We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein: protein interaction (PPI) target classes.

Prostate cancer is a complex disease primarily characterized by dependence on androgen receptor (AR) signaling. Androgen deprivation therapy is efficacious; however, prostate tumors which initially respond to castration or androgen antagonists eventually progress. These castration-resistant tumors may develop hypersensitivity to low levels of androgens and AR overexpression or may adapt to rely on alternate signaling pathways such as the PI3K/mTOR axis. Deregulation of the PI3K/mTOR axis is a feature of prostate cancer, as evidenced by the fact that 40% of primary and 70% of metastatic prostate tumors exhibit loss of the tumor suppressor PTEN. Recent publications have shown that crosstalk between the AR and PTEN/PI3K/mTOR pathways plays a role in prostate cancer development and progression (1,2). Building upon these data, we evaluated the effects of ASP7486 (OSI-027), a selective inhibitor of mTORC1/mTORC2 as monotherapy and combined with bicalutamide, an androgen receptor antagonist. ASP7486 inhibited proliferation across a panel of prostate cancer cell lines, including those refractory to bicalutamide, supporting the hypothesis that tumors which have acquired androgen independence may rely upon mTOR signaling for survival. ASP7486 but not rapamycin, an allosteric mTORC1 inhibitor, induced apoptosis, implying that mTORC2 signaling is an important regulator of survival in prostate cancer. ASP7486, but not rapamycin, significantly upregulated AR expression which may serve to sensitize cells to the effect of an antiandrogen while limiting sensitivity to mTOR inhibition as a monotherapy. Consistent with this, the combination of ASP7486 and bicalutamide synergistically inhibited proliferation in vitro. In prostate tumors which have acquired resistance to androgen antagonists, activation of alternate RTKs may provide a survival mechanism in the absence of functional AR. PI3K pathway alterations and increased expression of IFG-1R are observed in castrate-resistant tumors (3,4). Treatment of prostate cancer cells with ASP7486 leads to increased phosphorylation of multiple RTKs, including IGF-1R and IR. We reasoned that ASP7486-mediated activation of IGF-1R and IR would sensitize these cells to the effects of OSI-906, a selective IGF-1R/IR inhibitor. The combination of ASP7486 and OSI-906, a selective inhibitor of IGF-1R/IR, synergistically inhibited proliferation and induced apoptosis. The combination of the two drugs provided greater inhibition of key signaling effectors than either monotherapy. These effects are not limited to prostate cancer. We have observed ASP7486-induced upregulation of RTK expression and phosphorylation in multiple tumor types, and the combination of ASP7486 and OSI-906 synergistically inhibited proliferation in the majority of cell lines tested. Together these data demonstrate that combinations of ASP7486 with targeted inhibitors can attenuate crosstalk between signaling networks and provide synergistic efficacy in vitro. The importance of AR and IGF-1R/PI3K/mTOR signaling in prostate cancer provides a rationale for targeting the disease and its intrinsic mechanisms of resistance with inhibitors of these pathways or in combination.

Resistance to targeted therapies is emerging as a major theme in cancer research. As more therapies become used routinely in the clinic it is apparent that although significant responses are observed the majority of patients progress while on therapy. Drug resistance can occur through a number of distinct mechanisms and no single mechanism can account for all the resistance that occurs in response to a particular therapy. These mechanisms include acquisition of secondary drug-resistant mutations within the target and activation of alternate prosurvival signaling pathways through compensatory signaling or epithelial to mesenchymal transitions (EMT). The ability of cancer cells to undergo an EMT has been implicated as a major factor driving metastasis, through the acquisition of enhanced migratory and invasive properties. However it is also clear that by undergoing this process the cancer cells become resistant to a number of targeted therapies. Recent retrospective analysis of phase 3 clinical trial samples has revealed that a poorer response to Erlotinib in the 2/3rd line setting in NSCLC was associated with a loss of E-cadherin (an epithelial tumor marker), suggesting that tumors that had undergone EMT were less responsive to EGFR-directed therapy. In addition an EMT phenotype has been reported in a number of EGFR-mutant NSCLC patients who have progressed while on erlotinib therapy. These clinical observations suggest that EMT plays an important role in mediating response to targeted therapy. In order to understand the full impact of these clinical observations and identify mechanisms of resistance in mesenchymal tumor cells we have modeled EMT in a number of different