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J. Minna
Moderator of
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 14
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.01 - Inhibiting Tankyrase Prevents Epithelial-To-Mesenchymal Transition and Synergizes with EGFR-Inhibition in Wnt-Dependent NSCLC Lines (ID 2850)
16:45 - 18:15 | Author(s): H.A. Scarborough, B.A. Helfrich, M. Casas-Selves, Z. Zhang, D.C. Chan, A. Schuller, S. Grosskurth, P.A. Bunn, Jr, J.V. DeGregori
- Abstract
Background:
Despite their promise, therapies targeting driver receptor tyrosine kinases (RTKs) rarely produce complete responses and have shown modest clinical benefit in NSCLC. This suggests the presence of escape mechanisms that allow cells to survive and proliferate despite inhibition of an oncogenic driver.
Methods:
Using a genome-wide shRNA screen, we identified that the canonical Wnt/β-catenin pathway contributes to the survival of NSCLC cells during inhibition of the epidermal growth factor receptor (EGFR). In order to evaluate the effects of inhibiting the Wnt pathway on EGFR-inhibited cells, we categorized NSCLC cell lines as “Wnt-responsive” or “Wnt-non-responsive” based on their ability to upregulate β-catenin-dependent targets in response to treatment with exogenous Wnt3a. Using both shRNA knockdown and a novel tankyrase inhibitor, AZ1366, we evaluated the ability of tankyrase inhibition to synergize with EGFR-inhibition in multiple Wnt-responsive and Wnt-non-responsive cell lines. We then evaluated the effects of the combination of gefitinib and AZ1366 on the survival and tumor progression in an orthotopic mouse model. In order to comprehensively query transcriptional changes brought about by treatment, we performed RNA-seq on cells treated with gefitinib, AZ1366, or the combination of the two drugs.
Results:
We have demonstrated that inhibition of tankyrase, a key player in the canonical Wnt pathway, significantly increases the induction of senescense and/or apoptosis mediated by EGFR-inhibitors in cell lines with a Wnt-responsive phenotype, and that the ability of the tankyrase inhibitor to synergistically eliminate NSCLC cells is dependent on its actions within the canonical Wnt pathway. In Wnt-non-responsive cell lines, tankyrase inhibition did not synergize with inhibition of EGFR. We have further demonstrated that Wnt-responsive cell lines show evidence of EMT in response to Wnt ligand stimulation, and that this can be prevented with tankyrase-inhibitor treatment. Additionally, we have shown that mice orthotopically implanted with Wnt-responsive cell lines and treated with a combination of a tankyrase inhibitor and an EGFR inhibitor have a substantially reduced tumor burden and a significant improvement in survival when compared to treatment with an EGFR inhibitor alone. When Wnt-non-responsive cell lines were used, we noted no improvement in survival or reduction in tumor burden. RNA-seq analysis revealed that while most transcriptional changes present in the combination were driven by gefitinib, AZ1366 had the effect of significantly amplifying many of the changes thought to be instrumental in resistance to EGFR inhibition including increased expression of TP53 and apoptosis signaling machinery, increased expression of NF-kB signaling components, and a strong decrease in cell cycle drivers. Furthermore, treatment with AZ1366 alone resulted in decreased expression of Axl and its ligand, Gas6, a known mechanism of resistance to EGFR inhibition.
Conclusion:
Taken together, these results indicate that tankyrase inhibition impinges on multiple mechanisms of escape from EGFR-inhibition, and that its ability to synergize with EGFR-inhibition is dependent on its actions within the canonical Wnt pathway. As the goal of these studies is the development of combination therapies with EGFR inhibition, this suggest tankyrase as a promising target in the subset of NSCLC with known dependencies on signaling through the canonical Wnt pathway.
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MINI09.02 - Transcriptome-Metabolome Reprogramming of EGFR-Mutant NSCLC Contributes to Early Adaptive Drug-Escape via BCL-xL Mitochondrial Priming (ID 3085)
16:45 - 18:15 | Author(s): P.C. Ma, P. Thiagarajan, W. Zhang, X. Wu, P. Leahy, I. Shi, Y. Feng, M.L. Veigl, D. Lindner, D. Danielpour, L. Yin, Z. Zhang, R. Rosell, T. Bivona
- Abstract
Background:
Precision therapy using EGFR small molecular inhibitors is the current standard-of-care in treatment of advanced non-small cell lung cancer (NSCLC) patients (pts) with EGFR mutations. Nonetheless, emergence of acquired resistance to therapy invariably occurs despite effective initial response. Classical rebiopsy studies of EGFR-mutant pts at clinical tumor progression based on RECIST criteria have identified diverse resistance mechanisms involving T790M-EGFR, MET amplification or activation and AXL upregulation. Tumor cells within minimal or microscopic residual disease during drug response may constitute founder cells for future disease relapse. The mechanisms of molecular changes intrinsic to these early therapeutic survivors are not yet well-understood. Our studies focus on tumor cells adaptation early during therapy to map the initial course of molecular drug resistance emergence and evolution.
Methods:
Drug-sensitive model studies of EGFR-mutant lung cancer were performed using HCC827 and PC-9 cells (exon 19 deletions EGFR) under erlotinib, and H1975 (T790M/L858R-EGFR) cells under CL-387,785 inhibition. Affymetrix microarray profiling was performed in triplicate at 0h, 8h, 9d and 9d tyrosine kinase inhibitor (TKI) followed by 7d drug-washout. Both in vitro and in vivo xenograft analyses, immunofluorescence, immunohistochemistry, time-lapse video microscopy analysis were conducted. Mass-spectrometry based global metabolomics profiling was also conducted under similar conditions as in gene expression profiling.
Results:
We identified an early adaptive and reversible drug-escape within EGFR-mutant cells that could emerge as early as 9 days during course of effective therapy with molecular drug resistance. Principal component analysis (PCA) of gene expression profiling data identified distinct transcriptome signatures in each cell state. Of note, the prosurvival cell state was independent of MET pathway activation, and had a TKI cytotoxicity escape at 100x higher IC50. The drug resistant cell state was associated with reversible cellular quiescence, suppressed Ki-67 expression, and profoundly inhibited cellular motility and migration. Transcriptome gene expression profiling revealed a remarkable adaptive genome-wide signature reprogramming, centered on the autocrine TGFβ2 cascade that involved pathways of cell adhesion, cell cycle regulation, cell division, glycolysis, and gluconeogenesis. Global metabolomic profiling of cellular metabolites in HCC827 cells under erlotinib inhibition also revealed a concurrent adaptive reprogramming of cellular metabolism during the early drug-resistant cell state, with suppression of glycolysis, TCA cycle, amino acids metabolism, and lipid bioenergetics. Our studies identified a direct link of TGFβ2 within the drug escaping cells, with the metabolic-bioenergetics quiescence, reverse Warburg metabolism and mitochondrial BCL-2/BCL-xL priming. Furthermore, this adaptive drug-resistant cell state also displayed an increased EMT and cancer stem cell signaling as adaptation to the drug treatment and that could be overcome by broad BCL-2/BCL-xL BH3 mimetic ABT-263, but not BCL-2 only mimetic ABT-199.
