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G. Reid
Moderator of
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MO15 - Novel Genes and Pathways (ID 89)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 12
- Moderators:Y. Ohe, G. Reid
- Coordinates: 10/29/2013, 16:15 - 17:45, Parkside Ballroom A, Level 1
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MO15.01 - Pathway activation mapping of KRAS wild type and mutated adenocarcinomas of the lung: new implications for patient stratification for MAP kinase pathway inhibition (ID 2705)
16:15 - 16:20 | Author(s): E. Baldelli, E.B. Haura, L. Crino, W..D. Cress, V. Ludovini, M.B. Schabath, G. Bellezza, J. Vannucci, V. Tassi, L. Pistola, F.R. Tofanetti, A. Flacco, A. Siggillino, L.A. Liotta, E.F. Petricoin, M. Pierobon
- Abstract
- Presentation
Background
KRAS proto-oncogene is one of the most frequent mutated genes in Non-Small Cell Lung Cancer (NSCLC) with greater incidence among adenocarcinomas (AD). While the clinical importance of KRAS mutation as a negative predictor for anti-EGFR therapy is not clearly understood in NSCLCs, selection of targeted therapies for KRAS mutated (MUT) patients has often focused on the inhibition of its direct downstream effectors. The aim of this study was to explore the impact of the KRAS status on the cellular signaling network of ADs of the lung harboring different KRAS mutations with a focus on ERK signaling architecture.Methods
A total of 58 AD samples were collected from chemo-naïve patients at the H. Lee Moffitt Cancer Center & Research Institute (Tampa, FL) and at S. Maria della Misericordia Hospital (Perugia, Italy). Twenty-four tumors were KRAS wild type (WT) and 34 were KRAS MUT (G12C n=18, G12V n=9, G13D n=3 and G12D n=4, respectively). All samples were subjected to laser capture microdissection and reverse phase protein microarray to quantitatively evaluate the activation status of the MAP Kinase signaling network.Results
Statistical analysis of signaling protein activation based on KRAS status revealed an overall increase in activation level of the MAPK signaling network in the KRAS MUT tumors compared to tumors expressing KRAS WT: ERK 1/2 (T202/Y204), Elk-1 (S383), p90RSK (S380), Smad2 (S245/250/255) and p70S6K (p<0.01; p<0.01; p<0.01, p=0.04 and p<0.01 respectively). Nevertheless, 6 KRAS WT patients (25%) showed activation of ERK greater than the median of the entire population and an overall MAPK signaling activation comparable to tumors harboring KRAS MUT. Eleven of the KRAS MUT tumors (32%) had ERK activation lower than the median of the population as a whole. Interestingly a high activation level of Estrogen Receptor alpha (ERα) (S118) was detected in the KRAS MUT tumors compared to the KRAS WT one (p=0.02). Moreover the nonparametric test performed to establish the correlation of activated ERK 1/2, Raf, B-Raf, C-Raf and Mek 1/2 with the expression/activation levels of the 152 endpoints analyzed in this study, revealed the activation of distinct pathways in the KRAS MUT tumors when compared to KRAS WT tumors. Significant correlations were detected with Akt, KRAS, their downstream substrates and with several receptor tyrosine kinases (p<0.0003).Conclusion
Our results suggest that MAPK signaling activation was clearly observed in KRAS MUT tumors. However, the heterogeneity in the activation level of MAPK downstream substrates within KRAS MUT and WT tumors suggests that selection of patients for MAPK targeting might benefit from the evaluation not only of the mutation itself, but also from a direct analysis of the MAPK protein network architecture. In particular the role played by ERα in KRAS MUT tumors deserves further investigations as a possible novel therapeutic target in KRAS MUT adenocarcinomas of the lung.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.02 - Impact of co-occurring genetic events on the signaling landscape of KRAS-mutant lung adenocarcinoma. (ID 2936)
16:20 - 16:25 | Author(s): F. Skoulidis, L. Diao, Y. Fan, J.D. Minna, J.N. Weinstein, J. Wang, J.V. Heymach, L.A. Byers
- Abstract
- Presentation
Background