ways in vitro. We have used panels of NSCLC cell lines that are in a fixed epithelial or mesenchymal state, induced an EMT with TGFβ, or driven an EMT through prolonged exposure to EGFR-TKi targeted therapy (erlotinib). Using large-scale phosphoproteomic and transcriptomic datasets we used a systems biology approach to uncover important observations relating to the role of EMT as a drug-resistance mechanism. Firstly, these models confirm the clinical observations and show that tumor cells that have undergone EMT are less responsive to a number of targeted agents including EGFR and IGF1R-IR directed agents. Secondly, they reveal the plasticity of the EMT process where three distinct stages of EMT: epithelial, ‘metastable’ mesenchymal and ‘epigenetically-fixed’ mesenchymal are observed. Thirdly, upon undergoing EMT tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. These include receptor tyrosine kinase (RTK) autocrine and paracrine loops, such as PDGFR, FGFR, AXL and integrin α5β1 and up regulation of IL-6 and IL-11 mediated JAK-STAT signaling. Reciprocal activation of PDGFR signaling through EGFR inhibition was observed in the mesenchymal state. Lastly, these models indicate that as part of the EMT process the tumor cells display a CD44high/CD24low cancer stem cell phenotype and show enhanced colony formation. These observations reinforce the important role that EMT can have in driving drug resistance in tumor cells and highlight the wide diversity of mechanisms that can be used by tumor cells to evade targeted drug therapy. An understanding of these mechanisms and the contexts in which they are most likely to arise will have important implications in driving combinatorial drug therapy in cancer patients in the future.

Abstract The EGFR kinase inhibitor erlotinib is approved as a maintenance therapy in 1st line NSCLC as well as for treatment of 2nd/3rd line NSCLC and in combination with Gemcitabine for pancreatic cancer. It has been observed that the most pronounced responses to EGFR tyrosine kinase inhibitors (TKI's) were observed in patients whose tumors expressed a mutated form of the EGFR kinase. These mutations mapped to the kinase domain of the receptor and functionally have been shown to render tumors and cells lines onco-addicted to EGFR signaling. Although patients expressing a mutated EGFR show a dramatic initial response to EGFR TKI's, ∼50% of these patients will progress while on therapy after 1-2 years. The mechanisms that underlie this acquired resistance to EGFR TKI therapy have been intensively studied and include but are not limited to the presence of a second mutation, T790M, or increased HGF-MET signaling. Previously, the role of epithelial to mesenchymal transition (EMT) in resistance to EGFR kinase inhibitors has been described in the context of wild type EGFR. EMT. We were therefore interested in understanding whether EMT could play a role in resistance to EGFR TKIs in the context of an EGFR mutation. Here we show that a panel of NSCLC cell lines, expressing mutant EGFR, can undergo an EMT in response to TGFβ treatment. The cell lines take on a scattered and spindle-like morphology and also down regulate the expression of E-cadherin and up regulate expression of vimentin, classic protein markers of an EMT. Importantly we show that after undergoing EMT, the EGFR mutant line HCC827 has reduced sensitivity to erlotnib treatment which is regained after reversal of the EMT. To further explore whether EMT could play a role in acquired resistance to erlotinib, we generated in vitro cell line models that were resistant to EGFR inhibition through continued culturing in the presence of erlotinib over a 6 month period. Resistant clones generated from parental HCC4006 cells acquired a more scattered and spindle-like morphology consistent with an EMT. These clones had down-regulated E-cadherin and ErbB3 expression and upregulated vimentin, fibronectin and Zeb1 expression and also showed a gene expression pattern consistent with having undergone an EMT. In addition, the resistant H4006 clones were more migratory and invasive than their parental counterpart. Finally we show that the resistant clones are enriched for stem cell markers and have enhanced signaling through the Src family kinases and the JAK-STAT pathway suggesting a mechanistic rationale for their reduced sensitivity to EGFR inhibitors. Taken together. these data indicate that NSCLC cell lines that express a mutant version of EGFR are able to undergo an EMT which can influence the efficacy of EGFR TKIs, suggesting that this may be an additional mechanism underlying the acquired resistance of NSCLC patients to EGFR therapy in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2011-3370

Epithelial–mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and ‘epigentically-fixed’ EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell–cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49fhi/EpCAM-/lo and CD44hi/CD24lo, respectively.

