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L. Heasley
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MS 26 - Genomic Alterations and Drug Targets in Small Cell Lung Cancer (ID 44)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:D. Beer, J.W. Goldman
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 2c-3c
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MS26.04 - FGFR1 Co-Activation Networks in Lung Cancer (ID 1966)
14:15 - 15:45 | Author(s): L. Heasley
- Abstract
- Presentation
Abstract:
FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of FGFR-specific and selective tyrosine kinase inhibitors (TKIs) are underway. Our recent studies have demonstrated a role for unmutated FGFR1 as a driver in lung cancer cell lines of all histologies including small cell lung cancers (SCLCs), head and neck squamous cell carcinomas (HNSCCs) and mesotheliomas. Although potent in vitro growth suppression of lung cancer cell lines is observed in response to multikinase inhibitors such as ponatinib as well as FGFR-specific TKIs (AZD4547, BGJ398), the in vivo inhibitory effects of these drugs on xenografts propagated in immune deficient mice are more modest and short-lived in our hands. Thus, while treatment with single FGFR TKIs represents a logical entry point to personalized therapy of cancers bearing over-expressed FGFR1, we hypothesize that intrinsic mechanisms involving rapid kinome reprogramming events limit the therapeutic efficacy of these TKIs. In fact, ample precedent exists to support the signaling of receptor tyrosine kinases (RTKs) within "co-activation networks" where multiple RTKs engage multiple signal pathways to bring about robust and flexible activation of signal cascades. We deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and HNSCC cells to ponatinib, a multi-kinase FGFR-active inhibitor. Mammalian Target of Rapamycin (MTOR) was identified and validated as a synthetic lethal kinase with ponatinib in H157 and H1299 cells. In other FGFR1-expressing cell lines (Colo699, H520 and H1703), MTOR was an essential protein kinase as evidenced by high sensitivity to MTOR-targeting shRNAs and pharmacological inhibitors. Despite wide ranging MTOR dependencies observed among the FGFR1-dependent cell lines, synergistic in vitro growth inhibition was a general observation when FGFR inhibitors where combined with pharmacological inhibitors of MTOR or AKT. At the molecular levels, FGFR inhibitors potently inhibited MEK/ERK activity while MTOR inhibitors reduced the activity of TORC1 (p70S6K, S6) and TORC2 (AKT Ser473)-specific targets. In combination, FGFR TKIs and MTOR inhibitors simultaneously eliminated MEK/ERK and MTOR signaling. Xenografts generated from the FGFR1-dependent lung cancer cell lines, Colo699 and H1581, exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547. However, consistent with the in vitro findings, combination treatment with AZD4547 and the MTOR inhibitor, AZD2014, afforded significantly greater tumor growth inhibition and prolonged survival. The data support the existence of a signaling network wherein unmutated FGFR1 drives the ERK pathway and distinct receptors under investigation activate the MTOR/AKT pathway to induce full transformation. Combining MTOR inhibitors with FGFR-specific TKIs may yield greater clinical efficacy in FGFR1-driven lung cancers.
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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): L. Heasley
- 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 42 - Drug Resistance (ID 160)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:R.C. Doebele, J.V. DeGregori
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 4a-4f
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ORAL42.03 - Discussant for ORAL42.01, ORAL42.02 (ID 3441)
18:30 - 20:00 | Author(s): L. Heasley
- Abstract
- Presentation
Abstract not provided
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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: 2
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-006 - MiRNA Signature to Assess Sensitivity to FGFR Tyrosine Kinase Inhibitors (ID 1717)
09:30 - 17:00 | Author(s): L. Heasley
- Abstract
Background:
Increased signaling through the FGF/FGFR signaling pathway has been implicated as a driver in a number of different malignancies including lymphomas, prostate cancer, breast cancer, and lung cancer. This pathway also appears to play a role in conferring de novo and acquired resistance to cancers driven by EGFR mutations. Consequently, drugs that inhibit FGFRs are being investigated as potential therapeutics for cancer. Here we screened a large panel of miRNAs as potential predictors of sensitivity to FGFR tyrosine kinase inhibitors (TKIs).