Personalized medicine frameworks centered on identification and therapeutic targeting of dominant oncogenic driver mutations are rapidly becoming a standard of care in the clinical management of patients with lung adenocarcinoma. However, little is currently known about the nature and impact of co-occurring genetic events on signaling output downstream of initiating oncogenes. This lacuna in our understanding is particularly pertinent for the subgroup of KRAS-driven tumors, where mounting data point towards considerable heterogeneity in pathway activation and clinical response to targeted therapies. Here, we report a comprehensive analysis of genetic events that co-occur with or are mutually exclusive of mutant KRAS in a cohort of 230 lung adenocarcinomas and assess the impact of individual co-mutations on signaling streams using data derived from state of the art transcriptomic and (phospho)proteomic profiling of primary tumors.Methods
An integrated analysis of 230 lung adenocarcinomas from The Cancer Genome Atlas (TCGA) consortium was performed using mutation (whole exome sequencing), transcriptomic (RNASeq), and proteomic (reverse phase protein array) datasets. Fischer’s exact test was applied to identify secondary mutations that occurred more frequently in either KRAS-mutant (n=68) or KRAS-wild-type (n=162) tumors and (phospho)protein markers that associated with each co-mutation. Genes with a mutation rate of ≥3% in the overall cohort were included in the analysis.Results
Mutations in 18 genes were associated with KRAS mutational status in patient tumors (p≤0.01). Mutations in EGFR (p=0.0001), NF1 (p=0.001), and TP53 (p=0.001) were negatively correlated with the KRAS mutation. On the other hand, mutations in STK11 were significantly more frequent in the KRAS-mutant cohort (p=0.004), as were mutations in ATM (p=0.023) and MTOR (p=0.045). The most significant positive association involved mutations in ARHGEF11, a gene that encodes a Rho guanine nucleotide exchange factor (p=0.0004). Mutations in STK11 (29.4%) and TP53 (29.4%), the two most highly prevalent genetic events within the KRAS-mutant cohort were mutually exclusive. Unsupervised hierarchical clustering of transcriptomic and quantitative (phospho)proteomic profiles revealed separation of STK11-mutant tumors at the first branch of the cluster dendrogram, indicating activation of distinct signaling pathways downstream of this key tumor suppressor gene. Several less frequent genetic events had prominent and consistent effects on signaling output. We focused our attention on signaling via the MAPK pathway which may impact clinical sensitivity to MEK inhibitors, one of the most promising classes of targeted agents currently in clinical development for KRAS-mutant tumors. Preliminary analysis suggests that mutations in 3 individual genes can identify a subgroup of tumors (19% of the cohort) with profoundly suppressed MAPK signaling flux.Conclusion
Analysis of recurrent secondary genetic events may define distinct and clinically relevant subsets of KRAS-mutant lung adenocarcinoma. Efforts to refine the sub-classification further and assess the impact of co-mutations on sensitivity to molecularly targeted agents are underway and updated results will be presented at the meeting.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.03 - Deciphering the RASSF1A signaling pathway in lung cancer cells reveals a metastasis-suppressor role through YAP-dependent epithelial-mesenchymal transition (EMT) (ID 3189)
16:25 - 16:30 | Author(s): G. Zalcman, F. Dubois, M. Keller, E. Bergot, A. Hergovich, J. Camonis, G. Levallet
- Abstract
- Presentation
Background
RASSF1A gene promoter hypermethylation was previously shown to predict poor overall survival in the IFCT-0002 randomized phase 3 trial of neo-adjuvant platinum-based chemotherapy, in early stage (I & II) NSCLC. We investigated the molecular and cellular basis for such a dramatic influence.Methods
We studied isogenic immortalized bronchial, non-tumorogenic, HBEC3 cell lines only differing by their K-Ras status (wild-type or mutant K-Ras Val12 allele), and a panel of lung cancer cell lines recapitulating the main molecular alterations encountered in lung cancer. RASSF1A protein was depleted by 80% using 2 specific siRNAs, followed by the evaluation of EMT markers and cell motility regualors using qRT-PCR, Western blot or Immunofluorescence. Migration of transfected cells was assayed by 2D wound-healing migration assays or 3D migration assays using transwell devices with or without a matrigel coating mimicking basement membrane (invasion assay), or an endothelial cell monolayer (trans-endothelial cell invasion). Phenotypic rescue was studied by using plasmids encoding full-length RASSF1A or RASSF1C isoform, and a construct encoding a SARAH-deleted RASSF1A protein, unable to interact with the Hippo/MST kinase. We also tested co-transfection of RASSF1A siRNAs together with siRNAs directed against Hippo pathway members LATS1/2, WW45, YAP. Depletion of RASSF1A was finally combined with expression of wild-type, activated or dominant negative RhoA, RhoB, Rac1 or CDC42 constructs.Results