The progression of cancer from non-metastatic to metastatic is the critical transition in the course of the disease. The epithelial to mesenchymal transition (EMT) is a mechanism by which tumor cells acquire characteristics that improve metastatic efficiency. Targeting EMT processes in patients is therefore a potential strategy to block the transition to metastatic cancer and improve patient outcome. To develop models of EMT applicable to in vitro and in vivo settings, we engineered NCI-H358 non-small cell lung carcinoma cells to inducibly express three well-established drivers of EMT: activated transforming growth factor β (aTGFβ), Snail or Zeb1. We characterized the morphological, molecular and phenotypic changes induced by each of the drivers and compared the different end-states of EMT between the models. Both in vitro and in vivo, induction of the transgenes Snail and Zeb1 resulted in downregulation of epithelial markers and upregulation of mesenchymal markers, and reduced the ability of the cells to proliferate. Induced autocrine expression of aTGFβ caused marker and phenotypic changes consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype. In vivo, this manifested as tumor cell infiltration of the surrounding mouse stromal tissue. Overall, Snail and Zeb1 were sufficient to induce EMT in the cells, but aTGFβ induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.

Abstract In oncology, the goal of personalized medicine is to improve patient outcome by tailoring therapy to the biochemical signaling within the individual's tumor. This requires identifying effective predictors of response that can be measured in biopsy material. High expression of the epithelial marker E-cadherin is associated with increased sensitivity in cultured cells and an improved survival benefit in response to erlotinib in patients. Downregulation of E-cadherin is a critical event in epithelial to mesenchymal transition (EMT) and is associated with decreased sensitivity to erlotinib. In order to determine if a more complete characterization of the EMT state would better predict sensitivity to erlotinib as well as other epithelial-targeting drugs, we developed an 88-gene signature that can be used to calculate a numerical index value which represents the EMT state of cells or tumors. In a panel of human tumor cell lines, the EMT index predicted erlotinib sensitivity correctly in 21 out of 24 lines. The index also predicted sensitivity to the IGF1R inhibitor OSI-906. While the EMT index marginally improved sensitivity prediction in vitro compared to E-cadherin, the numerical index allowed for a comparison of relative EMT states that was not possible with E-cadherin alone. Using a microarray database of human tumor sections, we computed EMT index values across 8 solid tumor types in order to compare their relative EMT states. Breast and lung tumors had a fairly even distribution between epithelial and mesenchymal tumors, while colon tumors were more epithelial, and kidney tumors were more mesenchymal. When comparing EMT index values to E-cadherin mRNA levels, some tumor types showed good agreement between E-cadherin and the EMT index (colon, kidney, lung) while others showed less agreement (breast, pancreas). We next compared our EMT signature to other published signatures of EMT and EGFR inhibitor response. Interestingly, most signatures were equally predictive of erlotinib sensitivity in vitro, even though less than 20% of the genes overlaped between any two signatures. We also analyzed signatures developed for individual characteristics of EMT such as invasion or stem cells. These did not correlate with erlotinib sensitivity, suggesting the protection from current therapeutics is not controlled solely by these specific aspects of EMT. Finally, we characterized sub-types of lung cancer for EMT state to determine if the EMT classification might correlate with established response rates to erlotinib. Adenocarcinomas, which tend to respond well, had more epithelial index values while squamous cell carcinomas, which tend to be less sensitive, had more mesenchymal values. This suggests that more epithelial index values might predict for erlotinib response in patients, potentially providing clinicians with a tool to more effectively treat patients based on the EMT status of their tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2011-5063