Conclusion:
We identified and characterized the emergence of early adaptive drug-escape within EGFR-mutant NSCLC cells amid an overall precision therapy excellent response, through a MET-independent mechanism. The profoundly drug-resisting prosurvival cell state undertook remarkable cellular transcriptome-metabolome adaptive reprogramming coorindated through autocrine TGFβ2 signaling augmentation. Our study results have important implications in lung cancer drug-resistant minimal/microscopic disease and future therapeutic remedies in precision therapy.
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- Abstract
- Presentation
Background:
Non-small cell lung cancer patients harboring epidermal growth factor receptor (EGFR) mutation respond well to EGFR tyrosine kinase inhibitors (TKI). However, all patients develop resistance to EGFR TKI after long term use. EGFR T790M mutation can be found in about half of the resistant re-biopsy tumors. Afatinib is an irreversible EGFR TKI with in vitro activity against resistant T790M mutation. However, afatinib has little activity in EGFR TKI resistant patients whose tumors developed T790M mutation. A novel alinino-pyrimidine based WZ8040 has been developed to specifically inhibit phosphorylation of EGFR with T790M mutation and not on wild type EGFR. Similar compounds such as CO1686 or AZD9291 has demonstrated high activity against T790M mutations. We plan to develop afatinib or AZD9291, WZ8040 resistant PC9 cells to study afatinib or AZD9291 resistance.
Methods:
PC9 cells were grown in culture media containing escalating concentrations of afatinib, WZ8040 or AZD9291. When cells can grow in high concentrations of drugs, cells were cloned, expanded and grew in drug-free media for more than two weeks to obtain stable afatinib, WZ8040 or AZD9291 resistant clones. Gefitinib, afatinib, WZ8040, AZD9291. Cells viability were determined by surforodamine bromide method. PC9 parental and EGFR TKI resistant cells were treated with gefitnib, afatinib, WZ8040 or AZD9291 for one hour and EGFR, AKT, ERK phosphorylation were determined by Western blot. DNA repair capacity were compared between sensitive and resistant cells after exposure of cells to ultraviolet light and measured by pGL3-luciferase plasmid transfection methods. Epithelial mesenchyma transition of these cells were tested by snail, slug, vimentin and E-cadherin western blot. Autophagy was measured by LC3-II levels by Western blot. EGFR exon 18-21 sequence of each clones were determined by Sanger’s direct sequencing.
Results:
We developed afatinib resistant PC9 cells, PC9/AFAb2, PC9/AFAc3 and WZ8040 resistant PC9/WZd7, PC9/WZf6. PC9/AFA cells were more than 100-fold resistant to afatinib and PC9/WZ cells were more than 50-fold resistant to WZ8040. 10nM of afatinib treatment inhibits EGF-induced EGFR, AKT and EKR phosphorylation in PC9 cells, but phosphorylation of these kinases were only partially inhibited in PC9/AFA cells. Phosphorylation was completely blocked at 100nM afatinib. MEK inhibitor plus afatinib did not reverse resistance to afatinib in PC9/AFA cells. On the other hand, WZ8040 or AZD9291 alone completely reversed resistance in PC9/AFA cells. EGFR, AKT and ERK phosphorylation can be blocked by 100nM WZ8040 in PC9 and PC9/WZd7 cells. However, it is curious that phosphorylation of these proteins can be inhibited by 100nM gefitinib as well. EGFR T790M mutation was only detected in PC9/AFA cells and not in PC9/gef, PC9/WZ cells. None of the PC9/WZ cells have EGFR C797S mutation. We did not detect any other EGFR resistance mechanism in PC9/AFA cells. Other of comparing EMT, autophagy and DNA repair capacity of PC9 and their resistant cells are ongoing.
Conclusion:
We developed multiple gefitinib, afatinib, WZ8040, AZD9291 resistant PC9 cells. Only afatinib resistant cells develop EGFR T790M. We demonstrated that EGFR T790M was the predominant resistant mechanism in PC9/AFA cells. The characteristics of PC9/WZ and PC9/AZD9291 are still under investigation.
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MINI09.04 - Identification of Effective Drug Combinations to Prevent or Delay Resistance to the EGFR Mutant Selective Inhibitor Rociletinib (CO-1686) (ID 3010)
16:45 - 18:15 | Author(s): A. Simmons, H.J. Haringsma, M. Nguyen, L. Robillard, A. Allen, T.C. Harding
- Abstract
- Presentation
Background:
Rociletinib (CO-1686) is a novel, oral, irreversible tyrosine kinase inhibitor for the treatment of patients with mutant epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) that has demonstrated efficacy against the activating mutations (L858R and del19) and the dominant acquired resistance mutation (T790M), while sparing wild-type (WT) EGFR. Although rociletinib has generated compelling objective responses in heavily-pretreated T790M positive and negative NSCLC patients, acquired resistance to rociletinib monotherapy has also been observed. We are currently exploring preclinical combinations to delay or prevent resistance to rociletinib.
Methods:
To study acquired resistance in an unbiased fashion, rociletinib resistant populations and clones were generated from the EGFR mutant NSCLC cell lines PC-9 (del19 EGFR), HCC827 (del19 EGFR), and NCI-H1975 (L858R/T790M EGFR) by chronic in vitro and/or in vivo exposure of rociletinib. Compound library screening was performed with rociletinib resistant cell lines to identify drug combinations that could restore rociletinib sensitivity. In vitro and in vivo validation, mechanism of action, and combination studies were performed to evaluate the potency of these combinations in rociletinib sensitive and resistant preclinical models. In addition, combination studies with therapies commonly used in NSCLC, including radiotherapy (RT), an anti-EGFR antibody, and anti-PD-1/L1 antibodies were also explored.
Results:
Multiple mechanisms of resistance were observed in rociletinib resistant cell lines, including MET amplification and an epithelial-mesenchymal transition (EMT). In a PC-9 resistant population (designated 2A10) generated by in vitro and in vivo selection, multiple agents including the aurora kinase inhibitor MLN8237, the MEK inhibitor trametinib, and an anti-EGFR antibody restored rociletinib sensitivity in cell viability assays. Western blot analysis demonstrated that the levels of p-ERK in the parental PC-9 cell line were comparable to p-ERK levels in 2A10 cells grown in the presence of 1 mM rociletinib. The combination of rociletinib and trametinib in the 2A10 cell line suppressed p-ERK signaling, concomitant with increased levels of apoptotic markers such as PARP cleavage. The combination of rociletinib and trametinib also demonstrated potent in vivo activity in the 2A10 xenograft model. In vitro and in vivo studies performed with additional cell lines and combinations are ongoing and will also be presented.
Conclusion:
Resistance to all 3[rd] generation EGFR inhibitors is likely to be observed, and identifying tolerable and effective combinations to delay or prevent resistance is critical in extending the clinical benefit of these therapies. In vitro and in vivo studies reported here highlight multiple combinations that restore the activity of rociletinib in rociletinib resistant models. In particular, the combination of trametinib and rociletinib restored MAPK pathway suppression and anti-tumor activity in the rociletinib resistant 2A10 model. These nonclinical data support the ongoing Phase 1/2 evaluation of the combination of trametinib and rociletinib.
