Ines Pulido

Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition

Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy.

Abstract C75: Overcoming KRAS/LKB1 mutant NSCLC resistance to BET bromodomain inhibitors with gemcitabine or Mcl-1 inhibition

The purpose of our study was to define a method and mechanism for overcoming the resistance of clinically relevant KRAS-mutant/LKB1-deficient NSCLC cells to the BET-bromodomain inhibitor JQ1. LKB1 (Serine/threonine kinase 11) is mutated with loss of function in conjunction with mutated KRAS in 7-10% of NSCLC. Importantly, KRAS-mutant/LKB1-deficiency is associated with tumor aggressiveness and poor survival in human patients as well as in genetically engineered mouse models. Indeed, although the BET bromodomain inhibitor JQ1 dramatically reduces tumor volume in KRAS mutant mice, it has little effect in KRAS-mutant/LKB1-deficient mice. BET bromodomain proteins are chromatin readers that facilitate binding and activity of transcription factors such as the oncogene MYC. As such, JQ1 inhibits the transcriptional program of MYC and decreases MYC in mice. To overcome JQ1 resistance in KRAS-mutant/LKB1-deficient NSCLC, we proposed to test JQ1 combination with chemotherapy, as such a combination would be a necessary arm of a clinical trial testing a BET bromodomain inhibitor. Here, we present data showing that gemcitabine permits and potentiates JQ1-mediated apoptosis of multiple KRAS-mutant/LKB1-deficient human NSCLC cell lines above either monotherapy. Increased apoptosis was observed by AnnexinV and TUNEL flow cytometry, PARP cleavage, and Comet assays. Mechanistically, the drug combination had minimal effects on DNA damage and repair molecules in the KRAS-mutant/LKB1 deficient NSCLC cell line A549. Interestingly, however, JQ1 and combination with gemcitabine had profound effects on apoptotic molecules. JQ1 alone showed a robust increase in pro-apoptotic BimEL, BimL, and BimS that was balanced by a large increase in anti-apoptotic Mcl-1 24 hrs post-treatment. Similarly, MYC knockdown also increased Mcl-1, suggesting that JQ1 upregulation of Mcl-1 was at least partly dependent on JQ1 suppression of MYC. On the other hand, JQ1 combination with gemcitabine showed an increase in Bim that was not associated with increased Mcl-1. Given these findings, we reasoned that KRAS-mutant/LKB1-deficient cells were resistant to JQ1-induced apoptosis executed by Bim because of protection provided by increased Mcl-1. In support of this hypothesis, Bim knockdown prevented JQ1-induced PARP cleavage in JQ1-sensitive H441 cells. Furthermore, Mcl-1 knockdown cells showed increased Bim, decreased Bcl-2, and increased PARP cleavage 48 hrs after JQ1 treatment in otherwise JQ1-resistant A549 cells. Collectively, KRAS-mutant/LKB1-deficient cells are resistant to JQ1-induced apoptosis because of a compensatory increase in Mcl-1. This resistance can be overcome by combinatorial treatment with gemcitabine or direct Mcl-1 suppression, restoring apoptosis. Therefore, combinatorial treatment of a BET bromodomain inhibitor with gemcitabine or a Mcl-1 inhibitor may represent a potential novel strategy for treating the clinically relevant KRAS-mutant/LKB1-deficient subtype of NSCLC. Citation Format: Michael Kahle, Margaret Soucheray, Jeffrey Becker, Eiki Kikuchi, Ines Pulido, Esra Akbay, Camilla Christensen, Wei Qiu, Fatima Al-Shahrour, Neil Johnson, Julian Carretero, Kwok-Kin Wong, Takeshi Shimamura. Overcoming KRAS/LKB1 mutant NSCLC resistance to BET bromodomain inhibitors with gemcitabine or Mcl-1 inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C75.