Abstract Erlotinib is a tyrosine kinase inhibitor of EGFR approved for use in non-small cell lung cancer (NSCLC) and pancreatic cancer in combination with Gemcitabine. Within the context of NSCLC it has been observed that patients harboring an activating mutation within the kinase domain of EGFR demonstrate a high response to treatment. However, their disease often progresses within 1 year. OSI-906 targets IGF-1R and IR receptor tyrosine kinases and has shown combination efficacy with erlotinib in preclinical models of NSCLC with wild type EGFR. Here we report preclinical data suggesting that the combination of erlotinib and OSI-906 is more efficacious in NSCLC models expressing an EGFR mutation than either single agent. A panel of NSCLC cells lines with confirmed mutations in the EGFR kinase domain was screened for erlotinib and OSI-906 single agent drug sensitivities. All mutant EGFR models exhibited sensitivity to EGFR inhibition by erlotinib (IC50 7–33 nM) while none showed sensitivity to OSI-906 (IC50 7gt;10 uM). In vitro, the combination of erlotinib and OSI-906 resulted in synergistic inhibition of cell proliferation and induction of apoptosis in PC3 (JCP-1) cells. Mechanistically we found the combination had enhanced inhibition of signaling through the PI3K/AKT pathway compared to treatment with either single agent. We also observed that while OSI-906 showed no induction of cell death, erlotinib treatment caused almost complete cell death. However after 9 days of erlotinib treatment small colonies of viable cells still remained. Removal of drug allowed the cells to grow and expand. When cells were treated with both erlotinib and OSI-906, complete cell death occurred without any evidence of cell regrowth following removal of both drugs. In order to evaluate this combination effect in vivo the EGFR mutant human tumor xenograft models NCI-H1650 and PC-14 were employed. Both lines are highly sensitive to erlotinib single agent treatment but show no anti-tumor activity with OSI-906 single agent treatment. The combination of 100 mg/kg erlotinib and 10 mg/kg OSI-906 demonstrated enhanced tumor growth delay when compared to either single agent dosed at MTD. However, as a first step towards understanding preclinical mechanism of this interaction we investigated the combination of 25 mg/kg of erlotinib with 30 mg/kg of OSI-906 and identified potential in vivo synergy. These studies with 25 mg/kg of erlotinib and 30 mg/kg of OSI-906 showed initial tumor regressions, enhanced tumor inhibition and substantially prolonged tumor growth delay when compared to either single agent. Drug-drug interaction PK studies suggest that OSI-906 is not acting to enhance erlotinib exposure. This data provides preclinical proof-of-concept for the use of OSI-906 in combination with erlotinib to obtain greater anti-tumor activity in the mutant EGFR setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-388. doi:10.1158/1538-7445.AM2011-LB-388

Abstract Multiple signal transduction pathways can be concurrently active within a single cell, and extensive crosstalk can occur between RTKs. Additionally, tumor tissues can be comprised of a heterogeneous collection of cell states utilizing distinct RTKs for maintenance of tumor cell growth and survival. As a consequence of this complexity, many tumors may be only partially sensitive to single agent therapies and would require the interdiction of multiple RTKs and other protein signaling targets for optimal anti-cancer therapy. Understanding pathway crosstalk is vital to guide the rational combination of approved and experimental anti-cancer agents. Receptor tyrosine kinases (RTKs) are key mediators of tumor cell survival, proliferation and migratory pathways, and inhibitors of RTKs have demonstrated anti-tumor efficacy in both the preclinical and clinical settings. Multiple sensitivity and resistance mechanisms have been described for EGF receptor inhibitors for the treatment of NSCLC. In carcinomas with an epithelial phenotype, onco-addiction and tumor progression have been associated with autocrine ligand-dependent activation of EGFR (1, 2). Notably, EGFR dependence can also occur through genetic mutations in exons 19-21 of the EGFR gene (3-5) which renders the encoded protein less able to bind ATP. Resistance can occur, primarily in these patients with these primary activating mutations, from acquisition of a secondary mutation of EGFR at the T790M gatekeeper site (6, 7) which increases ATP affinity and thereby decreases binding of ATP competitive inhibitors (8). Importantly, resistance can also occur through up regulation of alternate receptor tyrosine kinases (RTKs), and therefore understanding crosstalk between RTKs is critical for optimizing the use of RTK inhibitors in the clinic. Tumor tissues progress from in situ to metastatic states through the reacquisition of developmental programs allowing invasion and metastasis. The acquisition of an invasive phenotype can occur by epithelial-mesenchymal transition (EMT). The molecular characteristics of epithelial and mesenchymal cell phenotypes were extensively characterized by intersection of proteomic, phosphoproteomic and gene expression profiling approaches. Tumor cells that have undergone EMT show a marked reduction in sensitivity to EGFR TKIs and anti-EGFR MAbs (9). In several instances, the EMT-derived mesenchymal-like tumor cells have gained sensitivity to PDGFR and/or FGFR1 inhibitors. Significant switching of receptor tyrosine kinases, from EGFR, Met/Ron and IGF-1R to cells utilizing PDGFR and FGFR was observed. The acquisition of autocrine fibronectin - integrin was also