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MINI09.05 - Discussant for MINI09.01, MINI09.02, MINI09.03, MINI09.04 (ID 3313)
16:45 - 18:15 | Author(s): C. Lovly
- Abstract
- Presentation
Abstract not provided
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MINI09.06 - Oncogenic Drivers including RET and ROS1 plus PTEN Loss and MET by IHC in Patients with Lung Adenocarcinomas: Lung Cancer Mutation Consortium 2.0 (ID 2114)
16:45 - 18:15 | Author(s): B.E. Johnson, M.G. Kris, I.I. Wistuba, L. Berry, M.A. Socinski, S.S. Ramalingam, B. Glisson, G. Otterson, J. Schiller, J. Cetnar, J.R. Brahmer, L.V. Sequist, C. Lovly, J. Minna, P.A. Bunn, Jr, D.J. Kwiatkowski, K. Kugler, S. Waqar, K. Politi, E.B. Garon, E. Haura
- Abstract
- Presentation
Background:
The Lung Cancer Mutation Consortium (LCMC) 1.0 demonstrated multiplexed genomic platforms can assay 10 oncogenic drivers in tumor specimens from patients with lung adenocarcinomas. 28% of the patients with oncogenic drivers could be effectively targeted. The survival of these 275 patients treated with targeted agents was longer than the patients who were not treated with a targeted agent (Kris and Johnson JAMA 2014). The efficiency of Next-Generation Sequencing enables more comprehensive testing of additional aberrations with less tumor tissue. LCMC 2.0 was initiated to test tumor specimens for 12 oncogenic drivers and to provide the results to clinicians for treatment decisions and research purposes.
Methods:
The 16 site LCMC 2.0 is testing tumors from 1000 patients with lung adenocarcinomas in CLIA laboratories for mutations in KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, and NRAS, MET DNA amplification, and rearrangements in ALK as done in LCMC 1.0. The new genes that were added because of emerging information about potential therapeutic targets include MAP2K1 mutations, RET and ROS1 rearrangements, PTEN (MAb 138G4) loss and MET (MAb SP44) overexpression by immunohistochemistry (IHC). All patients were diagnosed with stage IIIB/IV lung adenocarcinoma after May 2012, had a performance status 0-2, and available tumor tissue.
Results:
Of 1073 patients registered, data is now reported for 759. The median age of the patients is 65 (23-90). The population includes 369 (55%) women; 164 (24%) never smokers, 399 (59%) former smokers, and 73 (11%) current smokers; 26 (4%) Asians, 58 (9%) African American, 548 (81%) Caucasian, and 43 (6%) of other races. As of April 2015 information on genomic and immunohistochemical changes for 675 eligible patients were recorded in our database. Alterations in oncogenic drivers were found in 45% of samples as follows: 159 KRAS (24%), 88 EGFR (13%), 25 ALK (4%), 19 BRAF (3%), 17 PIK3CA (3%), 9 HER2 (1%), 4 NRAS (1%) 0 AKT1, 28 had ≥ 2 findings (4%) and 25 MET DNA amplification (4%). The new genes studied in LCMC 2.0 revealed 1 MAP2K1 mutation (<1%), 19 RET (3%) and 9 ROS (1%) rearrangements, 94 had PTEN loss (14%), and 362 with MET overexpression (54%). As expected, PIK3CA mutations and PTEN loss by IHC were mutually exclusive in 109 of 111 (98%) patients’ tumors. Seventeen of the 23 (74%) with MET DNA amplification studied thus far with IHC had MET overexpression. Next-Generation platforms were used at 13 of 16 LCMC 2.0 sites.
Conclusion:
Next-Generation Sequencing is rapidly becoming routine practice at LCMC 2.0 centers with use going from 0 to 81% of sites since 2012. LCMC 2.0 identified additional targets (RET and ROS1 rearrangements and PTEN loss). PIK3CA and PTEN were largely mutually exclusive and an actionable oncogenic driver has been identified in the 45% of initial lung adenocarcinoma specimens. Supported by Free to Breathe
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- Abstract
- Presentation
Background:
Fibroblast growth factor receptor (FGFR) tyrosine kinase plays a crucial role in cancer cell growth, survival, and resistance to chemotherapy. FGFR1 amplification occurs at a frequency of 10-20% and is a novel druggable target in squamous cell carcinoma of the lung (SCCL). A number of FGFR-targeted agents are currently being developed in SCCL harboring FGFR alterations. The aim of the study is to evaluate the activity of selective FGFR inhibitors (AZD4547, BAY116387) and the mechanisms of intrinsic and acquired resistance to these agents in SCCL.
Methods:
The antitumor activity of AZD4547 and BAY116387 was screened in a panel of 12 SCCL cell lines, among which 4 cell lines harbored FGFR1 amplification. To investigate mechanisms of acquired resistance, FGFR1-amplified H1581 cells which were exquisitely sensitive to FGFR inhibitors, were exposed to AZD4547 or BAY116387 to generate polyclonal resistant clones (H1581-AR, H1581-BR). Characterization of these resistant clones was performed using receptor tyrosine kinase (RTK) array, immunoblotting and microarray. Migration and invasion assays were also performed.
Results:
Among 12 SCCL cell lines, two FGFR1-amplified cells, H1581 and DMS114, were sensitive to FGFR inhibitors (IC~50~<250 nmol/L). Compared with resistant cells, sensitive cells showed increased phosphorylation of FRS2 and PLC-γ, but decreased phosphorylation of STAT3. There was no noticeable difference in FGFR1-3 protein expression level between sensitive and resistant cells. Importantly, phosphorylation of ERK1/2 was significantly suppressed upon treatment of FGFR inhibitors only in sensitive cells, suggesting phospho-ERK1/2 as a pharmacodynamic marker of downstream FGFR signaling. RTK array and immunoblots demonstrated strong overexpression and activation of MET in H1581-AR and H1581-BR, in comparison to almost nil expression in parental cells. Four different SCCL cells with intrinsic resistance to FGFR inhibitors also showed intermediate to high MET expression, suggesting that MET may be involved in both intrinsic and acquired resistance to FGFR inhibitors. Gene-set enrichment analysis against KEGG database showed that cytokine-cytokine receptor interaction pathway was significantly enriched, with MET contributing significantly to the core enrichment, in H1581-AR and H1581-BR, as compared with parental cells. Stimulation with HGF strongly activated downstream FGFR signaling or enhanced cell survival in the presence of FGFR inhibitors in both acquired and intrinsic resistant cells. Quantitative PCR on genomic DNA and fluorescent in situ hybridization revealed MET amplification in H1581-AR, but not in H1581-BR. MET amplification led to acquired resistance to AZD4547 in H1581-AR by activating ERBB3. The combination of FGFR inhibitors with ALK/MET inhibitor, crizotinib, or small interfering RNA targeting MET synergistically inhibited cell proliferation in both H1581-AR and H1581-BR, whereas it resulted in additive effects in SCCL cells with intrinsic resistance to FGFR inhibitors. Acquisition of resistance to FGFR inhibitors not only led to a morphologic change, but also promoted migration and invasion of resistant clones via inducing epithelial to mesenchymal transition phenotype, as documented by a decrease in E-cadherin and an increase in N-cadherin and vimentin.
Conclusion:
MET activation is sufficient to bypass dependency on FGFR signaling and concurrent inhibition of these two pathways may be desirable when targeting FGFR-dependent SCCL.