In each bronchial/lung cancer cell line tested, RASSF1A silencing led to EMT resulting in E-cadherin, Syndecan1, Zo-1, miR200 decrease and concurrent N-cadherin, vimentin, Twist1, miR-21 increase. RASSF1A silencing-induced EMT was associated with cytoplasmic to nucleus translocation of YAP transcription factor, the terminal effector of the Hippo signaling pathway. RASSF1A silencing reduced cell adhesion and increased 2D cell motility with collective migration features. RASSF1A knock-down increased 3D migration, invasion as trans-endothelial migration. These effects correlated with the up-regulation of RhoA, RhoC, CDC42, MMP2/14 mRNAs and down-regulation of RhoB, DIA1 and MMP9 mRNAs. We also observed an increase of adhesion/invasion signaling proteins, i.e. CD44v6, cofilin, ERM and NF2, cofilin being activated by inhibition of LIMK-induced phosphorylation. Finally we report that immortalized non-tumorogenic cell lines, unable to grow without adhesion, acquired the capacity to grow in soft agar when RASSFIA was knocked-down. Those effects were rescued by co-transfection of RASSF1A siRNAs with full-length RASSF1A cDNA, showing the specificity of the motile phenotype induced by RASSF1A silencing, but not by RASSF1C nor SARAH-deficient RASSF1A plasmids. SiRASSF1A-induced cell migration was inhibited by LATS1/LATS2/WW45 or YAP siRNAs, showing the involvement of the Lats/YAP signaling cascade. We finally show that RASSF1A knockdown-promoted migration was inhibited by using RhoB-Val14 constitutively active cDNA but not RhoBN19 dominant negative construct, and by specific RhoB GEFs or RhoB effectors (DIA1) constructs.Conclusion
In lung cells, the RASSF1A protein acts as a migration-suppressor protein by regulating the LIMK/cofilin pathway through RhoB signaling. RASSF1A prevents YAP induced EMT by inhibiting its nuclear accumulation through LATS1/2 signaling, whereas Hippo/MST kinase seemed dispensable. We thus provide evidence, for the first time in human lung cancer cells, for a direct connection between RASSF1A signaling and the LATS/YAP pathway.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.04 - Loss of Tumor Suppressor Hic1 Accelerates the Progression of Lung Adenocarcinoma Induced by Oncogenic KRas (ID 2655)
16:30 - 16:35 | Author(s): A. Szczepny, S. Jayasekara, A. Mudiyanselage, D.N. Watkins
- Abstract
- Presentation
Background
Hypermethylated in Cancer-1 (Hic1) is a novel tumour suppressor gene that is frequently epigenetically silenced in adult tumours. In non-small cell lung cancer (NSCLC), loss of Hic1 expression is associated with reduced patient survival, suggesting reduced Hic1 expression is associated with malignant progression of NSCLC. However, whether Hic1 silencing is causal in lung cancer is not known. Hic1 is a transcriptional repressor that can regulate p53 function by repressing the expression of SIRT1, a class III histone deacetylase. We therefore hypothesized that loss of Hic1 function could cooperate with an oncogenic mutation in KRas (KRasG12D) to promote lung cancer initiation and/or progression in a similar fashion to genetic deletion of p53.Methods
To address this question, we used a conditional genetic mouse model of lung adenocarcinoma in which administration of recombinant adenovirus expressing Cre can trigger recombination at loxP sites. When virus is administered to mice heterozygous for a conditional KRasG12D allele, mice develop multiple lung adenomas that progress to adenocarcinomas over 8-12 weeks. To test the function of Hic1 as a tumour suppressor, we created a conditional knockout mouse allele (Hic1[lox]), in which loxP sites flank the single coding exon.Results
Following administration of Cre adenovirus, mice carrying the KRasG12D allele and homozygous for the Hic1[lox] allele, developed aggressive lung adenocarcinomas at a markedly accelerated rate and had a significantly shortened survival compared to KRasG12D animals. Remarkably, these tumours exhibited a highly malignant phenotype with highly proliferative micropapillary and pleomorphic features.Conclusion
These data show that loss of Hic1 function can substitute for p53 mutation as a cooperating event in lung adenocarcinoma progression. Since the highly aggressive phenotype of KRasG12D/Hic1[lox/lox] lung tumours has not been reported in the KRasG12D/p53[lox/lox] lung cancer model, we speculate that Hic1 may function as a tumour suppressor beyond the regulation of p53 through Sirt1.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.05 - Oncogenic ARAF mutation in lung adenocarcinoma (ID 2860)