observed in several tumor lines and inducible models. Therefore, EMT can promote use of alternative signaling pathways. Resistance can also derive from over activation of partially redundant pathways concurrently active in a cell. For example, activation of the IGF-1 receptor (IGF-1R) or the HGF receptor (Met) has been shown to obviate the need for EGFR signaling in epithelial-derived lung tumors (10). Crosstalk has been well documented with EGF, HGF and IGF1 receptors. These receptors, when activated by ligand binding, can create network redundancies (for example by IGF stimulation of EGFR onco-addicted cells; (11)). Reciprocal activation of one receptor following the inhibition of a distinct receptor has also been observed. For example PDGFRα was observed to be activated and substrates phosphorylated when EGFR was inhibited in the NSCLC line H1703(12). These studies involved a quantitative anti-phosphotyrosine profiling (13) coupled to an LC-MS/MS shotgun approach. In a second example in the Ewings sarcoma line A673, IGF-1R inhibition, by kinase inhibition resulted in the reciprocal activation of the insulin receptor and cell survival (14). We have previously shown reciprocal activation of EGFR by IGF1R inhibition and activation of IGF1R through EGFR inhibition (15). The mechanisms by which reciprocity is achieved are under investigation. The ability of tumor cells to reciprocally induce alternative RTKs following the inhibition of a given receptor (in this case EGFR or IGF1R) highlights the need for rational combination anti-cancer therapy. One RTK may also positively affect the activity of another in a process that can be termed receptor co-option. Here, one dominant ligand-stimulated RTK, possibly amplified or mutated, directly or indirectly tyrosine phosphorylates additional RTKs to create functional signaling scaffolds and to engage signaling networks beyond the normal capabilities of the original dominant kinase. Where the co-option of RTK signaling networks is observed, markers of RTK activation (e.g. the extent of receptor tyrosine phosphorylation) are not necessarily predictive of onco-addiction. Direct or indirect crosstalk between EGFR, ErbB2, Met, Ron and other RTKs has been observed in a bidirectional manner. Crosstalk can occur on the RON Y1238/Y1239 autophosphorylation site where MET and/or EGFR are the active kinases. Such phosphorylation in trans (either direct or indirect) may promote RON kinase activity in the absence of ligand. Alternatively or in addition, cross phosphorylation of RTKs in trans can allow signaling scaffolds to be established, mimicking kinase activation in the absence of ligand. Three NSCLC models illustrate co-option of RTKs as described below: The NSCLC line H1650 harbors a gain-of-function exon 19 deletion that is strongly onco-addicting in adenocarcinoma of the lung and is often associated with exquisite sensitivity to EGFR blockade. Pharmacological inhibition of EGFR in H1650 by the EGFR-ErbB2 inhibitor erlotinib results in a rapid dephosphorylation of ErbB2, Src family kinases and the direct and downstream substrates Cbl-B and Erk2 respectively. Interestingly attenuation of Met tyrosine phosphorylation is also observed suggesting that hyperactive mutant EGFR (del19) can also co-opt additional RTKs. In the NSCLC line H292, ~50% of the tyrosine phosphate on Met and on Ron is inhibitable by EGFR blockade. Crosstalk between EGFR, Met and Ron kinases was measured by combined anti-phosphotyrosine immunoaffinity selection, stable isotope peptide labeling (iTRAQ) and LC-electrospray tandem MS. The phosphotyrosine content on Met and Ron was decreased following exposure to erlotinib relative to mock control cells. The inhibition of phospho-Met and phospho-Ron followed kinetics for the erlotinib-dependent decrease in the SH2 adapter proteins phospho-SHC, -Erk2 and Grb2. The data suggest either direct phosphorylation of Met and Ron by EGFR or rapid recruitment and activation of intermediary non-transmembrane tyrosine kinases. These non-transmembrane tyrosine kinases could include Src/Yes/Fyn family kinases or Brk family kinases, possibly in a cell-specific manner, but this remains to be tested. A more extreme example of co-option of diverse kinase signaling networks by a single RTK is observed in the NSCLC H1993 cell line. Here exposure to small molecule Met tyrosine kinase inhibitors attenuated both Met and Met-associated SH2/PTB domain adapter proteins as well as more distantly related RTK signaling networks. Exposure of H1993 epithelial carcinoma cells to Met inhibitors markedly inhibited ~250 Met substrates, both known and unknown, including the cell surface signaling proteins Met, Ron, EGFR, ErbB2, DDR1, CSFR2, ITG4, ITG6, EphA2, EphB4. This in turn results in a comparatively complete dephosphorylation of a wide array of signaling adaptors, cell-cell junction proteins, cytoskeletal reorganizing elements and folding chaperones. While marked attenuation of EGFR was observed in response to Met inhibition