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MINI09.08 - Secondary C805S Mutation in HER2 Gene Confers Acquired Resistance to HER2 Kinase Inhibitors in HER2 Mutant Lung Cancer (ID 2181)
16:45 - 18:15 | Author(s): T. Kosaka, H. Endoh, C. Repellin, J. Tanizaki, D. Ercan, M. Capelletti, P.A. Jänne
- Abstract
- Presentation
Background:
Activating mutations in the HER2 kinase domain are detected in 2-4% of non-small cell lung cancers (NSCLC), and are oncogenic in both in vitro and in vivo models. Current clinical strategies to target mutant HER2 include the use of covalent HER2 inhibitors afatinib, dacomitinib and neratinib; all of which have limited single agent activity. We evaluated how drug sensitive models of HER2 mutant lung cancer develop acquired resistance in vitro to gain biological insights and to predict how acquired resistance may develop in the clinic.
Methods:
Murin Ba/F3 cells expressing duplication/insertion of four amino acids (YVMA) between codon 775 and 776 in exon 20 of HER2 gene (A775_G776insYVMA (insYVMA)) were exposed to N-ethyl-N-nitrosourea mutagenesis and expanded in the presence of neratinib and dacomitinib. Total RNAs were extracted from resistant clones and sequencing of the HER2 tyrosine kinase domain was performed. Drug resistance was confirmed with cell growth assays and western blotting.
Results:
Total 5 clones for Neratinib and 7 clones for Dacomitinib were expanded from each 300 wells. Sequencing analysis revealed that all resistant clones retained original insertion mutation and acquired same substitution of Cysteine to Serine change in codon 805 (C805S) in exon 20 of HER2 gene. This mutation is analogous to the EGFR C797S mutation that mediates resistance to 3[rd] generation EGFR inhibitors. Next, we generated Ba/F3 cells co-expressing activating mutations; insYVMA and a dacomitinib hypersensitive insertion mutation (insertion of three amino acids (WLV) after codon 774 with deletion of M774 (M774del insWLV (insWLV))), in cis with the C805S mutation. Cell growth assay revealed these double mutants were resistant to all three second generation inhibitors for EGFR family; neratinib, dacomitinib, and afatinib, compared to parental cells which only have activating mutation. They were also resistant to 3[rd] generation EGFR inhibitors; WZ40002 and AZD9291. Phosphorylation of HER2 was not completely inhibited by these drugs. Resistant cells showed moderate sensitivity to mTOR inhibitor; rapamycin alone. Combination treatment with afatinib and rapamycin effectively inhibited growth of these cells.
Conclusion:
The C805S secondary HER2 mutation results in acquired resistance to covalent HER2 inhibitors in HER2 mutant NSCLC. Our results provide insights into drug resistance mechanisms and help predict likely clinical mechanisms of resistance to HER2 targeted therapies in HER2 mutant NSCLC.
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MINI09.09 - Discussant for MINI09.06, MINI09.07, MINI09.08 (ID 3314)
16:45 - 18:15 | Author(s): E.B. Garon
- Abstract
- Presentation
Abstract not provided
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MINI09.10 - Tumor Angiogenesis in LKB1-Mutant Non-Small Cell Lung Cancer (NSCLC) (ID 3059)
16:45 - 18:15 | Author(s): I. Guijarro, M. Nilsson, A. Poteete, P. Tong, H. Sun, W. Denning, D. Xia, F. Skoulidis, J. Wang, E. Roarty, J.V. Heymach
- Abstract
- Presentation
Background:
LKB1 is a critical regulator of cell growth, metabolism and EMT, and it is mutated in 20-30% of non-small cell lung cancers (NSCLC). LKB1 mutations co-occur with KRAS-activating mutations in 7%-10% of all NSCLC and results in an aggressive phenotype and a worse response to chemotherapy compared to KRAS-mutated tumors. Because LKB1 activates AMPK (AMP-activated protein kinase) which functions as a cellular energy sensor, LKB1-deficient cells are unable to appropriately sense metabolic and energetic stress. LKB1 is also known to regulate angiogenesis, but the mechanism(s) by which this occurs remains unclear. Bevacizumab, the human anti-VEGF antibody approved for the treatment of NSCLC, improves the progression-free and overall survival of NSCLC patients combined with chemotherapy, but often the benefit is transient, and therapeutic resistance occurs. Our laboratory has previously identified phenotypical differences in vasculature patterns in A549 NSCLC tumors resistant to bevacizumab (LKB1 mutant), when compared to H1975 tumors, (LKB1 wild-type). In addition, LKB1 mutant NSCLC cell lines are highly vulnerable to agents acting on energetic pathways. These results may indicate that loss of LKB1 in NSCLC could alter the tumor vasculature and regulate sensitivity to anti-angiogenic therapies. Here, we investigate the hypothesis that combinations of energetic-depleting compounds along with blockade of tumor angiogenesis would be more effective in NSCLC LKB1 mutant tumors.
Methods:
mRNA and protein expression of 584 angiogenesis-related genes were analyzed in wild-type and LKB1 mutant NSCLC (TCGA, RPPA and PROSPECT databases). In vitro validation was performed using qPCR, immunohistochemistry and western blot analysis as well as pairs of isogenic LKB1 mutant cell lines with overexpressed or silenced LKB1. Endothelial cells were incubated with conditioned medium of wild-type and LKB1 mutant NSCLC cell lines, and tube formation matrigel, proliferation and migration (Boyden chamber) assays were performed.
Results:
We identify a group of new and classic angiogenesis-related molecules: VEGFA, VEGFR1, KDR, NRP1, PDGFB, PDGFRA-B, HIF-1A, C-KIT, VCAM1, hypoxia related molecules: HIF1AN, EGLN1, HIF3A, CA12, EPAS1 and immune related molecules: TNFSF11, NFKB1, CD47, PDL1 differentially expressed in LKB1-wild type and LKB1 mutant NSCLC (p<0.05 and fold-change ≥ or ≤1.5). LKB1 mutant cell lines showed higher protein expression of phospho-cKIT, a tyrosine-kinase receptor involve in cell proliferation and angiogenesis, and CA12 (Carbonic anhydrase 12), a known HIF-1α regulated molecule, involved in maintaining cellular pH homeostasis. Also, LKB1 mutant cells exhibit different quantitative vascular patterns in matrigel assays like number of nodes, junctions, length and branching of the endothelial matrix (p<0.05). Human endothelial cells exhibited an increase rate of proliferation and migration when incubated with conditioned medium from LKB1 mutant NSCLC cell lines compared with conditioned media from LKB1-wild type NSCLC cell lines (p<0.05).
Conclusion:
There are biological differences in vasculature patterns in LKB1 mutant NSCLC tumors and in LKB1 mutant cell lines comparing with wild-type LKB1. These differences are translated in biological alterations of human endothelial cells in vitro suggesting an important role of LKB1 in resistance to anti-angiogenic treatments in vivo.