16:35 - 16:40 | Author(s): M. Imielinski, H. Greulich, B. Kaplan, L. Araujo, J. Amann, L. Horn, M. Villalona-Calero, M. Meyerson, D.P. Carbone
- Abstract
- Presentation
Background
Targeted cancer therapies often induce “outlier” responses in molecularly defined patient subsets.Methods
One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a complete clinical and radiographic remission for five years. Whole genome sequencing (WGS) and RNA sequencing (RNA-seq) on primary tumor and normal samples from this patient was performed.Results
We identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutants were shown to transform immortalized human airway epithelial cells and were associated with in vitro sorafenib sensitivity.Conclusion
These results suggest that mutant ARAF may be a novel oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.06 - A prospective internet-based study of patients with lung cancer harboring baseline EGFR T790M to identify germline carriers and characterize familial risk (ID 1667)
16:40 - 16:45 | Author(s): G.R. Oxnard, J.C. Heng, I.R. Rainville, A.L. Sable-Hunt, E.J. Root, G.L. Wiesner, D.P. Carbone, P.A. Jänne, J.E. Garber
- Abstract
- Presentation
Background
The EGFR T790M mutation, commonly seen with acquired resistance to EGFR kinase inhibitors, has also been described rarely as a germline mutation in association with familial lung cancer. In a prior study (Oxnard et al, JTO, 2012), the presence of EGFR T790M at diagnosis was associated with a 50% chance of carrying an underlying germline T790M mutation. This suggests that by studying patients whose cancer was shown to carry T790M at diagnosis, it is possible to efficiently screen for a germline allele that otherwise is rare among patients with non-small cell lung cancer. We therefore initiated a prospective trial to identify patients and families carrying germline EGFR mutations in order to characterize phenotype and cancer risk.Methods
Subjects are eligible if they (1) have a cancer harboring EGFR T790M (excluding acquired T790M), (2) are a relative of a known germline carrier, or (3) are already known to carry a germline EGFR mutation on prior testing. Subjects may present at a participating cancer center or may enroll remotely using a study website (www.dana-farber.org/T790Mstudy/). Eligible subjects receive genetic counseling in person or over the phone, and then submit a saliva and/or blood specimen for central testing in a CLIA lab. Results are disclosed to the subject if they wish but do not enter the medical record. Those subjects carrying germline EGFR mutations are given the option of inviting relatives to participate. Chest CT scans are collected from germline carriers and analyzed centrally to study nodule prevalence and characteristics. Available tumor specimens are collected for central pathology review and advanced genomic analysis.Results
The trial was registered to clinicaltrials.gov (NCT01754025) and began accrual in December 2012. To date, 7 subjects have been enrolled and 5 are actively being screened, including 4 kindreds. More than half of the subjects have participated remotely via the study website. Of 4 probands with lung cancer and germline T790M, 3 have a family history of lung cancer, 2 of whom have children with CT scans showing multiple sub-centimeter ground-glass nodules. The fourth proband has no family history of lung cancer, suggesting variable penetrance or a de novo germline event. All cancers in germline T790M carriers have also harbored secondary EGFR kinase domain mutations.Conclusion
Using a novel trial design, including remote accrual, genetic counseling by phone, and germline testing by mail, we have begun collecting a sizeable cohort of families affected by germline EGFR mutations. By leveraging referrals from commercial laboratories and contributing academic centers, we aim to study 100 patients over a three year period in order to better understand the natural history and risk associated with this unique familial cancer syndrome. Supported by grants from the Conquer Cancer Foundation of ASCO, the Bonnie J. Addario Lung Cancer Foundation, and the National Cancer Institute.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.07 - DISCUSSANT (ID 3898)
16:45 - 17:00 | Author(s): G. Riely
- Abstract
- Presentation
Abstract not provided