alone, the combination of both Met and EGFR inhibitors was required for full dephosphorylation of both Erk1/2 and STAT5A. These findings suggest that while Met can co-opt EGFR, phosphorylate EGFR, and establish initial SH2/PTB domain dependent complexes on EGFR via active Met kinase, the extent of Met-dependent EGFR tyrosine phosphorylation is insufficient for full Erk and STAT5 activation. Several conclusions can be drawn. First, Met is the principle source of phosphotyrosine in H1993, and the cell line is likely onco-addicted solely to Met. Second, Met essentially ‘highjacks’ other RTKs as signaling adapters. Third, effective blockade of Erk activation was only observed with dual Met and EGFR inhibition suggesting synergy relies on erlotinib directed Erk inhibition in the H1993 model. The quantitative measurement of cancer cell signaling under dynamic conditions of pharmacological or siRNA mediated inhibition of specific signaling nodes gives insight into the requirements for effective cellular blockade of survival and invasive networks associated with cancer progression. The use of quantitative shotgun LC-MS/MS methods incorporating stable isotopes provides a rapid means to identify proteins and phosphoproteins perturbed in particular cancer cell states. These methods have allowed interrogation of RTK crosstalk in tumor cell lines and xenografts so as to generate specific hypotheses relating to targeted drug combinations. In addition such data have better defined the role of EMT in survival signaling switching and anticancer drug resistance, again suggesting combination therapy options for further investigation. References 1. Han et al Br J Cancer 2009. 2. Jacobs et al J Clin Oncol 2009. 3. Lynch et al N Engl J Med 2004. 4. Paez et al Science 2004. 5. Pao et al Proc Natl Acad Sci 2004. 6. Bell et al Nat Genet 2005. 7. Pao et al PLoS Med 2005. 8. Carey et al Cancer Res 2006. 9. Barr et al Clin Exp Metastasis 2008. 10. Engelman et al Science 2007. 1.1 Thelemann et al Mol Cell Proteomics 2005. 12. Thomson et al Clin Exp Metastasis 2008. 13. F. Petti et al Mol Cancer Ther 2005. 14. E. Buck manuscript submitted. 15. E. Buck et al Cancer Res 2008. Citation Format: John D. Haley, Stuart Thomson, James Bean, Ruixi Xie, David Epstein, Mark Miglarese, Liz Buck. Adaptive protein and phosphoprotein networks which promote therapeutic sensitivity or acquired resistance [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY01-01

Abstract The phenotypic changes associated with epithelial to mesenchymal transition (EMT) give tumor cells at least two attributes that increase metastatic efficiency. First, mesenchymal cells have lost cell-cell contacts and are more invasive and can therefore escape the primary tumor, whereas epithelial cells remain anchored by cell-cell contacts in the primary tumor. Second, mesenchymal cells in vitro are less sensitive than epithelial cells to agents that inhibit the EGFR pathway. This appears to translate into the clinic, since patients with tumors that express mesenchymal markers do not respond to EGFR targeting therapeutics as well as patients with more epithelial tumors. To determine whether we can control sensitivity to EGFR inhibitors by manipulating a cell's epithelial/mesenchymal status, we examined the role of EMT and its reversibility in tumor cell sensitivity to the EGFR inhibitor erlotinib. We studied in vitro models of EMT driven by Transforming Growth Factor β (TGFβ), Hepatocyte Growth Factor + Oncostatin M (HGF+OSM), Snail or Zeb1 in the H358 non-small cell lung carcinoma model. EMT driven by TGFβ or HGF+OSM caused a significant decrease in sensitivity to erlotinib, while EMT driven by induced expression of Snail or Zeb1 was much less effective, suggesting a change in drug sensitivity requires more input than these canonical EMT transcription factors. Once mesenchymal tumor cells have localized to a metastatic site, it is thought the tumor cells must undergo a mesenchymal to epithelial transition (MET) in order to form a cohesive tumor. We examined the reversibility of EMT in our models and the corresponding erlotinib sensitivity, both by withdrawal of ligand and by pharmacological inhibition of the signaling pathways downstream of the drivers. Using these approaches, we found EMT driven by HGF+OSM was reversible as evidenced by morphology and marker changes, and this reversion correlated with an increase in erlotinib sensitivity, comparable to the parental H358 cells. EMT driven by TGFβ was partially reversed by both ligand withdrawal and pharmacological inhibition of the receptor, and the extent of reversion in erlotinib sensitivity correlated with the extent of marker reversion. These results demonstrate that the extent of reversibility of EMT is dependent on the driver. Furthermore, it may be possible to drive some erlotinib-insensitive tumors back to a more erlotinib-sensitive state with pharmacological agents, thus improving patient response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1463.

