Targeting PH domain proteins for cancer therapy

Targeted therapy has been one of the most promising treatment options for cancer during the past decade. Discoveries of potent and selective small molecule inhibitors are critical to new and promising targeted therapy. Pleckstrin Homology (PH) domain proteins are one of the biggest protein families in the human proteome. However, no drugs have been achieved to the late development stages, let alone getting to the market. Thus, a deeper understanding of this protein family is required and there is an urgent need to develop novel small molecule compounds targeting these proteins. Studies of PH domains began around two decades ago and a lot of efforts have been focused on their structures and functions. However, not much is known about their role in cancers, except a few proteins such as AKT. In order to delineate the roles of PH domain proteins in cancers, we performed a comprehensive analysis of 313 PH domain proteins using 13 types of most common cancers in TCGA. From this analysis, we identified the most frequently upregulated and mutated PH domain proteins. Interestingly, we found Tiam1, a guanine nucleotide exchange factor (GEF) specific for Rac1 activation, was overexpressed in several cancers, particularly neuroendocrine prostate cancer. Targeting PH domain proteins remains to be a significant challenge for multiple reasons. First, the binding pockets of most PH domain proteins are unknown due to lacking of PH-PIPs complex crystal structures. Second, these binding pockets are positively charged, which makes it really difficult to design small molecule inhibitors targeting these sites. In order to address these issues, we performed structural sequence alignment of available PH domain structures to identify conserved residues. Also, ensemble docking was performed in order to address the flexibility of the proteins. Through these efforts, we identified two scaffolds as Tiam1 small molecule inhibitors. These inhibitors showed binding affinity to the PH domain using surface plasmon resonance (SPR) assay and inhibition of Rac1 activation in prostate cancer cells. Also, these compounds inhibited prostate cancer cell proliferation and migration in vitro

Signal transduction in macrophages. Intracellular pathways activated by microbial constituents

Macrophages play an essential role in the defense against infection by phagocytosis and by production of inflammatory mediators. Such mediators are TNF? and eicosanoids. Many microbial agents elicit arachidonate release in macrophages that lead to the formation of eicosanoids. This thesis is based on studies performed with the use of microbial constituents which induce arachidonate release or/and TNF? production. Whole gram-positive bacteria S.aureus, the bacterial products LPS and peptidoglycan, the yeast preparation zymosan and ?-glucan were used to stimulate the cells. The main focus has been to elucidate the potential role of the non-receptor tyrosine kinases from the Src family (SFK) and the Tec kinase Btk. in signaling pathways induced by microbial constituents. SFK are important in bacteria and yeast induced arachidonate release and these studies bring forward that SFK has a pivotal role in acting proximally of several known members of the signaling like, ERK, p38 and PLC?2. Btk is also observed to be a part signaling pathway downstream of SFK. Differences were detected between bacteria and zymosan elicted responses, probably due to use of different receptors. Zymosan can bind to several receptors and our results suggest that zymosan-induced arachidonate release is mediated by the ?-glucan receptor dectin-1. Furthermore we show that the adaptor protein gab-2 is affected by ?-glucan and zymosan stimulation, indicating a role in dectin-1 signaling. SFK are also seen to be involved in TNF? production induced by microbial constituents, but their mode of action is still unknown. In summery this thesis has contributed to an increased understanding of the role of SFK in eicosanoid and TNF? production in macrophages. Furthermore has we contributed to knowledge about the signaling pathway resulting in arachidonate release