Abstract LB-C21: CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC

Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response, acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we have engineered EGFR-mutated NSCLC cell lines with mesenchymal phenotype by stably depleting E-Cadherin or by overexpressing Snail or chronically exposing the cells to TGFβ1. The resulting mesenchymal cells are resistant to EGFR TKIs. We employed genomic analyses to identify that C-X-C chemokine receptor type 7 (CXCR7) is commonly overexpressed in the engineered cells with mesenchymal phenotype. We also discovered that CXCR7 is overexpressed in EGFR-mutated HCC4006 NSCLC cells grown resistant to gefitinib that developed a mesenchymal phenotype (HCC4006 Ge-R). To extend our findings to in vivo, we assessed if CXCR7 is overexpressed in mouse lung cancers driven by human EGFR exon19 deletion/T790M (TD) that initially respond to the EGFR mutant-specific irreversible TKI WZ4002 to promote tumor regression but later develops tumors with acquired resistance. We discovered that the murine tumors with acquired resistance to WZ4002 present mesenchymal phenotype and overexpress CXCR7. Stable depletion of CXCR7 in HCC4006Ge-R mesenchymal gefitinib resistant cells promoted gradual mesenchymal to epithelial transition. Sustained depletion of CXCR7 in HCC4006Ge-R cells resulted in inactivation of PI3K and MAPK pathways upon gefitinib treatment and greatly restored the sensitivity to gefitinib. Furthermore, the depletion of CXCR7 in HCC4006GeR cells resulted in the downregulation of mesenchymal transcription factors essential including TWIST, ZEB1 and ZEB2, suggesting but not proving that CXCR7 maintains mesenchymal phenotype. To determine if the resistance mechanisms to gefitinib with mesenchymal cells expressing CXCR7 evolve under the selective pressure of gefitinib or pre-exist prior to treatment, we sorted EGFR-mutated NSCLC cells with CXCR7 and mesenchymal markers to find less than 5% of the cells express CXCR7 with mesenchymal phenotype. Interestingly, ectopic expression of CXCR7 in EGFR-mutated cells was not sufficient to confer resistance to EGFR TKIs or to promote EMT. Taken together, we discovered that CXCR7 is necessary for the maintenance of EGFR TKI resistance with mesenchymal phenotype and the population of mesenchymal cells that overexpress CXCR7 pre-exists and is selected upon chronic EGFR TKI treatment. Citation Format: Jeffrey H. Becker, Yandi Gao, Ines Pulido, Eiki Kikuchi, Margaret Soucheray, Rutu Gandhi, Camilla L. Christensen, Fatima Al-shahrour, Kwok-Kin Wong, Julian Carretero, Takeshi Shimamura. CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C21.

Abstract 753: Genomic alterations of autophagy genes disrupts autophagic flux in human lung adenocarcinomas

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Targeted therapy using EGFR tyrosine kinase inhibitor (TKI) is a standard therapy for a subset of non-small cell lung cancer (NSCLC) patients with lung adenocarcinomas (LADs) harboring EGFR kinase domain mutations; however, EGFR TKI therapy shows limited efficacy due to de novo and acquired resistance. Consequently, formulating strategies to potentiate the efficacy of EGFR TKI is of great interest. In EGFR TKI sensitive cells harboring EGFR mutation, it has been shown that EGFR inhibition induces autophagy to protect the cells from metabolic stress. Hydroxychloroquine (HQ), an inhibitor of autophagy, has been shown to potentiate EGFR TKIs in preclinical models, however, preliminary results from recent phase II clinical trials shows no added benefits to the development of resistance to erlotinib in patients with EGFR mutant NSCLC. The result suggests additional layers of mechanisms in controlling autophagy in EGFR mutated NSCLC. Furthermore, little is known about genomic alterations affecting these autophagy genes in lung cancer samples. The meta-analysis of The Cancer Genome Atlas (TCGA) database shows that essential genes for autophagic flux such as ATG5 and ATG7 tend to reduce their expression levels and/or are deleted in a significant proportion of LAD patients, suggesting a more complex scenario where the presence of these genetic alterations may lead to a structurally deficient autophagy. Besides, the detailed CNV analysis of the Cancer Cell Line Encyclopedia (CCLE) dataset suggests that a small subset of NSCLC cell lines may have lost ATG7 locus at chromosome 3. Our validation study showed that, as expected, EGFR-mutant H1650 cells exhibit an intragenic deletion at ATG7 and lack of protein expression. Moreover, functional assays of autophagic flow by western blot and confocal microscopy demonstrated that ATG7-deficient H1650 cells are not able to activate autophagy, whereas infection with lentivirus expressing ectopic ATG7 reconstituted autophagic flux and sensitivity to autophagy inhibitors including HQ. Taken together, our results suggest that genomic biomarkers based on autophagy genes could allow stratification of tumors, and selection of those candidates who could benefit from anti-autophagy therapy. Citation Format: Ines Pulido, Juan L. Pascual, Margaret Soucheray, Maria L. Rodriguez, Daniel T. Crespo, Salvador Aparisi, Joan A. Sirerol, Salvador Mena, Javier Pereda, Fatima Al-shahrour, Angel L. Ortega, Takeshi Shimamura, Julian Carretero. Genomic alterations of autophagy genes disrupts autophagic flux in human lung adenocarcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 753. doi:10.1158/1538-7445.AM2015-753

