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A. Tan
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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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MINI09.11 - Adaptor Re-Programming and Acquired Resistance in RET-Fusion Positive NSCLC (ID 2891)
17:45 - 17:50 | Author(s): A. Tan
- 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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MINI 23 - Lung Cancer Risk: Genetic Susceptibility and Airway Biology (ID 135)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:P.E. Postmus, R. Young
- Coordinates: 9/08/2015, 16:45 - 18:15, 401-404
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MINI23.10 - Subtraction of Allelic Fractions (Delta-θ): A Sensitive Metric to Detect Chromosomal Alterations in Heterogeneous Premalignant Specimens (ID 2434)
17:35 - 17:40 | Author(s): A. Tan
- Abstract
- Presentation
Background:
Lung squamous carcinoma is believed to arise from premalignant bronchial epithelial dysplasia, which demonstrates progressive histologic changes leading up to invasive cancer. However, only a small subset of these lesions progress to carcinoma. Recent studies have shown that somatic chromosomal alterations (SCAs) status is a better biomarker than premalignant histology alone. Single-nucleotide polymorphism microarray (SNP array) has been frequently used to delineate these genomic alterations across the whole genome. However, the cellular heterogeneity, from clinical samples such as endobronchial specimens, is a basic obstacle to perform sensitive and accurate detection of SCAs.
Methods:
We used: 1) a lung cancer cell line (NCI-H1395) and its matched lymphoblastoid (NCI-BL1395) cell line; 2) frozen lung tissues containing different percentage of invasive cancer cells surgically resected from a patient; and 3) biopsies and brushings obtained at the visually concerning areas during bronchoscopy. The histology of the clinical samples were graded by the study pathologist. Genomic DNA was isolated from each sample, quantified, and labeled for Illumina SNP array (HumanOmni 2.5-Quad BeadChip). Data analysis and visualizations were performed using Partek Genomic Suite 6.6 software.
Results:
Our study focused on the detection of SCAs by the comparison of genomic DNAs from cancer/premalignant cells (subject) to blood/normal cells (reference) from the same individual. We tested a B allele frequency metric, the subtraction of allelic fractions (delta-θ), on a standardized mixture of genomic DNAs from a lung cancer cell line and its matched lymphoblastoid cell line. Delta-θ proved to be a sensitive parameter to clearly delineate SCAs present in the tumor cell line even with a large proportion of normal cells (up to 90%). To explore the utility of using delta-θ for heterogeneous samples, we used clinical lung cancer specimens with known cancer cell content. In comparison to the other publicly available analytical metrics/algorithms (conventional Log R Ratio plot, mirrored B Allele Frequency plot, and GAP algorithm), delta-θ performed as well or better (with lower computational power needed), and enabled the detection of SCAs even in highly heterogeneous clinical samples (<30% tumor cell content). In addition, we completed a study using a number of bronchial biopsies and brushings with histologic grade ranging from normal to squamous cell carcinoma. SCAs were rarely detected in those of low to mild dysplasia, while they were detected in approximately 25% of moderate or severe dysplasia, and in all carcinoma in situ (CIS) and squamous cell carcinoma specimens. Longitudinal, repeated samplings from a high risk patient who persistently showed high grade dysplasia across the bronchus, revealed that delta-θ could identify SCAs continuously across the whole genome. The fact this individual had highly overlapping SCAs between different bronchial locations indicates genomic field cancerization may occur, along with the histological field effect in premalignant epithelium.
Conclusion:
In SNP microarray studies, delta-θ is a highly sensitive metric for detecting SCAs even in heterogeneous dysplastic bronchial specimens. SNP array may be a powerful tool to understand premalignant genetic alterations and field cancerization.
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.14 - The Aurora Kinase B Inhibitor AZD1152-HQPA Inhibitor in Small Cell Lung Cancer (SCLC) (ID 2161)
18:00 - 18:05 | Author(s): A. Tan
- Abstract
- Presentation
Background:
Aurora kinase expression has been associated with a poor prognosis in non-small cell lung cancer (NSCLC) and aurora kinase inhibitors have activity in preclinical lung models. Aurora kinases are required for mitosis and cell division. Small cell lung cancer cells have rapid proliferation and higher rates of MYC family amplification, which makes aurora kinase inhibition a natural target.
Methods:
23-SCLC lines with known MYC family amplification and MYC family gene expression were exposed to varying concentrations of the specific aurora kinase B inhibitor AZD1152-HQPA. The percentage growth inhibition compared to control was determined in MTS assays at 120 hours. Cell lines were classified as “sensitive” if the GI50 concentration was < 50 nM and with ≥ 80% growth inhibition at 100 nM. Fisher’s exact test was used to determine the correlation between amplification of MYC family members and sensitivity of the cell lines to growth inhibition by AZD1152-HQPA. A two-group t-test (gene expression as a continuous variable) and an odds ratio estimate (dichotomized gene expression level) were used to determine a correlation between MYC family gene expression and growth inhibition by AZD1152-HQPA. To determine whether growth inhibition correlated with the published MYC-signature gene expression, we used Fisher’s exact test. In vivo growth inhibition by AZD1152 (prodrug) was evaluated on SCLC xenografts in nude mice.