Feedback regulation of Gab2-dependent signaling by the Ras/MAPK pathway

La voie de signalisation des Récepteurs Tyrosine Kinase (RTK) occupe un rôle central dans la régulation de la croissance cellulaire, la prolifération, la différentiation et la motilité. Une régulation anormale des RTKs mène à plusieurs maladies humaines telles que le cancer du sein, la seconde cause de mortalité chez les femmes à cause de l’amplification et la mutation fréquente de la protéine tyrosine kinase HER2 (ERBB2). Grb2-associated binder (Gab) 2 est une protéine adaptatrice qui agit en aval de plusieurs RTKs, y compris HER2, pour réguler de multiples voies de signalisation. En réponse à la stimulation par de nombreux facteurs de croissances et cytokines, Gab2 est recruté à la membrane plasmique où il potentialise l’activation des voies de signalisation Ras/mitogen-activated protein kinase (MAPK) et PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B). En plus d’occuper un rôle essentiel durant le développement du système hématopoïétique, Gab2 est souvent amplifié dans les cancers, notamment le cancer du sein et les mélanomes. Cependant, les mécanismes moléculaires qui régulent le fonctionnement de Gab2 sont peu connus. Il est établi que lors de l’activation des RTKs, Gab2 est phosphorylé au niveau de plusieurs résidus Tyrosine, menant à l’association de différentes protéines comme p85 et Shp2. En plus de la phosphorylation en Tyrosine, notre laboratoire ainsi que d’autres groupes de recherche avons identifié que Gab2 est aussi phosphorylé au niveau de résidus Ser/Thr suite à l’activation de la voie de signalisation MAPK. Cependant, la régulation des fonctions de Gab2 par ces modifications post-traductionnelles est encore peu connue. Dans le but de comprendre comment Gab2 est régulé par la voie de signalisation MAPK, nous avons utilisé différentes approches. Dans la première partie de ma thèse, nous avons déterminé un nouveau mécanisme démontrant que la voie de signalisation Ras/MAPK, par le biais des protéines kinases RSK (p90 ribosomal S6 kinase), phosphoryle Gab2. Ce phénomène se produit à la fois in vivo et in vitro au niveau de trois résidus Ser/Thr conservés. Des mutations au niveau de ces sites de phosphorylation entrainent le recrutement de Shp2 menant à l’augmentation de la motilité cellulaire, ce qui suggère que les protéines RSK restreignent les fonctions dépendantes de Gab2. Ce phénomène est le résultat de la participation de RSK dans la boucle de rétroaction négative de la voie de signalisation MAPK. Dans la seconde partie de ma thèse, nous avons démontré que les protéines ERK1/2 phosphorylent Gab2 au niveau de plusieurs résidus pS/T-P à la fois in vitro et in vivo, entrainant l’inhibition du recrutement de p85. De plus, nous avons établi pour la première fois que Gab2 interagit physiquement avec ERK1/2 dans des cellules lors de l’activation de la voie de signalisation MAPK. Par ailleurs, nous avons montré un nouveau domaine d’attache du module ERK1/2 sur Gab2. Des mutations sur les résidus essentiels de ce domaine d’attache n’entrainent pas seulement la dissociation de ERK1/2 avec Gab2, mais diminuent également la phosphorylation dépendante de ERK1/2 sur Gab2. Ainsi, nos données montrent que la voie de signalisation MAPK régule les fonctions de la protéine Gab2 par le biais des kinases RSK et ERK1/2. Cette thèse élabore par ailleurs un schéma complet des fonctions de Gab2 dépendantes de la voie de signalisation MAPK dans le développement de nombreux cancers.Receptor tyrosine kinase (RTK) signaling plays an essential role in modulating cell growth, proliferation, differentiation and motility. Abnormal regulation of RTKs results in many human diseases, including breast cancer, the second leading cause of cancer mortality in women by the frequent amplification and mutation of the HER2 (ERBB2) tyrosine kinase. The Grb2-associated binder (Gab) 2 is an adaptor protein that acts downstream of several RTKs, including HER2, to regulate multiple signaling pathways. In response to the stimulation of a number of growth factors and cytokines, Gab2 is recruited to the plasma membrane, where it potentiates the activation of the Ras/mitogen-activated protein kinase (MAPK) and PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B) pathways. In addition to playing an important role in the hematopoietic system during development, GAB2 is often amplified in cancers including breast cancer and melanoma, however, little is known about the molecular mechanisms that regulate Gab2 function. It has been well established that upon RTKs activation, Gab2 becomes phosphorylated on several Tyr residues leading to diverse adaptor protein associations, such as p85 and Shp2. Aside from the tyrosine phosphorylation, our lab and other groups noticed that Gab2 is also phosphorylated on Ser/Thr residues upon activation of MAPK signaling. However, less is known about this post-translational modification in the regulation of Gab2 functions. In order to understand how Gab2 is regulated by the MAPK pathway, we used different approaches. In the first part of my thesis, we determined a new mechanism by which the Ras/MAPK pathway through RSK (p90 ribosomal S6 kinase) phosphorylated Gab2 on three conserved Ser/Thr residues, both in vivo and in vitro. Mutation of these phosphorylation sites promoted Shp2 recruitment leading to increased cell motility, suggesting that RSK restricts Gab2-dependent functions as a result of participation in the negative feedback loop of MAPK signaling. In the second part of the thesis, we found that ERK1/2 phosphorylated Gab2 on several potential pS/T-P residues, both in vivo and in vitro, resulting in inhibited p85 recruitment. In addition, to the best of our knowledge, we established for the first time that Gab2 physically interacted with ERK1/2 in cells upon activation of the MAPK pathway. Furthermore, we revealed a novel ERK1/2 docking domain in Gab2. Mutation of the essential residues in this docking domain not only disrupted ERK1/2-Gab2 interaction, but also diminished ERK1/2-dependent phosphorylation on Gab2. Taken together, our data showed that the MAPK pathway regulates Gab2 functions through both RSK- and ERK1/2-dependent manners. Given that Gab2 is overexpressed in several cancers, this thesis decodes a complete figure of modulating actions of Gab2 by MAPK signaling in cancer development