Abstract 766: Suppression of gefitinib-induced EMT in EGFR mutant NSCLC preferentially selects for acquired T790M

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we attempted to develop a strategy to prevent the emergence of EGFR TKI resistance with EMT phenotype. In order to mimic the development of acquired EGFR TKI resistance in NSCLC patients, TKI-sensitive HCC4006 cells harboring mutated-EGFR were exposed to increasing concentrations of gefitinib to generate resistant cells with mesenchymal phenotype. After 6 months, the cells became resistant to gefitinib (HCC4006GeR) with no MET copy number increase and with no apparent gain of secondary T790M mutation. Subsequent genomics and proteomics analyses of HCC4006GeR confirmed enrichment of genes distinctive to mesenchymal cells. Multiplex Luminex growth factor assays identified increased secretion of TGFβ1 from HCC4006Ge-R cells. We discovered that the depletion of EGFR by shRNA or inactivation of mutated EGFR activity by EGFR TKI promoted TGFβ1 secretion and subsequent induction of EMT, which modulate signaling and apoptosis pathways contributing to the development of the resistance. Consequently, we hypothesized that concurrent inhibition of EGFR and TGFβ receptor in HCC4006 cells should prevent the emergence of gefitinib resistance with mesenchymal phenotype. After culturing HCC4006 cells in EGFR (gefitinib) and TGFβ receptor (SB431542) inhibitors, we successfully prevented EMT, although those cells were still resistant to gefitinib (HCC4006GeSB-R). Interestingly, the cells remain sensitive to irreversible EGFR TKI AZD9291 and CO-1686, suggesting the presence of T790M mutation. DNA sequencing of EGFR expressed in HCC4006GeSB-R detected secondary T790M mutation. Droplet digital PCR analysis detected that the frequencies of EGFR allele coding for T790M in HCC4006Ge-R and HCC4006GeSB-R cells are 1.2% and 18.3%, respectively, which were increased from 0.015% in HCC4006 cells harboring heterozygous Del L747-E749+A750P/T790M mutation. The frequency of T790M allele in HCC4006 was higher than the frequency in PC9 cells (0.001%) which reproducibly develop EGFR T790M as a mechanism of EGFR TKI resistance. Taken together, we discovered that suppression of gefitinib-induced EMT in EGFR mutant HCC4006 NSCLC cells preferentially selects for the previously unreported and rare subpopulation of HCC4006 harboring T790M. These results also underscore heterogeneity within HCC4006 cells that give rise to the divergent resistance mechanisms according to treatment. Citation Format: Margaret Soucheray, Marzia Capelletti, Ines Pulido, Yunan Kuang, Cloud P. Paweletz, Jeffrey H. Becker, Eiki Kikuchi, Chunxiao Xu, Tarun B. Patel, Fatima Al-shahrour, Julian Carretero, Kwok-Kin Wong, Pasi A. Janne, Geoffrey I. Shapiro, Takeshi Shimamura. Suppression of gefitinib-induced EMT in EGFR mutant NSCLC preferentially selects for acquired T790M. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2015-766

Abstract 1605: Pterostilbene, a natural phytoalexin, effectively protects against UVB-induced skin carcinogenesis by increasing antioxidant cellular defenses and preventing mutagenesis