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MO15.08 - KDR (VEGFR-2) copy number gains and mutations are targetable alterations in non-small cell lung cancer (ID 1466)
17:00 - 17:05 | Author(s): M.B. Nilsson, T. Cascone, U. Giri, J. Gudikote, L. Diao, A. Koo, H. Lu, T. Dogruluk, E. Riquelme, X. Tang, H.T. Tran, K. Scott, I.I. Wistuba, D. Carbone, M.A. Socinski, J.V. Heymach
- Abstract
- Presentation
Background
Therapeutic regimens targeting the vascular endothelial growth factor (VEGF) pathway have been extensively tested in the treatment of malignancies including non-small cell lung cancer (NSCLC). VEGF pathway inhibitors including bevacizumab or VEGF receptor (VEGFR) tyrosine kinase inhibitors (TKIs) have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, occurring only in subsets of patients. Therefore, predictive markers to identify patients likely to derive benefit are critically needed. Although expression of VEGFR-2, also known as KDR, was initially thought to localize primarily on endothelial cells, VEGFR-2 has been detected on malignant cells. We recently observed that KDR copy number gains (CNGs) were detectable by FISH in ~30% of both adenocarcinoma and squamous cell carcinoma and were associated with poor clinical outcome in early stage NSCLC patients treated with adjuvant chemotherapy. In addition to CNGs, mutations and polymorphisms within the KDR gene were also observed. The impact of these alterations is unknown. Here, we investigated KDR CNGs, polymorphisms, and mutations in NSCLC and their effects on sensitivity to VEGFR targeting agents in preclinical models and in NSCLC patients.Methods
Cell migration was evaluated by Boyden chamber assay. NSCLC cell lines were treated with VEGF pathway inhibitors for 24 hours, and protein lysates where collected. HIF-1α levels were evaluated by ELISA assay. VEGFR, p38, and p70s6K were evaluated by Western blotting. Tumor DNA and peripheral blood DNA, were analyzed in duplicate using Affymetrix Genome-Wide SNP Array 6.0. Transformation of Ba/F3 cells was evaluated by an IL-3-independent growth assay.Results
In tumor cells with KDR CNG, VEGF stimulation induced activation of p38 and p70S6K, and VEGFR TKIs including sorafenib and vandetanib effectively inhibited VEGF-mediated signal transduction. In tumor cell lines with KDR CNG, exogenous VEGF ligand increased cell motility and this was inhibited by VEGFR blockade with TKIs including sunitinib, sorafenib, and axitinib. Various receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxia compared to NSCLC cell lines without KDR CNG. In NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG had a partial response to the VEGFR inhibitor, sorafenib. In addition to gene amplification, mutations and polymorphisms within the KDR gene were also observed. KDR mutation 1586A>T and polymorphism 1416A>T effectively transformed Ba/F3 cells. Finally, we report two clinical cases in which NSCLC patients with the 1416A>T polymorphism had a partial response the VEGF pathway inhibitor, bevacizumab.Conclusion
Collectively, our data indicate that KDR amplification promotes downstream signaling events including activation of the p38, mTOR, and HIF pathways and are targetable by VEGF pathway inhibitors. KDR gene alterations may be predictive markers for VEGF pathway inhibitors.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.09 - Amplification of YEATS4, a novel oncogene in NSCLC, inhibits the p53 pathway and increases resistance to cisplatin (ID 1073)
17:05 - 17:10 | Author(s): L. Pikor, W.W. Lockwood, K.L. Thu, E.A. Vucic, R. Chari, A.F. Gazdar, S. Lam, W.L. Lam
- Abstract
- Presentation
Background
Characterization of lung cancer genomes has revealed a number of genes critical to tumorigenesis (e.g. EGFR, KRAS, EML4-ALK), resulting in significant changes to the treatment of lung cancer and an increase in survival for a subset of patients. These successes have prompted the search for additional driver alterations, leading to the discovery of a number of recurrently mutated or amplified genes and gene fusions with promising clinical utility. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also to new molecular markers and therapeutic targets. Using an integrative analysis of gene expression and copy number data to identify novel candidate oncogenes, we identified the chromosomal region at 12q13-15, and more specifically, the putative transcription factor YEATS4 (YEATS domain containing 4) as frequently amplified and overexpressed in NSCLC. Amplification of YEATS4 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma.Methods