Crk and CrkL adaptor proteins: networks for physiological and pathological signaling

The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses

Signaling and Feedback Networks Underlying Senstivity and Resistance to Kinase Inhibitors in Oncogene Addicted Cancers

Targeted therapies have begun to be developed and approved in the clinic over the past several decades to treat cancers with specific genetic alterations. In non-small cell lung cancer (NSCLC), patients harboring EGFR activating mutations often respond to the EGFR inhibitors gefitinib/erlotinib, exhibiting down-regulation of central oncogenic pathways and dramatic tumor regressions. Despite initially promising results, the vast majority of patients develop resistance to targeted therapies. Thus far, several mechanisms of resistance including T790M mutation in EGFR, amplification of the MET receptor tyrosine kinase (RTK), activating mutations in downstream signaling molecules, and loss of negative regulators have been identified. As a result, next generation inhibitors and combination therapies continue to be developed and tested in the clinic. There are still many cases in which the cause of resistance to a particular targeted therapy is unknown, or the subset of patients most likely to benefit has not been identified. This thesis describes the ability of the MET ligand, HGF, to activate PI3K signaling and cause gefitinib resistance in EGFR-driven cancers. In addition, detection of a preexisting subpopulation of MET amplified cells (present before treatment with an EGFR inhibitor) is shown to successfully predict the development of MET amplification as a resistance mechanism. These results suggest that it may be possible to prospectively identify patients who will benefit from combined MET/HGF and EGFR inhibitors as initial therapies. Further, this thesis highlights the importance of both PI3K/AKT and MEK/ERK signaling as drivers of cell proliferation and viability, and describes a novel feedback network regulating these pathways. In multiple cancer models, treatment with a single agent MEK inhibitor leads to feedback up-regulation of ERBB3/PI3K/AKT signaling. The mechanism for this feedback involves loss of an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2 following MEK inhibition, which leads to increased ERBB receptor activation. These results further elucidate the complex feedback networks that regulate signaling in cancer cells, and suggest possible limitations for the efficacy of single agent RAF/MEK pathway inhibitors. Collectively, this work describes multiple resistance mechanisms to kinase inhibitors, and suggests new biomarkers to define those patients who are likely to benefit from specific targeted therapies

AXL receptor tyrosine kinase in breast cancer : defining novel substrates and pathways involved in cell motility and invasion