Clinical and laboratory studies have demonstrated that skin exposure to ultraviolet radiation (UV) is the main cause of non-melanoma skin cancer (NMSC) (≈ 99%) and melanoma (≈ 95%) development. The procarcinogenic effects of solar exposure are mainly due to UVB radiation (290-320 nm range), the same tight range that produces burning in human skin (erythema), inflammation, oxidative stress, DNA damage, etc. The number of skin cancers around the world keeps increasing and, thus, it is an urgent need to find effective protection remedies. Phytoalexins of polyphenolic structure are naturally occurring compounds involved in the defense against pathogens and environmental stresses in plants. Epidemiological studies confirm that diets enriched in plant polyphenols may offer protection against chronic diseases related to oxidative stress. In this sense polyphenols have shown anticarcinogenic and photoprotective effects (e.g. Clifford JL & DiGiovanni J. Cancer Prev Res 3:132-5, 2010). Seminal studies showed that resveratrol (trans-3,5,4′-trihydroxystilbene; Resv) exerted chemopreventive activity in a model of mouse skin carcinogenesis where topic administration of this polyphenol inhibited multistage carcinogenesis [Jang M et al. Science 275:218-20, 1997]. Pterostilbene (3,5-dimethoxy-4′-hydroxy-trans-stilbene; Pter), a Res analog, shows a higher half-life and more potent anticancer effects in vivo than Res. We compared the potential anti-UVB protection of both polyphenols in a murine model of chronic irradiation (180 mJ UVB/cm2; 3 doses/week; for a total of 30 weeks) that experimentally resembles long-term chronic human exposure to damaging sun radiations. We observed that topical pretreatment of the skin with Pter (1-2 μmol/cm2 per dose, administered right before UB exposure), but not with Resv, fully prevents chronic UVB-induced skin carcinogenesis. The molecular mechanism underlying this protective effect involves a Pter-induced increase in antioxidant skin defenses and a decrease in the rate of mutagenesis. Citation Format: J. Antoni Sirerol, Ines Pulido, Miguel Asensi, Angel Ortega, Jose M. Estrela. Pterostilbene, a natural phytoalexin, effectively protects against UVB-induced skin carcinogenesis by increasing antioxidant cellular defenses and preventing mutagenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2014-1605

Abstract A292: Salinomycin, an anti-cancer stem cell antibiotic, overcomes acquired resistance to BRAF inhibitors in BRAF-mutant human melanoma cell lines.

Advanced malignant melanoma is one of the most lethal cancers, because it is highly metastatic and refractory to conventional chemotherapy. About 60% of melanomas harbor oncogenic BRAF mutations which aberrantly activate MEK/ERK signaling pathway. BRAF and MEK inhibitors have been shown efficacious in patients with BRAF-mutant melanoma, but there is not effective target therapy for BRAF wild type melanomas. Unfortunately acquired resistance to BRAF targeted therapies is a common event: 50% of treated patients progressed within 6 to 7 months after the initiation of treatment. Resistance is associated with reactivation of the MAPK pathway (through development of de novo NRAS, NF1 or MEK mutations) or activation of parallel pathways, such as the PI3K/AKT pathway. Cancer stem cells (CSCs) represent a subpopulation of tumor cells that possess self-renewal and tumor initiation capacity. It has been postulated that CSC could be linked to acquired resistance to both conventional and targeted therapies. Salinomycin, an ionophore antibiotic, has been shown to selectively kill CSCs in several types of human cancers, most likely by interfering with ABC drug transporters, the Wnt/β-catenin signaling pathway, and other CSC pathways. In this study, we have analyzed a wide panel of human melanoma cell lines with a known mutational status of BRAF, tumor suppressors, and other frequently mutated oncogenes in melanoma, and their sensibility to several protein kinase inhibitors (PKIs) in vitro, including the BRAF inhibitor vemurafenib. We have combined these PKIs with the CSC inhibitor, salinomycin, in order to find therapeutically relevant synergies. To better characterize CSC phenotype, we carried out tumorsphere formation assays in vitro, FACS analysis of CSC canonical markers, to find that salinomycin sensitizes BRAF-wild type melanoma cell lines to the cytotoxic effect of particular PKIs. Importantly, we demonstrated that salinomycin-PKI combination therapy supresses the emergence of vemurafenib-resistant subpopulations in BRAF-mutant melanoma cell lines. Overall, we find that targeting CSC property of wild-type BRAF inhibitor with salinomycin significantly potentiates therapeutic activity of PKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A292. Citation Format: Daniel Crespo, Ines Pulido, Maria L. Rodriguez, Salvador Aparisi, Alejandro Lopez, Margaret Soucheray, Fatima Al-Shahrour, Takeshi Shimamura, Angel Ortega, Julian Carretero. Salinomycin, an anti-cancer stem cell antibiotic, overcomes acquired resistance to BRAF inhibitors in BRAF-mutant human melanoma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A292.