Copy number profiles were generated for 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) and expression profiles for a subset of tumors with matched non-malignant tissue. Recurrent DNA amplifications were identified using the GISTIC algorithm. Copy number data were integrated with gene expression data to identify genes frequently amplified and overexpressed (defined as a 2-fold difference in expression between tumor and matched non-malignant tissue). The functional significance of YEATS4 was assessed by lentiviral knockdown in lung cancer cell lines with and without YEATS4 amplification and ectopic expression in human bronchial epithelial cells (HBECs). In vitro and in vivo assays measuring proliferation, anchorage independent growth, senescence, apoptosis, drug sensitivity and tumor growth were used to assess the phenotypic effect of YEATS4 gene expression manipulation.Results
YEATS4 is gained or amplified and concomitantly overexpressed in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC and SqCC. Although frequently co-amplified with MDM2, amplification of YEATS4 was observed to occur in the absence of MDM2 amplification, suggesting it is not merely a passenger event. Overexpression of YEATS4 in HBECs abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence in cell lines with YEATS4 amplification. Western blotting revealed increased p21, cleaved PARP and p53 in knockdown lines compared to empty vector controls, implicating YEATS4 as a negative regulator of the p21-p53 pathway. Moreover, YEAST4 expression was found to correlate with cisplatin sensitivity, as overexpression increased resistance and knockdown conferred sensitivity. Consistent with our in vitro findings, tumor size and growth were significantly reduced in mice injected with YEATS4 knockdown cells relative to control mice. Furthermore, survival analysis revealed that patients expressing high levels of YEATS display poorer outcomes.Conclusion
Our findings reveal YEATS4 as a novel candidate oncogene frequently amplified and overexpressed in NSCLC. Gene expression manipulation resulted in distinct phenotypic changes consistent with oncogenic function, and suggesting YEATS4 amplification is a novel mechanism contributing to NSCLC tumorigenesis.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.10 - ELF3 is a novel oncogene frequently activated by genetic and epigenetic mechanisms in lung adenocarcinoma (ID 1024)
17:10 - 17:15 | Author(s): K.S. Enfield, D.A. Rowbotham, D.D. Becker-Santos, R. Chari, M. Fuller, M. Zhang, M. Suzuki, C.E. Macaulay, A. Karsan, S. Lam, W.L. Lam
- Abstract
- Presentation
Background
Lung cancer remains the cause of the most cancer-related deaths each year, with a 5 year survival rate of less than 15%. The predominant type of lung cancer is non-small cell lung cancer, and the majority of these cases consist of the adenocarcinoma (AC) histology. Oncogenes such as EGFR and KRAS are well defined drivers of AC, but in approximately 50% of cases the driver alterations are unknown. Furthermore, not all defined drivers are drugable. Additional oncogenes are clearly involved in driving this subtype, and must be elucidated to better understand AC biology and improve treatment. ELF3 is an member of the E-Twenty Six (ETS) transcription factor family, which includes several well known oncogenes such as ETS1. Expression of ELF3 is uniquely epithelial-specific, with high expression in fetal but not adult lung tissue. ELF3 overexpression has been reported in a handful of clinical AC cases and cell lines, however a comprehensive analysis of the extent and impact of this overexpression is lacking. Therefore we conducted a multi-'omic, functional analysis of ELF3, and hypothesize ELF3 represents a novel oncogene in lung AC.Methods
ELF3 was interrogated in a multidimensional integrative manner by assessing copy number (SNP 6.0), methylation (Illumina HM27), and expression (Illumina) data from a panel of 83 AC tumors and matched adjacent non-malignant tissues. ELF3 expression was also assessed in The Cancer Genome Atlas (TCGA) public database. Stable ELF3 mRNA knock-down models were established in AC cell lines with high ELF3 expression, and these models were used to assess the role of ELF3 in cell viability and proliferation via MTT and BrdU incorporation assay, respectively. Knock-down models were also used to assess the impact of ELF3 overexpression on tumor growth in vitro and in vivo by soft agar colony formation assay and flank injections of NOD-SCID mice. Subcellular localization of ELF3 was determined by western blot and confirmed with immunofluorescence. In addition, an ELF3 overexpression model was established in immortalized Human Bronchial Epithelial Cells (HBECs) to assess proliferation and soft agar colony formation in a non-malignant model system.Results