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MINI09.11 - Adaptor Re-Programming and Acquired Resistance in RET-Fusion Positive NSCLC (ID 2891)
16:45 - 18:15 | Author(s): S. Nelson, L. Schubert, A.T. Le, K.A. Ryall, S. Kako, A. Tan, M. Varella-Garcia, R.C. Doebele
- Abstract
- Presentation
Background:
RET gene fusions were identified as a novel oncogenic driver of ~1-2% of non-small cell lung cancer (NSCLC) patients and clinical trials investigating the use RET TKI therapy are underway. Like all NSCLC patients treated with TKI therapies, it is expected that drug resistance will emerge in this patient population. The mechanisms that drive acquired resistance to RET TKI therapy are still unknown. The objective of this study is to advance current understanding of RET signaling in NSCLC and to identify the cellular mechanisms of acquired RET TKI resistance that will eventually emerge in RET fusion positive NSCLC patients by using in vitro models of drug resistance.
Methods:
The LC-2/ad is a lung adenocarcinoma cell line that harbors the CCDC6-RET fusion. We created three distinct ponatinib resistant (PR) LC-2/ad cell lines (PR1, PR2, PR3) derived from three different dose-escalation strategies. RET break-apart fluorescence in situ hybridization (FISH) was performed on the parental LC-2/ad and PR-derivatives. Interactions between the RET kinase domain and known adaptor signaling molecules were assessed via proximity ligation assay (PLA) in parental LC-2/ad cells and resistant lines. Formation of RET-adaptor signaling complexes were confirmed via immunoprecipitation and western blot analysis. Next-generation RNA sequencing in conjunction with a high-throughput small molecule inhibitor screen were performed to elucidate the signaling pathways that drive resistance to RET-inhibition. Pathways and candidate molecules identified by these screens were validated using siRNA knockdown and pharmacologic inhibition in the context of a cell-proliferation MTS assay. Western blot analysis was utilized to identify the downstream signaling programs responsible for proliferation and survival in the RET-inhibition resistant cell lines.
Results:
MTS cell proliferation assay confirmed that all three ponatinib resistant cell lines are significantly less sensitive to ponatinib than parental LC-2/ad cells. RET FISH analysis demonstrated that the CCDC6-RET gene was retained in the PR1 and PR2 cell lines, but lost in the PR3 cell line. RT-PCR and western blot analysis confirmed the loss of the CCDC6-RET fusion in the PR3 cell line. DNA sequencing demonstrated no RET kinase domain mutations in either the PR1 or PR2 derivatives. Further, profound changes in the RET-signaling program have emerged in the PR1 and PR2 cell lines. Using a RET-GRB7 PLA, we have demonstrated that PR1 cells no longer form RET-GRB7 signaling complexes, while PR2 cells retain RET-GRB7 complexes even in the presence of ponatinib. Next-generation RNA sequencing of the PR1 cell line revealed an increase in expression of several known EMT markers including caveolin-1, vimentin, and ADAMTS1.
Conclusion:
Like many other targeted therapeutic strategies, resistance to small molecule Ret-inhibition in RET-fusion positive lung cancer cells can be driven by multiple mechanisms. Changes in the RET-adaptor programming appear to mitigate resistance in both the PR1 and PR2 cell lines, suggesting that RET-resistant cells may have successfully undergone an oncogenic switch to rely upon another known oncogenic driver in lieu of the CCDC6-RET fusion. Further, EMT reprogramming of the LC-2/ad cell may have contributed to the resistance phenotype in the PR1 cell line.
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MINI09.12 - HDAC Inhibitors Overcome New Generation EGFR-TKI-Resistance Caused by Homozygous BIM Polymorphism in EGFR Mutant Lung Cancer (ID 885)
16:45 - 18:15 | Author(s): S. Yano, A. Tanimoto, S. Arai, K. Fukuda, S.T. Ong, S. Takeuchi
- Abstract
Background:
The BIM deletion polymorphism in intron 2 was found in a significant percent (~13%) of the Asian population, with 0.5% of individuals being homozygous for this deletion. Patients with EGFR mutant lung cancers harboring this BIM polymorphism have shorter progression free survival and overall response rates to 1[st] generation EGFR-TKIs, gefitinib and erlotinib. We recently reported that the histone deacetylase (HDAC) inhibitor vorinostat can epigenetically restore BIM function and death sensitivity of EGFR-TKI, in cases of EGFR mutant lung cancer where resistance to 1[st] generation EGFR-TKI is associated with a heterozygous BIM polymorphism. Here, we examined 1) whether BIM polymorphism associated with resistance to new generation EGFR-TKIs and 2) whether vorinostat could overcome EGFR-TKI resistance in EGFR mutant lung cancer cells with a homozygous BIM polymorphism.
Methods:
We used EGFR mutant lung cancer cells lines, PC-9, PC-9[i2BIM-/-] (a genetically engineered subclone that was homozygous for BIM deletion polymorphism), and PC-3 (heterozygous for BIM deletion polymorphism). These cell lines were treated with gefitinib, afatinib (2[nd] generation), and AZD9291 (3[rd] generation). Apoptosis was evaluated by FACS and expression of cleaved-caspase 3/7 and PARP by western blot.
Results:
While PC-9 cells were sensitive to all EGFR-TKIs in terms of apoptosis induction, both of PC-3 and PC-9[i2BIM-/- ] cells were resistant to 1[st] generation EGFR-TKIs and new generation EGFR-TKIs as well. Vorinostat combined with new generation EGFR-TKIs induced apoptosis of PC-3 and PC-9[i2BIM-/- ] cells in vitro. In the subcutaneous tumor model, AZD9291 regressed the tumors produced PC-9 cells but not PC-9[i2BIM-/- ] cells, indicating in vivo resistance of PC-9[i2BIM-/- ] cells to EGFR-TKIs. Combined use of vorinostat with AZD9291 successfully decreased the size of tumors produced by PC-9[i2BIM-/-] cells by inducing tumor cell apoptosis.
Conclusion:
These observations indicated that BIM deletion polymorphism is associated with apoptosis resistance caused not only by 1[st] generation EGFR-TKIs but also by new generation EGFR-TKIs. Moreover, combined use of HDAC inhibitor may overcome EGFR-TKI resistance associated not only with heterozygous deletion but also with homozygous deletion in the BIM gene.
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MINI09.13 - Neuropilin-2 Promotes Acquired Resistance to EGFR-TKI Associated with the Epithelial–Mesenchymal Transition in Lung Cancer (ID 1271)
16:45 - 18:15 | Author(s): P. Nasarre, J. Nair-Menon, A. Dimou, T. Yoshida, H. Uramoto, E. Haura, R.M. Gemmill, H.A. Drabkin
- Abstract
Background:
Lung cancer accounts for one-fifth of cancer deaths worldwide with invasion, metastases and drug resistance representing major causes of mortality and barriers to cure. While lung cancers with activating mutations in the EGF receptor (EGFR) are susceptible to tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib, the efficacy of these agents is limited by the inevitable development of resistance. The epithelial-mesenchymal transition (EMT), by which epithelial cells acquire a mesenchymal and invasive phenotype, is one mechanism promoting EGFR-TKI resistance, including resistance to 3[rd] generation T790M-specific inhibitors. However, the molecular connections between EMT and resistance are not well understood. Here we report that upregulation of Neuropilin-2 (NRP2) is crucial for development of EGFR-TKI resistance associated with the EMT. NRP2 is a cell surface receptor for SEMA3F, a secreted semaphorin with tumor suppressor activity that is down-regulated during EMT. NRP1 and NRP2 are also co-receptors and signaling enhancers for several growth-promoting ligands such as VEGF, HGF and FGF. We previously reported that NRP2 was induced by TGFβ as part of an EMT response in lung cancers and that NRP2 knockdown suppressed the EMT phenotype, including local tumor invasion in a subcutaneous xenograft model.