Abstract 1280: Pterostilbene exerts full protection against UVB-induced skin carcinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epidemiological, clinical and laboratory studies have demonstrated that solar ultraviolet (UV) radiation is the main cause of nonmelanoma skin cancer (i.e., basal cell and squamous cell carcinoma) and it is also a prime factor in the etiology of cutaneous melanoma. The cancer-causing effects of solar exposure on the skin are mainly produced by UV-B radiation in the 290- to 320-nm range, the same range that produces burning in human skin (erythema), inflammation, oxidative stress, DNA damage, etc. Thus, and considering the alarming numbers of skin cancers being diagnosed around the world, it is increasingly evident the need of an effective protection against UV radiation. Polyphenols (PFs) are abundant in many fruits and vegetables, wine, tea, and various dietary supplements. The role of these natural molecules as potential anticarcinogens and photoprotectors has been postulated (e.g. Clifford JL & DiGiovanni J. Cancer Prev Res 3: 132-5, 2010). Resveratrol (trans-3,5,4′-trihydroxystilbene; RES) is a phytoalexin present in a wide variety of plant species, where its synthesis is induced by stress conditions. The cancer chemopreventive activity of RES was first reported by Jang et al [Science 275, 218 - 220 (1997)] in a model of skin carcinogenesis where topic administration of this polyphenol inhibited multistage mouse skin carcinogenesis. Equally promising action is exerted by resveratrol analogues, mainly pterostilbene (3,5-dimethoxy-4′-hydroxy-transstilbene: PTER), which shows a higher half-life and more potent anticancer effects in vivo than RES. The long-term UV-B administration (180 mJ/cm2; 3 doses/week; for a total of 30 weeks) reproduces, in an animal model, which are the consequences in humans of receiving chronic UV-B radiations. We observed that pretreatment of the skin with PTER prevented UV-B-induced skin tumorigenesis (∼90 % of tumor free-mice at the end of treatment, n=20; P<0.01). Our aim was to study the key molecular mechanisms involved in this strong anticancer effect elicited by PTER. Our results indicate that PTER efficiently avoid skin carcinogenesis induced by solar radiation by decreasing DNA mutagenesis, and protein and lipid oxidation. Citation Format: Angel Ortega, Maria L. Rodriguez, Joan A. Sirerol, Ines Pulido, Daniel Crespo, Julian Carretero, Miguel A. Asensi, Salvador Mena, Jose M. Estrela. Pterostilbene exerts full protection against UVB-induced skin carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1280. doi:10.1158/1538-7445.AM2013-1280

Abstract 1690: Differential autophagy activation in KRAS and EGFR mutant lung adenocarcinomas.

Lung cancer is the leading cause of cancer deaths in western countries, and adenocarcinomas (LADs) are the most frequent histological subtype. The aberrant activation of the kinases promotes plethora of tumorigenic processes, mainly through PI3K and MAPK oncogenic pathways leading to oncogene addiction. The activation of PI3K pathway deregulates mTOR, a master kinase for cell growth and autophagy. Autophagy can be pro- or anti- tumorigenic, however its roles in protecting tumors exposed to metabolic stress under chemotherapy are considered as a survival mechanism for the tumors leading to acquired resistance. Consequently, the inhibition of autophagy is an attractive therapy to prevent the emergence of acquired resistance. Activating mutations in EGFR and KRAS are mutually exclusive and are the most frequent oncogene activation in LAD. There are considerable differences in the control of signalling pathways including PI3K-mTOR axis. Therefore, it is hypothesized that the susceptibility to autophagy differs depending on EGFR or KRAS mutational status. To test the hypothesis, we applied nutrient-starvation conditions, the most well-known inducer of autophagy, to a panel of LAD cell lines bearing known EGFR or KRAS mutations. As a positive control, cells were treated with rapamycin, which also induced autophagy inhibiting MTOR activity. Autophagy activation was analyzed by: i) Western blot of LC3-II and p62/SQSTM1 protein levels, that measures autophagy flux and autophagic protein degradation, respectively; and ii) autophagolysosome detection and lysosomal activity by fluorescent microscopy. Our data suggest that KRAS-mutant LAD cell lines are able to activate autophagy, whereas EGFR-mutant are not, under starvation stress. Furthermore, we found a potential interaction between mutant EGFR and Beclin 1 (BECN1, a protein required for the initiation of the autophagosome) at the cell membrane proximity, using co-immunolocalization by confocal microscopy and immunoprecipitation followed by Western blot. Together, these data provide a compelling rationale to investigate anti-autophagy therapy in mutant KRAS LADs and warrant further investigation in the regulation of autophagy via a novel link between Beclin1 and mutant EGFR. Citation Format: Maria L. Rodriguez, Ines Pulido, Margaret Soucheray, Daniel Crespo, Fatima Al-shahrour, Takeshi Shimamura, Angel Ortega, Julian Carretero. Differential autophagy activation in KRAS and EGFR mutant lung adenocarcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1690. doi:10.1158/1538-7445.AM2013-1690