Le cancer du sein est le cancer le plus fréquemment diagnostiqué et le plus mortelle chez la femme, où sa progression vers le stade métastatique constitue une menace pour la vie des patientes. La présence de métastases représente le défi clinique central de l'oncologie des tumeurs solides, de sorte que les mécanismes et les voies sous-jacents au processus métastatique doivent être mieux définis. L'expression aberrante du récepteur tyrosine kinase (RTK) AXL a été liée cliniquement à la formation de métastases et à l'acquisition d'une résistance aux médicaments contre le cancer. AXL est un membre de la sous-famille des récepteurs tyrosine kinase TAM et intervient dans plusieurs processus biologiques tels que l'atténuation de la réponse immunitaire, l'élimination des cellules apoptotiques et la promotion de la survie cellulaire. L'expression d'AXL dans les tumeurs primaires humaines corrèle avec la faible survie des patients. Malgré sa régulation positive préférentielle dans les lignées cellulaires triple négatives / basales B, des études ont montré que l’expression d’AXL est indépendante du sous-type de la tumeur mammaire des patients. AXL peut être activé par son ligand GAS6 ou par d'autres RTK. Lors de son activation, AXL induit une signalisation en aval entraînant l'activation d'intermédiaires de signalisation canoniques, notamment MAPK, AKT et PI 3-kinases. Cependant, les voies de signalisation spécifiques engagées par AXL pour conférer un tel pouvoir pro-invasion ne sont pas connues. Ainsi, le but de cette thèse est d'identifier des substrats spécifiques d’AXL et des voies en aval qui jouent un rôle important dans le maintien d'un état « EMT » et d'un renforcement du phénotype mésenchymal dans les cellules cancéreuses. À la recherche de régulateurs en amont du complexe ELMO/DOCK1 impliqués dans l’activation de RAC, nous présentons au chapitre 2 les protéines d’échafaudage ELMO en tant que substrats directs et partenaires de liaison d’AXL. Grâce à des approches de protéomique et de mutagenèse, nous révélons que la kinase AXL phosphoryle ELMO1/2 sur un résidu tyrosine carboxy-terminal conservé. Dans les cellules cancéreuses du sein, l'activation d'AXL dépendante de GAS6 a conduit à la phosphorylation endogène d'ELMO2 sur Tyr-713, menant ainsi à la formation du complexe AXL/ELMO. En outre, l'activation de RAC induite par GAS6 dans les cellules cancéreuses du sein dépendait de l'expression d'ELMO2. Semblable au blocage d’AXL, l'inhibition d’ELMO2 ou l'inhibition pharmacologique de DOCK1 supprime l'invasion des cellules du cancer du sein, qui, selon nous, dépendait de l'état de phosphorylation d'ELMO. Notre travail au chapitre 2 définit un nouveau mécanisme par lequel AXL favorise la prolifération et l'invasion cellulaire et identifie l'inhibition de la voie ELMO/DOCK comme une cible thérapeutique potentielle pour arrêter les métastases induites par AXL. Bien qu'il soit encore difficile de savoir comment les signaux d’AXL induisent son phénotype pro-invasif, notre travail au Chapitre 3 vise à identifier des substrats et des voies de signalisation spécifiques qui sont significativement modulés lors de l'activation d'AXL. Pour y remédier, nous avons défini le phosphoprotéome de la régulation d’AXL dans des cellules cancéreuses du sein triple-négatives en utilisant une approche quantitative. Nous révélons qu’AXL module de manière robuste, parmi de nombreux processus et voies biologiques importants, la phosphorylation d'un réseau de protéines d'adhésion focale (FA) aboutissant à un désassemblage plus rapide des FA. De manière intéressante, nous avons trouvé que la modulation de la voie FA était unique à AXL par rapport à d'autres RTK tels que l'EGFR. En particulier, nous avons trouvé qu’AXL phosphoryle la protéine NEDD9, modulant la formation du complexe NEDD9/CRKII/DOCK3, qui orchestre la phosphorylation de la pseudo-kinase PEAK1 médiée par AXL. Nos données révèlent un mécanisme distinct par lequel les complexes PEAK1 avec la kinase CSK médient la phosphorylation de PXN et le renouvellement des FA induit par AXL. En utilisant l'injection orthotopique de cellules cancéreuses du sein dans le tissu adipeux mammaire des souris et dans la veine de la queue, nous révélons que l'inactivation de PEAK1 par CRISPR diminue la croissance tumorale et les métastases in vivo. De plus, notre travail au chapitre 3 révèle une contribution unique et inattendue de la signalisation d’AXL à la dynamique des FA, révélant un mécanisme longtemps recherché sous-tendant l'activité invasive d'AXL. Cette compréhension approfondie des réseaux de signalisation régulés par