ELF3 was found to be frequently overexpressed in our cohort (72%) and the TCGA cohort (80%). This upregulation correlated significantly with high frequencies of sequence gain (49%) and hypomethylation (71%), often seen within the same tumor. In fact, 82% of tumors with ELF3 overexpression had concurrent gain and/or hypomethylation of the ELF3 locus. Knock-down of ELF3 in cell models led to significantly reduced cell viability and proliferation. Western blot and IF revealed ELF3 to be predominantly located in the nucleus, indicating ELF3 likely behaves through its transcription factor activity. A similar hyperproliferative phenotype was seen in the HBEC ELF3 overexpression models.Conclusion
The high frequency of ELF3 overexpression (>70%) observed in lung AC is accompanied by frequent DNA-level selection events. The affect of ELF3 on cell proliferation suggests that ELF3 is a novel oncogene in lung AC. Further studies are warranted to determine the mechanism by which ELF3 drives hyperproliferation and potentially other oncogenic functions to define novel drugable targets for this disease.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.11 - Using synthetic lethal screening to identify therapeutic targets for innately platinum resistant lung cancer (ID 2629)
17:15 - 17:20 | Author(s): K. Marini, F. Rossello, L. Martelotto, N. Watkins
- Abstract
- Presentation
Background
Although platinum-based chemotherapy is the standard of care for most cases of advanced lung adenocarcinoma, its effectiveness is limited by the frequent incidence of innate chemoresistance. As a result, response rates rarely exceed 20%, even though cis-platinum and carboplatin are highly effective in other settings such as small cell lung, ovarian and testicular cancers. We hypothesized that innate chemoresistance in lung adenocarcinoma is mediated by one or more signalling pathways dependent on the expression of a single gene, and that these pathways could ultimately be targeted therapeutically.Methods
To address this question, we developed a synthetic-lethal high throughput siRNA screen using the innately resistant A549 lung adenocarcinoma cell line. Optimisation of the screen was performed using a siRNA death control (PLK1), which induced cell death in the absence of platinum, and a sensitization control (MTOR), which enhanced cell death only in combination with a sublethal concentration of carboplatin. These independent controls revealed that the screening protocol performed within acceptable limits of variability, quality and reproducibility as determined by Z’ factor analysis. Screening was then performed using a pool of four siRNAs targeting a single gene in conjunction with vehicle treatment, or with carboplatin.Results
After screening siRNAs targeting the 720 kinases, 256 phosphatases and 4794 “druggable” targets of the human genome, we identified 50 candidate targets based on fold change difference between platinum and vehicle treatments, and statistical significance determined by multiple t-test corrected for false discovery rate. Preliminary pathway analysis revealed a highly significant enrichment for genes in previously identified pathways as well as novel pathways.Conclusion
These data demonstrate that a synthetic-lethal approach can be used to identify therapeutic targets that could potentially sensitize lung adenocarcinoma to platinum-based chemotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO15.12 - DISCUSSANT (ID 3899)
17:20 - 17:35 | Author(s): P. Yang
- Abstract
- Presentation
Abstract not provided
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Author of
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MO20 - Preclinical Therapeutic Models II (ID 93)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 1
- Moderators:P. Waring, M. Kohonen-Corish
- Coordinates: 10/30/2013, 10:30 - 12:00, Bayside Gallery B, Level 1
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MO20.10 - DISCUSSANT (ID 3908)
11:25 - 11:40 | Author(s): G. Reid
- Abstract
- Presentation
Abstract not provided
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P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Moderators:
- Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P1.05-006 - Targeted delivery of RRM1-specific siRNA leads to tumour growth inhibition in malignant pleural mesothelioma (ID 1508)