Abstract Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR– insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR–directed Akt signaling, where all affect feedback loops converging at the level of IRS-1. [Cancer Res 2008;68(20):8322–32]

The EGFR pathway is a critical signaling pathway regulation cell proliferation and survival. As such, it is frequently deregulated in cancer through over expression of both EGF family ligands and receptors and by mutation of critical components within the pathway. These characteristics have made this signaling axis an attractive target for the development of molecularly targeted therapies in the treatment of cancer. To date there are numerous small molecule inhibitors and antibodies, either already in clinical use or in late stage clinical trials, that specifically target EGFR. These inhibitors have achieved great success in treating cancer patients and have generated a large amount of interest in identifying molecular markers that predict clinical benefit and mechanisms of resistance to such treatments. The first major breakthrough in this line of research was the identification of mutations in the EGFR kinase domain, which rendered the receptor hypersensitive to the actions of small molecule kinase inhibitors. However, the mutation rate was insufficient to explain the overall clinical benefit observed with these inhibitors, suggesting patients with wild-type EGFR also received some benefit. Subsequently, numerous efforts have been made to identify biomarkers of response and resistance other than EGFR mutational status. Here we will summarize the current literature describing attempts to identify such markers, with particular emphasis on markers of sensitivity and resistance to small molecule EGFR tyrosine kinase inhibitors (TKIs). These approaches have encompassed the analysis of expression levels, both at the protein and genomic level, of EGFR and the closely related family members HER2 and HER3 and the analysis of the mutational status of downstream components of the EGFR pathway. In addition, we will highlight the role of the epithelial to mesenchymal transition (EMT) in sensitivity to small molecule EGFR TKIs and finally the potential role of alternative signaling cascades as a mode of cellular resistance to EGFR inhibition.

NSCLC cells with a mesenchymal phenotype have shown a marked reduction in sensitivity to EGFR inhibitors, though the molecular rationale has remained obscure. Here we find that in mesenchymal-like tumor cells both tyrosine phosphorylation of EGFR, ErbB2, and ErbB3 signaling networks and expression of EGFR family ligands were decreased. While chronic activation of EGFR can promote an EMT-like transition, once having occurred EGFR family signaling was attenuated. We investigated the mechanisms by which mesenchymal-like cells bypass EGFR signaling and acquire alternative routes of proliferative and survival signaling. Mesenchymal-like NSCLC cells exhibit aberrant PDGFR and FGFR expression and autocrine signaling through these receptors can activate the MEK-ERK and PI3K pathways. Selective pharmacological inhibition of PDGFR or FGFR receptor tyrosine kinases reduced cell proliferation in mesenchymal-like but not epithelial NSCLC cell lines. A metastable, reversible EMT-like transition in the NSCLC line H358 was achieved by exogenous TGFbeta, which served as a model EMT system. The H358/TGFbeta cells showed many of the attributes of established mesenchymal-like NSCLC cells including a loss of cell-cell junctions, a loss of EGF-family ligand expression, a loss of ErbB3 expression, increased EGFR-independent Mek-Erk pathway activation and reduced sensitivity to EGFR inhibition. Notably an EMT-dependent acquisition of PDGFR, FGFR and TGFbeta receptors in H358/TGFbeta cells was also observed. In H358/TGFbeta cells both PDGFR and FGFR showed functional ligand stimulation of their intrinsic tyrosine kinase activities. The findings of kinase switching and acquired PDGFR and FGFR signaling suggest investigation of new inhibitor combinations to target NSCLC metastases.