Methods:
Immunohistochemistry (IHC) was performed for NRP2 on patient biopsies, before and after development of gefitinib resistance. EGFR mutant NSCLC cell lines, transfected with control or NRP2-specific shRNAs, were selected for gefitinib/erlotinib resistance in vitro, using progressively increasing concentrations or continuous exposure to IC~50~ levels of EGFR TKIs. Western blot analysis confirmed changes in NRP2 expression along with selected markers of EMT. MTS viability assays determined drug sensitivity while migration and invasion were assessed using Boyden chambers. Growth as spheroids was assessed in 1% methylcellulose medium in low-adherence plates.
Results:
Increased NRP2 was observed in lung tumor biopsies following acquisition of EMT-associated gefitinib-resistance, and in HCC4006-ER cells, which acquired a stable erlotinib-resistant EMT phenotype. In vitro, using multiple EGFR mutant cell lines, NRP2 knockdown blocked acquired gefitinib-resistance, arising both spontaneously following growth in IC~50~ concentrations or after exposure to TGFβ. Of interest, spontaneously-resistant cells exhibited increased migration similar to cells stimulated with TGFβ. NRP2 knockdown also blocked tumorsphere formation, which has been associated with stem-cell characteristics and drug resistance.
Conclusion:
Collectively, our results demonstrate that NRP2 is a mediator of acquired EGFR-TKI resistance. The results also suggest that NRP2 blocking antibodies could be useful for enhancing the duration of response to EGFR inhibitors, including those targeting the T790M mutation.
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MINI09.14 - Discussant for MINI09.10, MINI09.11, MINI09.12, MINI09.13 (ID 3315)
16:45 - 18:15 | Author(s): C. Mathias
- Abstract
- Presentation
Abstract not provided
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Author of
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MINI 02 - Immunotherapy (ID 92)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:P. Forde, S.J. Antonia
- Coordinates: 9/07/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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MINI02.09 - ERK Activation Mediates Increased PD-L1 Expression in KRAS Mutated Premalignant Human Bronchial Epithelial Cells (ID 1620)
10:45 - 12:15 | Author(s): J. Minna
- Abstract
- Presentation
Background:
Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in tumor evasion from the immune system. Elevated PD-L1 expression in tumor cells inhibits tumor-infiltrating T cell function and may be associated with poor prognosis in lung cancer patients. There is increasing interest in developing immunotherapies that block the immunosuppressive effects of checkpoint pathways such as PD-L1, and identifying patients who may benefit from PD-L1 blockade. Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. The effect of cancer cell driver mutations on immune checkpoint immune regulation is poorly understood. While recent clinical trials have suggested better response to PD-1 blockade in KRAS mutation subjects, it is unclear if this clinical finding is directly driven by KRAS regulating the PD-1/PD-L1 pathway with resultant improved efficacy to anti-PD-L1 immunotherapy or if the presence of a KRAS mutation is merely a surrogate marker of the overall mutational load and tumor immunogenicity. KRAS mutations are known to activate the RAF-MEK-ERK pathway. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation.
Methods:
Immortalized human bronchial epithelial cells (HBEC-vector control), KRAS–mutated (KRAS[v12]) HBEC cells (HBEC-KRAS), p53 knockdown HBEC cells (HBEC-p53), and p53 knockdown/KRAS mutated cells (HBEC-p53/KRAS) were used to assess mRNA and/or surface protein expression levels of immune checkpoints including Lag-3, Tim-3, PD-L1 and PD-L2 by real time-qPCR (RT-qPCR) and flow cytometry, respectively. HBEC-vector and HBEC-KRAS cells were treated with MEK (ERK kinase) inhibitor (PD0325901) at 1µM for 24hrs and evaluated for mRNA and surface protein expression of PD-L1. The premalignant HBEC cell lines were used instead of human lung cancer cell lines in order to assess the role of KRAS mutation in isolation without other mutations.
Results:
PD-L1 and PD-L2 mRNA levels increased 2.4 fold (p<0.001) and 3.6 (p<0.001) fold in comparing HBEC-KRAS to HBEC-vector (wild-type) cells, while Lag-3 and Tim-3 mRNA expression levels were unchanged. Based on mean fluorescence intensity on flow cytometry, cell surface PD-L1 protein expression level was 2.2 and 1.6 fold higher in HBEC-KRAS and HBEC-p53/KRAS, respectively, compared to HBEC-vector cells. There was no increase in surface PD-L1 expression in HBEC-p53 cells compared to HBEC-vector control, suggesting that p53 mutation did not alter PD-L1 expression in HBEC-p53/KRAS cells. With MEK inhibition, PD-L1 mRNA levels decreased 10 and 11 fold in HBEC-vector and HBEC-KRAS cells, respectively. Analogously, PD-L1 surface protein levels were reduced 2.7 fold in HBEC-vector and HBEC-KRAS cells, respectively. These findings suggest that ERK activation mediates intrinsic expression and KRAS mutation mediates over-expression of PD-L1 mRNA and protein.
Conclusion:
Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates constitutive and KRAS mutation driven up-regulation of PD-L1 in these cells. Our findings suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential combinational drugs to PD-L1 inhibition.
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.06 - Oncogenic Drivers including RET and ROS1 plus PTEN Loss and MET by IHC in Patients with Lung Adenocarcinomas: Lung Cancer Mutation Consortium 2.0 (ID 2114)
16:45 - 18:15 | Author(s): J. Minna
- Abstract
- Presentation
Background:
The Lung Cancer Mutation Consortium (LCMC) 1.0 demonstrated multiplexed genomic platforms can assay 10 oncogenic drivers in tumor specimens from patients with lung adenocarcinomas. 28% of the patients with oncogenic drivers could be effectively targeted. The survival of these 275 patients treated with targeted agents was longer than the patients who were not treated with a targeted agent (Kris and Johnson JAMA 2014). The efficiency of Next-Generation Sequencing enables more comprehensive testing of additional aberrations with less tumor tissue. LCMC 2.0 was initiated to test tumor specimens for 12 oncogenic drivers and to provide the results to clinicians for treatment decisions and research purposes.
Methods:
The 16 site LCMC 2.0 is testing tumors from 1000 patients with lung adenocarcinomas in CLIA laboratories for mutations in KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, and NRAS, MET DNA amplification, and rearrangements in ALK as done in LCMC 1.0. The new genes that were added because of emerging information about potential therapeutic targets include MAP2K1 mutations, RET and ROS1 rearrangements, PTEN (MAb 138G4) loss and MET (MAb SP44) overexpression by immunohistochemistry (IHC). All patients were diagnosed with stage IIIB/IV lung adenocarcinoma after May 2012, had a performance status 0-2, and available tumor tissue.
