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H.A. Scarborough
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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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MINI09.01 - 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
- 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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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-056 - Use of Pooled shRNA Synthetic Lethal Screens within an In Vivo Murine Model to Identify Microenvironment-Dependent Lung Cancer Genes (ID 3140)
09:30 - 17:00 | Author(s): H.A. Scarborough
- Abstract
Background:
Lung cancer remains the leading cause of cancer-related deaths worldwide. While significant knowledge has been gained regarding the characterization of mutational drivers in NSCLC, much less is known regarding interactions between tumor cells and the surrounding microenvironment that are critical for tumor progression. Additionally, a significant limitation in current understanding is the lack of knowledge regarding which tumor gene products are necessary for promoting cell survival in the context of the tumor microenvironment. We hypothesize that the use of pooled shRNA synthetic lethal screens within an in vivo murine model will allow for the elucidation of targetable microenvironment-dependent genes.
Methods:
We generated a custom murine shRNA lentiviral library targeting 250 genes implicated in the communication between cancer cells and the microenvironment, which was used to transduce two murine cell lines: Lewis Lung carcinoma (LLC) and CMT167 cells. Following puromycin selection of cells harboring incorporated shRNA’s of interest, populations were expanded and designated for in vitro versus in vivo replication and growth. Selected cells were allocated to either in vitro passage vs direct in vivo injection into the lungs of 18 week-old syngeneic C57BL6 mice. After 4 weeks, cells were harvested and gDNA was isolated. Sequencing and quantitation of shRNA was performed using an Illumina deep-sequencing platform. Both raw and normalized read counts were assessed and analyzed to determine the relative representation of a particular shRNA within an in vitro or in vivo sample. Following quality control assessments which demonstrated adequate read count numbers per sample, and appropriate correlation of sample similarity per groups, direct comparisons between in vitro and in vivo samples were performed.
Results:
Multiple gene candidates were identified and largely reproducible via either rank analysis, mean, or t-test analyses. Candidate genes included multiple chemokines, and their receptors, matrix proteases, complement factors, and growth factor receptors.
Conclusion:
These results suggest a list of genes that are both intriguing and diverse, pointing toward gene products that would not have been previously predicted to influence cancer cell survival and growth through a lung cancer cell-autonomous fashion. Furthermore, these genes appear to potentially interact with multiple compartments of the tumor microenvironment including the extracellular matrix, cytokine milieu, vascular structures (complement factors), and the adaptive immune system. Validation of specific gene targets are ongoing through assessment of tumor growth comparing murine cell lines transfected with individual shRNA’s of interest vs control tumor cells. Furthermore, parallel pooled shRNA synthetic lethal screens within selectively adaptive immune-deficient models are currently in progress.