AXL identifie PEAK1 comme une nouvelle cible thérapeutique dans les tumeurs AXL positives. En conclusion, cette thèse a identifié, pour la première fois, le phosphoprotéome d’AXL et des voies de signalisation spécifique à AXL, pouvant justifier le rôle du récepteur en tant que promoteur de métastases et de résistance aux médicaments. Notre travail révèle de nouvelles cibles thérapeutiques qui pourraient avoir un grand potentiel si elles sont utilisées en thérapie combinatoire avec l’inhibition d’AXL pour prévenir la formation de métastases des tumeurs AXL positives.Breast cancer is the most frequently diagnosed cancer in women where its progression to the metastatic stage poses a threat to the life of patients. The metastatic disease represents the central clinical challenge of solid tumor oncology such that mechanisms and pathways underlying the metastatic process must be better defined. The aberrant expression of the receptor tyrosine kinase (RTK) AXL has been linked clinically to metastasis and acquisition of drug resistance. AXL is a member of the TAM subfamily and functions in several biological processes such as dampening the immune response, clearing apoptotic cells and promoting cell survival. Despite its preferential upregulation in triple negative/basal B cell lines, studies have shown AXL expression in the clinic to be subtype independent. AXL can be activated by its ligand GAS6 or by a crosstalk with other RTKs. Upon its activation, AXL induces downstream signaling resulting in the activation of canonical signaling intermediates including MAPKs, AKT and PI 3-kinases. However, the specific signaling pathways engaged by AXL to confer such enhanced pro-invasion power are not known and the goal of this thesis is to identify AXL-specific substrates and downstream pathways that are behind AXL’s significant role in maintaining an EMT state and reinforced mesenchymal phenotype in cancer cells. In search of upstream regulators of ELMO/DOCK1 complex involved in RAC activation, we reported ELMO scaffolds as direct substrates and binding partners of AXL. Through proteomics and mutagenesis approaches, we revealed phosphorylation of ELMO1/2 by AXL kinase on a conserved carboxyl-terminal tyrosine residue. In breast cancer cells, GAS6-dependent activation of AXL led to endogenous ELMO2 phosphorylation on Tyr-713 and AXL/ELMO complex formation. In addition, GAS6-induced RAC activation in breast cancer cells was dependent on ELMO2 expression and phosphorylation. Our work in chapter 2 defines a new mechanism by which AXL promotes cell proliferation and invasion and identifies inhibition of ELMO/DOCK pathway as a potential therapeutic target to stop AXL-induced metastases. While it still remains elusive how AXL signals to induce its pro-invasive phenotype, our work strove to identify specific substrates and signaling pathways that are significantly modulated upon AXL activation using a quantitative phosphoproteomics approach. By generating GAS6-induced AXL phosphoproteome, we found that AXL robustly modulates, among many different significant biological processes and pathways, the phosphorylation of a network of focal adhesion (FA) proteins culminating in faster FA disassembly. Interestingly, we found AXL modulation of FA pathway to be unique to AXL in comparison with other RTKs such as EGFR. NEDD9 FA protein was identified to be a direct substrate of AXL, where its phosphorylation modulates its complex formation with CRKII/DOCK3, and this subsequently orchestrates the AXL-mediated phosphorylation of the pseudo-kinase PEAK1. Our data revealed a distinct mechanism by which PEAK1 complexes with CSK kinase, mediating PXN phosphorylation and AXL-induced FA turnover. Using in vivo assays such as tail-vein metastasis assay and tumor growth assay, we revealed that gene inactivation of PEAK1 by CRISPR CAS9 decreased tumor growth and metastasis. Furthermore, our work in chapter 3 uncovers an unexpected and unique robust contribution of AXL signaling to FA dynamics revealing a long sought-after mechanism underlying AXL pro-invasive activity. This in-depth understanding of AXL regulated signaling networks identifies PEAK1 as a new therapeutic target in AXL positive tumors. In conclusion, this thesis identified, for the first time, AXL phosphoproteome and AXL specific downstream signaling pathways that may justify AXL’s role as a promoter of metastasis and drug resistance. Our work reveals novel therapeutic drug targets that may hold a great potential if used in combinational therapeutics with AXL inhibition to prevent metastasis of AXL positive tumors