Results:
Of 1073 patients registered, data is now reported for 759. The median age of the patients is 65 (23-90). The population includes 369 (55%) women; 164 (24%) never smokers, 399 (59%) former smokers, and 73 (11%) current smokers; 26 (4%) Asians, 58 (9%) African American, 548 (81%) Caucasian, and 43 (6%) of other races. As of April 2015 information on genomic and immunohistochemical changes for 675 eligible patients were recorded in our database. Alterations in oncogenic drivers were found in 45% of samples as follows: 159 KRAS (24%), 88 EGFR (13%), 25 ALK (4%), 19 BRAF (3%), 17 PIK3CA (3%), 9 HER2 (1%), 4 NRAS (1%) 0 AKT1, 28 had ≥ 2 findings (4%) and 25 MET DNA amplification (4%). The new genes studied in LCMC 2.0 revealed 1 MAP2K1 mutation (<1%), 19 RET (3%) and 9 ROS (1%) rearrangements, 94 had PTEN loss (14%), and 362 with MET overexpression (54%). As expected, PIK3CA mutations and PTEN loss by IHC were mutually exclusive in 109 of 111 (98%) patients’ tumors. Seventeen of the 23 (74%) with MET DNA amplification studied thus far with IHC had MET overexpression. Next-Generation platforms were used at 13 of 16 LCMC 2.0 sites.
Conclusion:
Next-Generation Sequencing is rapidly becoming routine practice at LCMC 2.0 centers with use going from 0 to 81% of sites since 2012. LCMC 2.0 identified additional targets (RET and ROS1 rearrangements and PTEN loss). PIK3CA and PTEN were largely mutually exclusive and an actionable oncogenic driver has been identified in the 45% of initial lung adenocarcinoma specimens. Supported by Free to Breathe
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MS 26 - Genomic Alterations and Drug Targets in Small Cell Lung Cancer (ID 44)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:D. Beer, J.W. Goldman
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 2c-3c
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MS26.03 - Targeting ASCL1 in Neuroendocrine Lung Cancers via a MAPK-Regulated Double-Negative Feedback Loop (ID 1965)
14:15 - 15:45 | Author(s): J. Minna
- Abstract
- Presentation
Abstract:
ASCL1 is a lineage-specific transcription factor responsible during development for the formation of pulmonary neuroendocrine cells. ASCL1 is highly expressed in the majority of neuroendocrine lung tumors including small cell lung cancer (SCLC) and non-small cell lung cancer with neuroendocrine features (NSCLC-NE). Others have shown that SCLC survival depends on continued ASCL1 expression while we showed that ASCL1 is also required for the survival of NSCLC-NEs; that ASCL1 down-stream targets predict for poor survival in NSCLC patients; and that BCL2 is a therapeutically actionable ASCL1 target gene (PNAS 2014;111(41):14788-93). Thus, we are trying to target ASCL1 and its “druggable” downstream genes by developing ASCL1 based ChIP-Seq datasets in SCLC and NSCLC-NE tumors. We have now discovered a way to reliably regulate ASCL1 protein expression through “upstream” targeting. Phorbol 12-myristate 13-acetate (PMA) is an agonist of the MAPK pathway via specific activation of Protein Kinase C. Treatment of ASCL1(+) HCC1833 cells for 24 hours with nM quantities of PMA resulted in a robust down-regulation of ASCL1 mRNA and protein. Tumor cell death was apparent and apoptosis confirmed via induction of cleaved PARP. ASCL1 down-regulation was associated with activation of the MAPK pathway, measured by increased protein levels of phosphorylated ERK (pERK), and decreased ASCL1 mRNA expression was found to be at least partly due to mRNA degradation. These data indicate that activation of the MAPK pathway in high-grade neuroendocrine tumors has potential for therapeutic intervention and also provides a reason for the previously unexplained low levels of MAPK activation (pERK) in SCLC. Unexpectedly, we also found that siRNA mediated knockdown of ASCL1 resulted in activation of the MAPK pathway. In addition, pERK was significantly induced with ASCL1 knockdown even when we also knocked down MEK1 (MEK1 knockdown by itself completely eliminated pERK expression). The MAPK pathway depends on active phosphorylation/dephosphorylation and this is regulated in part by dual-specificity phosphatases (DUSPs). Using our ASCL1 ChIP-Seq data, we identified a conserved ASCL1 binding site in the promoter region of DUSP6. DUSP6 mRNA was found to be dramatically elevated in ASCL1(+) lines HCC1833 and H889, while by contrast there was little or no DUSP6 expression in ASCL1(-) SCLC lines H82 and H526, and knockdown of ASCL1 resulted in a decrease of DUSP6 protein suggesting transcriptional regulation. This led us to try a DUSP6 allosteric inhibitor (E/Z-BCI, Sigma-Aldrich) which induced pERK, decreased ASCL1 protein expression, and inhibited soft agar colony forming ability of H889 SCLC cells. In conclusion: Our data indicate that the MAPK pathway regulates ASCL1 expression, where activation of pERK signaling is correlated with decreased ASCL1 mRNA and protein. In addition, ASCL1 in turn, actively down-regulates the MAPK pathway. Our hypothesis is that high-grade neuroendocrine lung cancers down-regulate the MAPK pathway in order to maintain ASCL1 expression, which promotes cell survival and maintenance of the neuroendocrine lineage. This points to a double-negative feedback loop involving the MAPK pathway, ASCL1, and at least one DUSP. Targeting components of the MAPK pathway regulating ASCL1 expression is thus a new therapeutic avenue for high-grade neuroendocrine lung cancers. (Lung Cancer SPORE P50CA70907, NIH 1F30CA168264, CPRIT).
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ORAL 21 - Biology - Moving Beyond the Oncogene to Oncogene-Modifying Genes (ID 118)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:A. Katz, M.S. Tsao
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 4a-4f
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ORAL21.02 - Landscape and Functional Significance of KRAS Co-Mutations in Lung Adenocarcinoma (LUAC) (ID 3224)
10:45 - 12:15 | Author(s): J. Minna
- Abstract
- Presentation
Background:
The biological heterogeneity of KRAS-mutant LUAC represents a major impediment to the successful implementation of targeted therapeutic strategies for this clinically challenging group of lung cancer patients. Through integrative, multi-platform analysis of large scale omics data we recently identified three major subsets of KRAS-mutant LUAC defined on the basis of co-occurring genomic alterations in STK11/LKB1 (KL subgroup), TP53 (KP) and CDKN2A/B (KC), the latter coupled with low expression of the TTF1 transcription factor. We further demonstrated subset-specific molecular dependencies, patterns of immune system engagement and therapeutic vulnerabilities. Here, we extend these findings through comprehensive analysis of a wide panel of KRAS co-mutations and assess the impact of key co-mutations on facets of the malignant phenotype including flux through the MAPK and PI3K/AKT pathways and heterotypic interactions with the host immune system.
Methods:
Our datasets consisted of 431 tumors from TCGA (122 KRAS-mutant), 41 additional chemo-naive KRAS-mutant LUACs (PROSPECT dataset) and 36 platinum-refractory KRAS-mutant LUACs from the BATTLE-2 clinical trial. Significant KRAS co-mutations were identified on the basis of a P value threshold of ≤0.05 (Fisher’s exact test) coupled with a baseline prevalence of ≥3%. RNASeq data were downloaded directly from the TCGA site. Expression profiling of PROSPECT tumors was performed using the Illumina Human WG-6 v3 BeadChip Array whereas BATTLE-2 tumors were profiled using the GeneChipâHuman Gene 1.0 ST Array from Affymetrix. Generation of MAPK and PI3K proteomic scores, based on Reverse Phase Protein Array (RPPA) data, has been previously reported.