09:30 - 09:30 | Author(s): G. Reid
- Abstract
Background
Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy with poor prognosis. MPM is typically recalcitrant to treatment and new therapies are urgently needed. Multiple genes involved in proliferation and metabolic activity are upregulated in MPM and these represent attractive targets for an siRNA-based therapeutic intervention.Methods
We carried out an RNAi-based screen of 40 target genes previously shown to be upregulated in MPM to identify candidate genes with roles in cell growth and survival in MPM cell lines. Effects of target gene silencing were measured using standard in vitro proliferation assays. Lead candidates were further assessed with siRNA dose response experiments. The specificity of siRNA-mediated growth inhibition was confirmed by assessing gene knockdown by real-time qPCR and Western blotting. The effects of the most potent siRNAs on xenograft tumour growth were assessed in vivo by delivery using EGFR-targeted, siRNA-loaded, minicells.Results
All 40 genes were effectively silenced, and for 6 genes (PLK1, CDK1, NDC80, RRM1, RRM2 and BIRC5) knockdown with 2 independent siRNAs resulted in significant growth inhibition over time in multiple cell lines. Dose response experiments revealed that siRNAs specific for RRM1 and RRM2 were the most effective at inhibiting growth with IC50 values in the low nanomolar range. Intravenous administration of RRM1 siRNA packaged in minicells targeted with EGFR-specific antibodies (2x10[9] minicells per dose, 4 times per week for 3 weeks) led to consistent and dose-dependent inhibition of MPM tumor growth compared with treatment with an inactive siRNA. Reducing the dose and number of administrations did not reduce growth inhibition; as little as 1x10[9] minicells administered once a week were sufficient to completely inhibit MPM tumour growth.Conclusion
RRM1 is an attractive target for siRNA-based inhibition, and siRNA delivery with EGFR-targeted minicells represents a novel therapeutic approach for MPM.
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P3.01 - Poster Session 3 - Cancer Biology (ID 147)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Moderators:
- Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P3.01-008 - The importance of the Secreted Frizzled-Related Protein (SFRP) tumour suppressor gene family and the effect of long-term asbestos exposure on SFRP expression in malignant pleural mesothelioma (MPM) (ID 3495)
09:30 - 09:30 | Author(s): G. Reid
- Abstract
Background
The etiology of malignant pleural mesothelioma (MPM) is closely linked with asbestos exposure. Asbestos is capable of inducing chronic inflammation which potentiates tumour suppressor gene silencing. Epigenetic silencing of the Wnt pathway, well characterized in the progression of colon cancer, is associated with chronic inflammation. As antagonists of Wnt pathways, the SFRPs are functional tumour suppressors of colon, gastric, breast, ovarian and lung cancers, with some members methylated in mesothelioma. In this study, we aimed to investigate the functional significance of the SFRP2 and 5 in MPM, and the effect of long-term asbestos exposure on epigenetic alteration in the immortalised mesothelial cell MeT-5A.Methods
Gene expression and promoter DNA methylation of the SFRP family were analysed in MPM lines and MeT-5A with and without 5’Azacitidine treatment using RT-qPCR, MSP and COBRA. The effect of SFRP2 and SFRP5 re-expression on MPM cells was determined by cell growth and clonogenic assays in 2D and 3D culture. The expression and promoter DNA methylation of SFRP genes was also assessed in MPM patient samples using RT-qPCR and MSP. MeT-5A cells were cultured long-term (12 months) in the presence of asbestos, and SFRP mRNA expression and promoter DNA methylation was analysed.Results
SFRP2 and SFRP5 were either absent or down-regulated MPM lines, and restored after 5’Azacitidine treatment. SFRP1 was highly expressed and unmethylated in MeT-5A line. Expression of the SFRP family was down-regulated in MPM patient samples and this correlated with methylation of promoter CpG islands. Ectopic expression of SFRP2 or SFRP5 inhibited MPM cell growth and colony formation in both 2D and 3D culture. SFRP1 was down-regulated and methylated following prolonged asbestos exposure in MeT-5A cells.Conclusion
Our results indicate that both SFRP2 and SFRP5 function as tumour suppressor genes in MPM and long-term asbestos exposure induce gene silencing via DNA hypermethylation.
