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S.B. Keysar
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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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ORAL21.01 - Adaptive Survival Signaling in Oncogenic Fusion Kinase Addicted NSCLC (ID 864)
10:45 - 12:15 | Author(s): S.B. Keysar
- Abstract
Background:
Gene fusions involving the proto-oncogenes ALK, ROS1, RET and NTRK1 are established or potential drug targets in cancer. Although targeted kinase inhibitors induce significant tumor shrinkage, complete patient responses are rare, and it is from that residual tumor burden that drug resistant clones eventually emerge. We have previously shown a role for WT EGFR signaling in ROS1+ cancer cells and their drug resistant derivatives. We hypothesized that EGFR performs a similar role in cancer cells harboring other gene fusions.
Methods:
Fusion oncogene NSCLC cell lines were treated as described and analyzed through immunoblot analyses or fixed onto chamber slides and assayed using kinase-adaptor proximity ligation assays (PLA). FFPE from NSCLC patients treated at the University of Colorado Hospital were also analyzed using kinase-adaptor PLAs. Nu/nu mice were injected with fusion oncogene positive NSCLC cell lines, treated as described, and volumes were measured 3x/week. FFPE tumors from mice were analyzed using various immunohistochemical markers or kinase-adaptor PLAs.
Results:
Stimulation of NSCLC cells that harbor an oncogenic fusion with EGF not only increased downstream signaling, but also rapidly increased phosphorylation of the fusion kinase itself. Additionally, EGFR signaling can dictate the engagement of different downstream signaling effectors, diversifying the signaling and cell fate responses in certain cancer cells. Proximity ligation assays (PLA) were employed to visualize wild-type EGFR-GRB2 signaling complexes in NSCLC cells driven by an oncogenic fusion kinase. We observed two modes of EGFR-GRB2 complex formation, the first in unperturbed cells, and the second only when the fusion kinase was inhibited. The kinetics of the induction of EGFR-GRB2 signaling revealed EGFR can take over the signaling in these cells as quickly as 5 minutes, and this kinase inhibitor-induced rewiring can be reversed by simply washing out the drug, suggesting a preference for the fusion kinase in the signaling circuit of these cells. Analysis of fusion-positive patient samples acquired at the time of progressive disease from treatment with an oncogene targeted monotherapy revealed the presence of EGFR-GRB2 signaling complexes. Additional analyses of patient samples revealed evidence of potentially non-cell autonomous responses to these therapies that may enable the survival of cells that would otherwise be drug-sensitive. The combination of a fusion kinase inhibitor with anti-EGFR therapy provided superior blockage of EGFR and ALK signaling complexes, as well as improved reduction in tumor volume and prolonged survival in an ALK+ xenograft model.
Conclusion:
Collectively, these results demonstrate a previously unknown role for an unmutated kinase, EGFR, in modulating the oncogenic phenotype in cells addicted to oncogenic fusion kinases. The activation of the EGFR signaling pathway can quantitatively augment fusion kinase signaling, but also diversify it by regulating the engagement of alternate signaling effector proteins. This data provides evidence for a novel role for EGFR as an oncorequisite signaling partner in certain cancer cell populations that harbor an oncogenic fusion kinase. Combination therapy of a fusion kinase targeted inhibitor with anti-EGFR therapy may improve initial tumor cell killing, and delay or prevent the onset of drug resistance in these patient populations.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.01 - FISHing TRK Activation by Gene Rearrangements in Non Small Cell Lung Cancer (ID 834)
16:45 - 18:15 | Author(s): S.B. Keysar
- Abstract
- Presentation
Background:
The tropomyosin-receptor kinase (TRK) family includes genes important in nervous system development, NTRK1 (N1), NTRK2 (N2) and NTRK3 (N3). Oncogenic activation was identified long ago as N1 fusions in colon cancer and numerous fusions have been recently identified affecting all family members in multiple tumor types. This study developed FISH reagents for molecular diagnosis of NTRK rearrangements and investigated their prevalence in NSCLC. The ultimate goal is to validate a clinical assay for selection of patients who may benefit from novel tyrosine kinase inhibitors (TKIs) targeting these fusion proteins.
Methods:
Three FISH break-apart (BA) probe sets (LDTs) were tailored for diagnosis of rearrangements in N1, N2 and N3 and tested in specimens with known genomic status for these genes: cell lines KM12 (N1), CUTO3 (N1), MO-91 (N3), xenograft CULC001 (N1), and clinical specimens, and used to screen resected NSCLC. The LSI NTRK1 Cen and Tel probes (Abbott Molecular) were also tested. A specimen was positive for individual rearrangement when ≥15% tumor cells had split or single 3’,5’ signals. Moreover, a 6-target, 2-color FISH probe including the 3’N1, 3’N2 and 3’N3 sequences labeled in red and the 5’N1, 5’N2 and 5’N3 sequences labeled in green (TRKombo) was designed for rapid screening of TRK rearrangements in clinical specimens.
Results:
Results were obtained in 443, 410, and 434 examined NSCLC and positive patterns were detected in 5, 5 and 1 specimens, respectively for N1, N2, and N3. These 11 positive patients had age ranging from 38y to 76y, gender 6 male:5 female, and were current (4), former (5) or never (2) smokers. Histology was predominantly adenocarcinoma (7) but also included squamous cell (3) and neuroendocrine morphology (1). Unique to the N1 assay was the observance of FISH signal fusions where the 5’N signals appeared as doublet in >20% of the NSCLC specimens, which was determined to be copy number variation due to segmental duplication. Other atypical patterns were observed for all three targets and included doublets of the FISH fusion signals (18%, 14% and 9% respectively) and gene clusters (~5% for each). Twenty specimens (pre-clinical models and clinical cases) characterized as positive by the LDT N1 and by next generation sequencing (NGS) or atypical by the LDT NTRK1 BA were blindly analyzed with the LSI NTRK1 probe set and the results were reproducible, with brighter intensity of the fluorescent signals for the LSI probe. These specimens (positive by FISH and several atypicals) are currently under investigation to characterize the sequence specific genomic rearranged region by using a custom targeted, capture-based NGS panel (NimbleGen, Roche). The TRKombo screening probe performed well in blinded experiment using validation set including pre-selected positive and negative specimens and is under testing in clinical tissue sections.
Conclusion:
N1, N2 and N3 fusions were detected by FISH in a subset of lung carcinomas including adeno, squamous and neuroendocrine tumors. Optimization of molecular panels for diagnosis of these rearrangements is relevant since they represent a sizeable number of cases across multiple tumor types and there are numerous targeted inhibitor agents under development.
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