1025 The epithelial-mesenchymal transition (EMT) can convert epithelial tumor cells to a more metastatic phenotype characterized by enhanced cell migration and invasion, increased resistance to anoikis and apoptosis, and greater resistance to chemotherapy or molecular targeted therapeutics. PAK1 was reported to phosphorylate Snail, a transcriptional repressor that acts as a master regulator of the epithelial-mesenchymal transition, on serine 246. Ser246 phosphorylation by PAK1 in MCF7 breast cancer cells was reported to be important for Snail translocation to the nucleus where it could repress target genes such as E-cadherin, aromatase, and occludin. Here we present supporting evidence for a positive role of both PAK1 and PAK2 kinases in a TGFβ-driven EMT model. We show that TGFβ treatment of the epithelial H358 NSCLC cell line results in downregulation of epithelial markers (E-cadherin, ErbB3) and upregulation of mesenchymal markers (N-cadherin, Snail, ZEB1). siRNA oligos targeting both PAK1 and PAK2 abolished many of the EMT marker changes induced by TGFβ, whereas siRNA oligos targeting only PAK1 or PAK2 alone were insufficient to suppress these EMT marker changes. Importantly this effect was correlated with the inhibition of Snail phosphorylation on Ser246, translocation to the nuclear compartment, and protein accumulation. In addition, expression of the PAK1 auto-inhibitory domain (AID), which acts as a dominant negative inhibitor of both PAK1 and PAK2 function, was demonstrated to inhibit Snail phosphorylation and nuclear localization in HeLa cells. These findings indicate that dual inhibition of PAK1 and PAK2 may be a promising approach to inhibit Snail-mediated EMT for treatment of cancer.

A128 Both antibody and small molecule inhibitors of the epidermal growth factor receptor (EGFR) are currently clinically approved. Erlotinib, a low molecular weight kinase domain inhibitor, is approved for advanced NSCLC and pancreatic cancers, and cetuximab, a neutralizing antibody of EGFR, is approved for colorectal and head and neck cancers. Although both classes of therapeutics are specific inhibitors of EGFR, their differing modes of inhibition have been shown to convey differential effects on tumor cell growth and survival. In vitro studies have shown that neutralizing antibodies against EGFR produce less pronounced tumor cell growth inhibition compared with small molecule kinase domain inhibitors. The efficacy for neutralizing antibodies against EGFR observed in vivo and in the clinic has been proposed to be augmented, at least in part, by the ability of the antibody to recruit immune components. Herein we sought to determine the differential effects on cellular signal transduction for erlotinib compared with the EGFR neutralizing antibody C225 in pancreatic and NSCLC tumor cells. Previous reports have shown that EGFR mediated activation of Akt requires transactivation of HER3 for NSCLC, and we have recently extended these observations for pancreatic and colorectal tumors. Specifically, we have shown that HER3 mediates EGFR-directed Akt activity, but not MAPK activity, for epithelial tumor cells but not for tumor cells that have undergone an epithelial-mesenchymal-transition. In our current study we show that both erlotinib and C225 achieve similar inhibition of EGFR phosphorylation over a 24 hour period for a panel comprised of pancreatic (BxPC3) and NSCLC (NCI-H292 and NCI-H358) epithelial tumor cell lines. Both classes of inhibitors also similarly inhibited activity within the MAPK pathway, as assessed by the extent of inhibition of Erk phosphorylation. However, erlotinib differed from C225 in its ability to affect signaling through the EGFR-HER3-Akt cascade. Whereas erlotinib could potently inhibit EGFR-directed HER3 activity, C225 did not show inhibition of HER3. Consistent with the role of HER3 in conveying EGFR-directed Akt activity, only erlotinib and not C225 could inhibit Akt activity and downstream activation of S6 in a BxPC3 model. Several studies have presented data suggestive of ligand independent activation and propagation of EGFR signaling, and we hypothesize that EGFR transactivation of HER3 may be independent of ligand binding and insensitive to cetuximab. Collectively, these results suggest that the different, but complementary growth inhibitory mechanisms employed by small molecule kinase inhibitors and anti-EGFR antibodies could be combined to yield greater efficacy.