekularbiologische Unterschiede untersucht. Um die biologischen Hintergründe der identifizierten genetischen Besonderheiten zu verstehen, wurden anschließend Zelllinien genetisch modifiziert und in vitro modelliert. Es wurden 1086 SNVs und 4157 CNAs identifiziert. Dabei wiesen 99% aller Patienten mindestens eine genetische Veränderung auf, mit durchschnittlich 18 Aberrationen pro Patient. In Übereinstimmung mit früheren Berichten waren KRAS, TP53, CDKN2A und SMAD4 die am häufigsten betroffenen Gene. Alterationen in diesen Genen konnten in 63-93 % der Fälle nachgewiesen werden. Basierend darauf konnte ich fünf Patientengruppen identifizieren die sich in ihren biologischen Charakteristika unterscheiden und Angriffspunkte für gezielte Therapien bieten. Mittels NMF wurden zudem SMAD4alt MAPK9low als prognostische Biomarker für Erlotinib identifiziert. Anschließende in vitro Experimente zeigten, dass dies nicht auf eine Erhöhung der Erlotinib-Zelltoxizität zurückzuführen ist. Zuletzt definiere ich einen prognostischen Score der genutzt werden kann um das Überleben von R0-resizierten PDAC Patienten abzuschätzen. / In contrast to other cancer entities, PDAC patients have not benefited from recent improvements in precision medicine. To address this gap, I embarked on a comprehensive molecular study to identify predictive biomarkers and refine risk stratification. I performed targeted sequencing and targeted RNA expression analysis of 293 R0-resected patients from a multicenter phase III trial comparing adjuvant chemotherapy of gemcitabine with or without erlotinib. Patients were clustered using non-negative matrix factorization (NMF) based on their single nucleotide variant (SNV) and copy number alteration (CNA) statuses. Overall (OS) and disease-free survival (DFS) were analysed with the multivariate cox hazard and log rank tests. Finally, using a method based on CRISPR/Cas, findings from the patient cohort where modeled in vitro to assess their biological backgrounds. A total of 1,086 SNVs and 4,157 CNAs were found with at least one genetic alteration in 99% of all patients, and an average of 18 aberrations per patient. In line with previous reports, KRAS, TP53, CDKN2A, and SMAD4 were the most frequently affected genes, detected in 63–93 % of cases. In this thesis, I identified five biologically distinct patient subgroups with different actionable lesions that may serve for refined PDAC classification and tailored treatment approaches. NMF based clustering and subsequent differential expression analysis revealed SMAD4alt (SNV and/or CAN in SMAD4) MAPK9low (MAPK9 expression below median) as prognostic erlotinib biomarker. Modeling of SMAD4alt MAPK9low status in vitro showed that the effect is not based on increased erlotinib toxicity. Finally, I proposed a genetic risk score for prognostic evaluation of newly diagnosed R0-resected PDAC patients.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.