Results:
Our analysis identified somatic mutations in 31 genes as significantly co-mutated with KRAS in LUAC samples. Among them, co-mutations in STK11/LKB1 (P=0.00011) and ATM (P=0.0004) predominated. Somatic mutations in ERBB4 (P=0.0059), encoding a member of the ErbB family of receptor tyrosine kinases and MAP3K4 (P=0.0017) were also enriched in KRAS-mutant LUAC. We assessed the impact of KRAS co-mutations on the amplitude and directionality of signaling downstream of mutant KRAS using the proteomic “MAPK score“ and “PI3K score” as surrogates of effector pathway activation. Interestingly, co-mutations in ERBB4 were associated with significantly suppressed flux through the MAPK pathway (P=0.0024, t-test). Somatic mutations in other genes, including CAMSAP2, were associated with suppressed signaling through both the MAPK (P=0.00876, t-test) and PI3K-AKT (P=0.0032, t-test) cascades. Finally, within KRAS-mutant tumors, co-mutations in NLRC5, a master transcriptional regulator of MHC Class I molecules were associated with reduced mRNA expression of several of its classical target genes. In addition, low mRNA expression of NLRC5 correlated strongly with reduced expression of key components of the antigen presentation pathway across multiple independent datasets of chemotherapy naïve and platinum refractory KRAS-mutant tumors and cell lines. Thus, in addition to cell autonomous effects, co-mutations can also impinge on the reciprocal relationship between malignant cells and their immune microenvironment.
Conclusion:
Our work identifies a compendium of KRAS co-mutations that impact classical and emerging cancer hallmarks, including evasion of the host immune response. Systematic interrogation of the functional impact of prevalent KRAS co-mutations is essential for the development of personalized treatment approaches for this heterogeneous group of tumors.
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ORAL 22 - Moving Beyond a Smoking Related-Cancer to the Young, Never-smokers and Inherited Disease (ID 117)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:K. Suda, H.A. Wakelee
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL22.01 - Increasing Incidence of Never Smokers in Non Small Cell Lung Cancer (NSCLC) Patients (ID 707)
10:45 - 12:15 | Author(s): J. Minna
- Abstract
- Presentation
Background:
It is estimated that 10-15% of lung cancer cases occur in never smokers. The cause of lung cancer in these patients includes many possible environmental factors but the precise cause in a given case is often uncertain. Additionally, there has been significant debate about whether the rate of lung cancer in these never smokers is increasing. Using our institutions’ cancer registry data, our objective was to determine if the proportion of never smokers with lung cancer is increasing.
Methods:
We conducted a retrospective study using lung cancer registry data from The University of Texas Southwestern Medical Center in Dallas, Parkland Hospital in Dallas, and Vanderbilt University in Nashville. These registries were queried between 1990 and 2013 for demographic information including gender, age at diagnosis, diagnosis [non small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)], and self-reported smoking history. A total of 10,568 NSCLC cases and 1504 SCLC cases were analyzed. Logistic regression analysis was performed to assess the incidence of never smokers with lung cancer.
Results:
The percentage of never smokers increased among NSCLC pts between 1990 and 2013 [Table 1]. Univariate logistic regression demonstrated an increasing proportion of never smokers among NSCLC cases (p < 0.0001 for year) and multivariate logistic regression also demonstrates this increase (p < 0.0001 for year) after controlling for age and gender. Never smokers with NSCLC were more likely to be female (65.3%, p < 0.0001) than males. The increase in the percentage of NSCLC never smokers was seen at both university hospitals and the Dallas county hospital. In contrast, the percentage of never smokers among SCLC cases did not significantly increase during this time period. Table 1: Percentage of never smokers Figure 1
Conclusion:
This multi-institution study demonstrates an increasing proportion of never smokers with NSCLC between 1990 and 2013 in a large, geographically and demographically diverse population. Because the biology and, thus, often the treatment options of lung cancer in never smokers differs from that of smokers, further investigation is warranted as to the etiology of the increasing incidence of never-smoker lung cancer.
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ORAL 42 - Drug Resistance (ID 160)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:R.C. Doebele, J.V. DeGregori
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 4a-4f
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ORAL42.04 - Rictor Alterations Elicit Mechanisms of Survival Advantage and Resistance to Targeted Therapy in Non-Small Cell Lung Cancer (NCSLC) (ID 2991)
18:30 - 20:00 | Author(s): J. Minna
- Abstract
- Presentation
Background:
Rictor (RPTOR independent companion of MTOR, complex 2) is a highly conserved protein and is a critical component for assembly and functionality of the mTORC2 complex. Alterations of the PI3K/mTOR/AKT pathway are hallmark of many cancer types, underscoring the potential important role of Rictor. The goal of our current study was to characterize the functional consequences of genomic alterations of Rictor in advanced refractory NSCLC. Our preliminary data suggest that Rictor alterations have the potential to, not only signal canonically (via activation of AKT), but also provide cancer cells with alternate, more advantageous oncogenic signaling via non-canonical mechanisms.
Methods:
We correlated genomic data (DNA next generation sequencing (NGS), Foundation Medicine, Inc) gene expression profiling, and clinical outcome in the context of the ongoing BATTLE-2 clinical trial of targeted therapies in chemo-refractory NSCLC(198 cases). We further (1) surveyed early stage NSCLC cases(230 cases) in The Cancer Genome Atlas (TCGA) database to perform two-way hierarchical clustering comparing gene expression profiling in amplified vs diploid cases; (2) utilized a single-nucleotide polymorphism array to select Rictor amplified and diploid NSCLC cell lines; (3) assessed Rictor protein and RNA expression by Western blot and qRT-PCR, respectively; (4) performed Rictor knockdown (siRNA), and (5) performed drug sensitivity to targeted therapies by MTS assay.
Results:
In the Battle-2 cases, we identified 15% of Rictor alterations (9% gene amplifications, 6.6% mutations, non-concomitant). Among the mutations, 1 was mapped to an N-terminal phosphorylation site, while all others are of unknown significance to date. Rictor alterations were significantly associated with lack of 8-week disease control in the AKTi+MEKi therapeutic arm. In the TCGA we found: (1) 10% Rictor amplifications and 3% mutations; (2) significant correlation between amplification and elevated Rictor gene expression; (3) a putative functional gene expression signature associated with Rictor amplification. In diploid cell lines we found concordance between AKT phosphorylation and activation of other downstream mTORC2 targets (i.e. SGK1 and PKCα), but in Rictor amplified cell lines we witnessed a discordant activation of these pathways. Furthermore, following Rictor knockdown in our amplified cell lines, a significant reduction of colony formation, migratory, and invasive potential was seen in a pathway-differential manner. Thus, suggesting that Rictor amplifications may provide survival advantage in select cancer cells by tipping the signaling balance toward a non-canonical oncogenic pathway (AKT-independent[I1] ).Also in a differential pathway manner, Rictor gene amplification and overexpression contributed to resistance to a number of targeted therapies
Conclusion:
Rictor alterations may constitute a potential novel mechanism of targeted therapy resistance via the activation of non-canonical signaling pathways. These alterations could define new molecular NSCLC subtypes with distinct biology that expose unique avenues for therapeutic implication. Ongoing studies are exploring therapeutic vulnerabilities, non-canonical signaling and Rictor mutations.
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