Results:
Nine (39%) of the 23 cell lines were sensitive to AZD1152-HQPA with IC50 values < 50 nM. There was a significant association between sensitivity to growth inhibition by AZD1152-HQPA and cMYC amplification (p = 0.018). The odds of being sensitive is 16 (95% CI, 1.4, 183) times higher for cMYC amplified compared to non-cMYC amplified cell lines. By a two-group t-test, the mean cMYC gene expression of 10.9 (std 4) in sensitive lines compared to 7.2 (std 3.3) in resistant lines was also significant (p = 0.026). Cell lines were separated into two groups based on cMYC gene expression > 12.9 vs < 12.9. The odds of being sensitive is 11 (95% CI, 1.2, 103) time higher for cell lines with cMYC gene expression > 12.9 compared to cell lines with cMYC gene expression < 12.9. Sensitive cell lines were enriched in a published MYC-signature of gene expression (p = 0.042). AZD1152 (prodrug) caused significant growth delay in vivo in two of these lines. The doses of AZD1152-HQPA used in this study are within the range reported to be clinically achievable.
Conclusion:
Aurora kinase inhibitors have promise in SCLC therapy. Questions that currently need answering in translating aurora kinase inhibitors in the clinical setting are: (1) the dosing schedule to avoid myelosupression, (2) should aurora kinase inhibitors be used in maintenance therapy and (3) should the aurora kinase inhibitors be evaluating in combination with chemotherapy.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-046 - Kinome RNAi Screens Identify Essential Genes and Therapeutics In 'Driver Negative' Non-Small Cell Lung Cancer (ID 2970)
09:30 - 09:30 | Author(s): A. Tan
- Abstract
Background:
Lung adenocarcinoma is a leading cause of cancer-related death worldwide. The discovery of “driver” mutations in genes such as the epidermal growth factor receptor (EGFR) which are required for malignant transformation have revolutionized lung cancer care as chemotherapy directed against these proteins has resulted in dramatic improvements in survival. Approximately 75% of the non-small cell lung cancer (NSCLC) patients harbor a driver gene (KRAS, EGFR, ALK, ROS etc) from the recent molecular characterization of lung adenocarcinomas by The Cancer Genome Atlas. Unfortunately, ~25% of the patients are “driver negative”. Therefore, identifying genes essential for malignant pathogenesis in these “driver negative” NSCLC may serve as new therapeutic targets for these patients.
Methods:
Four “driver negative” NSCLC cell lines – H292, H1703, H228, and H322C – were used in the kinome RNAi essential screen. Cells were transduced with a short-hairpin loop lentiviral kinome library (~3700 shRNAs targeting ~600 kinases) developed by The RNAi Consortium (TRC 1.0/1.5). Cells were cultured and harvested after 2, 7, and 14 days of transduction. ShRNAs from surviving cells were extracted, reverse transcribed and barcoded for individual replicates. These samples were sequenced on the Illumina HiSEQ 2000. BiNGS! software was used for analyzing and interpreting the essential screen. Kinases were considered essential if were present at day 2 but they were lost (i.e. knocked out causing cell death) at both day 7 and day 14. We then queried these essential kinases to K-Map, a bioinformatics platform that systematically connects a kinase profile with a reference kinase inhibitor database and predicts the most effective inhibitor for a queried kinase profile.
Results:
All samples from four cell lines had on average 85% of mapping rates (70% - 92%) with 5 to 24 million mapped reads per sample. In total, twenty kinases were identified as essential kinases for these “driver negative” cell lines. For example, MAPK4 for H292; ERBB3 for H322C; ATR for H228; and KDR for H1703. We queried the K-Map using the twenty essential and potentially transformative kinases to connect them to drugs. One of the top kinase inhibitors connected by K-Map was sunitinib. From published papers, we found that H1703 has been validated to be sensitive to sunitinib, supporting the K-Map prediction of inhibitors on the essential kinases. Further validation will be performed and presented in the conference.
Conclusion:
Functional genetic screens have the potential to identify genes essential for cancer cell survival and proliferation, providing a “functional” map in “driver negative” NSCLC. Using a series of novel bioinformatics analyses, specifically connecting the essential kinases with small molecules based on inhibition activities, we have identified that candidate drugs effectively inhibits the essential kinases in “driver negative” NSCLC cell lines resulting in cell death. Further investigation of these candidate drugs and the functional role of these essential kinases could provide personalized treatment for the “driver negative” lung cancer patients.