Novel mechanisms of resistance to EGFR inhibitory drugs in non-small cell lung cancer

EGFR activating mutations are present in 10-40% of non-small cell lung cancer. Such mutations render tumour cells sensitive to EGFR tyrosine kinase inhibitors (EGFR TKIs), with responses of up to 80% in populations selected for the presence of an activating mutation. Unfortunately, almost all patients develop resistance after about a year. Clinically described mechanisms of resistance include the presence of a secondary mutation (T790M) in EGFR which prevents EGFR TKIs binding to the EGF receptor, and amplification MET which permits survival signalling via the ERBB3 receptor. However in 30% of cases, the mechanism of acquired resistance to EGFR TKIs is still unknown. My aim was to carry out a genome-wide siRNA screen to identify novel mechanisms of resistance to EGFR TKIs. I identified two genes that have not been implicated in EGFR TKI resistance previously, NF1 and DEPTOR, which are negative regulators of RAS and mTOR respectively. Depletion of NF1 or DEPTOR leads to increased resistance to EGFR TKIs via upregulation of MAPK signalling by direct and indirect mechanisms

Mechanisms of IKBKE Activation in Cancer

Cancer is the second leading cause of death in the USA and it is expected to surpass heart diseases making it important to understand the underlying mechanisms of cancer. The efforts to target single signaling molecule showed little success in increasing the patient survival and it can be due to increased compensation for cell survival by alternative pathway activations. Hence comprehensive understanding of the alternative signaling pathways may help us treat cancer better. Chronic inflammation is attributed to increased risk of cancer and emerging studies show the growing importance of both canonical and non-canonical IκB kinases such as IKKα, IKKβ, IKBKE and TBK1 in human cancer pathogenesis. Initially identified as activator of NFκB pathway, IKBKE was shown to play an important oncogenic role by regulating multiple pathways downstream. Although IKBKE is implicated in tumorigenesis for over a decade, therapeutic targeting of this pathway has been a challenge. Recently, amlexanox and CYT387, which are in clinical trials for Type II diabetes and myeloproliferative disorders respectively, were identified as potential IKBKE inhibitors. In this study, we uncovered specific novel mechanisms of activation of IKBKE in tumor cells and the outcomes of targeting IKBKE pathway. Oncogenic mutations are a cause of several human malignancies. Mutations in EGFR are observed in 15% of non-small cell lung cancer patients. While cells expressing these mutations respond better to the first generation TKIs, patients become resistant to these inhibitors due to secondary mutations in EGFR. These mutations were shown to make EGFR constitutively active even in the absence of ligands. Direct targeting of EGFR with secondary mutations has been challenging as EGFR acquires novel mutations upon inhibitor treatment, which confer resistance to the EGFR-TKIs. Hence, it is important to improve our knowledge of the downstream signaling pathways of EGFR. Although PI3K, MEK signaling are well established, mutant EGFR was shown to activate several novel signaling pathways such as miRNA processing and autophagy that are implicated in resistance to EGFR-TKIs. Here, we show that IKBKE acts downstream of mutant EGFR to activate the NFκB and AKT pathways. In addition, we show that mutant EGFR but not wildtype EGFR can directly phosphorylate IKBKE at Tyrosine 153 and Tyrosine 179 residues that are important for activation of IKBKE kinase. We also found that the IKBKE/TBK1 inhibitor Amlexanox exhibits increased efficacy in inhibiting cell viability in NSCLC cells with activating EGFR mutations. Furthermore, we also found that IKBKE inhibitors activate the MAPK pathway, and EGFR-TKI resistant NSCLCs exhibit enhanced response to co-treatment with IKBKE inhibitors and MEK inhibitors. Similar to lung cancer, pancreatic cancer is a challenging disease due to lack of direct inhibitors of the KRas mutations that are observed in more than 95% of pancreatic cancer patients. IKBKE/TBK1 pathway is important for KRas signaling, but the efficacy of IKBKE inhibitors in pancreatic cancers is not well studied. Here, we show that IKBKE is an important target in pancreatic cancers that regulates pancreatic cell viability, cell migration and cancer stem cells. Importantly, we provide mechanistic insights into the effects of IKBKE inhibitors on specific signaling pathways. We found that IKBKE inhibition results in significantly increased expression of RTKs, such as ErbB3 and IGF1-R, which increases ERK1/2 activation. Our findings provide support for novel combination strategies for pancreatic cancer. Metastasis is a poor prognostic factor for ovarian cancer. Although patients with early stage ovarian cancer with no distal metastasis exhibit a 70% 5-year survival rate, Stage IV patients with distal metastasis exhibit only 20% 5-year survival rate. Hence, ongoing efforts are aimed at targeting the pathways that regulate metastasis in ovarian cancers. IKBKE is upregulated in ovarian cancer patients, and IKBKE expression is known to regulate the expression of several genes important for cell motility in ovarian cancers. IKBKE is also implicated in chemo-resistance in ovarian cancer, and siRNA knockdown of IKBKE increases sensitivity towards chemotherapy. However, the mechanistic role of IKBKE in chemo-resistance in ovarian cancer is not known. EphA2 is another well studied oncogene in ovarian cancer as 70% of ovarian cancer patients exhibit elevated levels of EphA2. By activating Focal Adhesion Kinases (FAK), EphA2 can induce metastasis in ovarian cancers. In this study, we show that the clinical PARP inhibitor Olaparib (AZD2281) activates IKBKE by EphA2-mediated tyrosine phosphorylation. We also found that phosphorylation of EphA2 or IKBKE expression can be used as a biomarker for olaparib resistance. Together, these studies have shed light on novel mechanisms of regulation of IKBKE and their importance in therapy resistance. These observations form a strong pre-clinical proof-of-concept to study the inhibitors further in the clinic