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S. Nelson
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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)
16:45 - 18:15 | Author(s): S. Nelson
- 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 14 - Pre-Clinical Therapy (ID 119)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Fernandez-Cuesta, A.F. Gazdar
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI14.04 - In Vitro and in Vivo Evaluation of the Kinase Inhibitor, MGCD516, in TRK and RET Fusion Cancer Cells (ID 2756)
10:45 - 12:15 | Author(s): S. Nelson
- Abstract
- Presentation
Background:
The paradigm of treating oncogene-selected patients with non-small cell lung cancer (NSCLC) and other malignancies using targeted kinase inhibitors has significant improved patient outcomes, specifically for patients harboring ALK, ROS1, and EGFR oncogenes. Additional oncogene targets that may benefit from this therapeutic strategy are therefore of immense interest. NTRK1 (TRKA) and RET gene fusions are recently identified oncogenes in NSCLC (and other malignancies) without approved kinase inhibitors. MGCD516 is a spectrum-selective tyrosine kinase inhibitor with activity against TRKA, RET, MET, VEGFR, PDGFR, AXL, and Eph family of receptors. In this report, we evaluated MGCD516 in vitro activity in cell lines with an NTRK1, NTRK3, or RET gene rearrangements. Additionally, we used a mouse xenograft model to assess the in vivo effects of MGCD516 on tumors harboring TRKA and RET fusions.
Methods:
Gene fusion positive cell lines, KM12 (TPM3-NTRK1), CUTO-3 (MPRIP-NTRK1), MO-91 (ETV6-NTRK3) and LC-2/Ad (CCDC6-RET) were used for the in vitro evaluation of MGCD516 inhibitory activity against these oncogenic fusion kinases. Cell lines were assessed for cell viability (MTS-base proliferation assay) and downstream signaling pathways (immunoblot analysis) upon treatment with MGCD516. For in vivo studies, xenograft models of TRKA fusion tumors (CUTO-3 and KM12) and RET fusion tumors (LC-2/Ad and a tumor biopsy from a KIF5B-RET patient) were generated in athymic nude mice. Once tumors reached ~200cm[3], a single daily dose of 5mg/kg, 10mg/kg or 20mg/kg of MGCD516 was given to mice by oral gavage. Mice in the control arm of the study were gavaged with vehicle at similar volume. Tumor size and weight measurement of mice were assessed 3 times per week.
Results:
MGCD516 had notable in vitro effects on the proliferation of cell lines with either RET fusion (LC-2/Ad), TRKA fusion (KM12 and CUTO-3) or TRKC fusion (MO-91) with low nanomolar IC~50~. Western blot analyses showed specific loss of phosphorylated CCDC6-RET or TRKA/C fusion protein and decreased activation of the AKT and MAPK signaling pathways when cells were treated with MGCD516. In mouse xenograft studies, tumors with TRKA fusion displayed dose-dependent growth inhibition at 5mg/kg and 10mg/kg daily doses of MGCD516 compared to controls. Notably, we observed tumor regression in the mice originally assigned to the vehicle control arm once we enrolled the mice on a 10mg/kg or 20mg/kg daily regimen of MGCD516. Comparable to the TRKA fusion xenografts studies, RET fusion tumors were growth inhibited with a 20mg/kg daily dose of MGCD516.
Conclusion:
The spectrum-selective tyrosine kinase inhibitor, MGCD516, demonstrates potent in vitro activity in multiple TRKA/C and RET fusion cancer cell line models and in vivo activity against TRKA and RET fusion kinase in murine xenograft models. A phase I clinical trial of MGCD516 is ongoing and the inclusion of patients with TRK and RET fusion is planned.
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