Methods:
A panel of 377 miRNAs (Megaplex Card A, Life Technologies) was screened for expression level differences between four lung cancer cell lines that are sensitive (IC~50~< 50 nM) and four lines that are resistant (IC~50~ > 100 nM) to ponatinib (non-specific FGFR TKI) and AZD4547 (FGFR-specific TKI). Expression levels were assayed by RT-qPCR and analyzed using the Statistical Analysis of Microarrays (SAM) method. Thirty-nine miRNAs having an estimated false discover rate (FDR) of zero and large median fold differences (> 8) between the sensitive and resistant lines were selected for signature development. RT-qPCR assays were incorporated into a custom microfluidics card (Life Technologies), which was used to profile the original 8 cell lines and 10 additional sensitive lines and 16 additional resistant lines (34 lines total). Logistic regression was then used to identify the best signature panel for distinguishing sensitive cell lines from resistant.
Results:
Univariate analysis indicated three miRNAs (let-7c, miR-338, and miR-218) that differed between the sensitive and resistant lines at p < .05. The best signature panel consisted of let-7c, miR-200a and miR-200b, which gave an area under the receiver operator characteristic (AUROC) curve of 0.90 (95% CI = 0.8 to 1). This performance was nearly as good as using FGFR1 mRNA alone (AUROC = 0.94). The predominant miRNA in our 3-miRNA signature was let-7c, which also exhibited a suggestive additive effect to using FGFR1 as a biomarker (p = 0.09). We also tested whether cell lines with high sensitivity to ponatinib can be made resistant by reducing the high level of let-7c in these lines. We have found that transient transfection of let-7c silencing RNA (Life Technologies) produces a decrease in FGFR1 mRNA levels for some cell lines but not others.
Conclusion:
It appears possible to predict sensitivity to an FGFR1 inhibitor using miRNA expression signatures. More studies, however, are needed to confirm the 3-marker signature developed in this study. Modulating let-7c, the predominant predictor within the signature, appears to modulate FGFR1 levels in a manner consistent with altering ponatinib sensitivity. This effect is most likely indirect as the mRNA of FGFR1 does not contain predicted binding sites for let-7c.
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P2.04-007 - In Vitro and in Vivo Efficacy of AZD9291 Is Enhanced by Combination with AZD4547 in EGFR Mutant Lung Cancer Cells (ID 2456)
09:30 - 17:00 | Author(s): L. Heasley
- Abstract
Background:
EGFR-specific tyrosine kinase inhibitors (TKIs) provide marked clinical responses in patients bearing EGFR mutated lung tumors, although acquired resistance limits the durability of the response. In light of the frequent emergence of erlotinib and gefitinib-resistant EGFR T790M mutations upon tumor progression, 3[rd] generation EGFR-specific TKIs have been developed that specifically inhibit gain-of-function EGFR mutants irrespective of T790M status. Recently, we reported a distinct mechanism of acquired resistance whereby specific EGFR mutant lung cancer cell lines including H1650 and HCC4006 cells, but not PC9 cells, undergo an epithelial-mesenchymal transition (EMT) upon chronic in vitro treatment with gefitinib. As a result, the adapted cells acquire vulnerability to FGFR inhibitors by virtue of EMT-mediated FGF2 and FGFR1 induction. Herein, we have tested the hypothesis that combination of the FGFR inhibitor, AZD4547, with the 3[rd] generation EGFR TKI, AZD9291 will yield superior anti-tumor activity relative to AZD9291 alone.
Methods:
Lung cancer cell lines bearing gain-of-function EGFR mutations (HCC4006, H1650 and PC9) were submitted to in vitro clonogenic growth assays in the presence of AZD9291 and/or AZD4547 over concentration ranges for 1 to 300 nM for each drug. For in vivo measurement of the activity of these drugs, flank xenografts were established in Nu/Nu mice with the 3 lung cancer cell lines and treated by daily oral gavage (5 days on, 2 days off) with diluent, AZD9291 (5 mg/kg), AZD4547 (12.5 mg/kg) and the combination of the two drugs at these doses. Tumor size was measured with calipers and volume was calculated using the formula, Volume=3.14(short diameter)[2](long diameter)/6.
Results:
HCC4006, H1650 and PC9 cells were highly sensitive to ZD9291 in vitro with IC~50~ values of 1.6, 7.4 and 3.3 nM, respectively. In a 2 week clonogenic growth assay, AZD9291 reduced growth of all cell lines by >95%, although viable drug resistant persisters clearly remained. While none of these cell lines exhibited significant growth inhibition in response to AZD4547 alone, combination of AZD9291 and AZD4547 further reduced clonogenic growth of HCC4006 and H1650 cells, but not PC9 cells. In flank xenograft studies, AZD9291 monotherapy induced marked tumor shrinkage (H1650, ~80% at day 10; HCC4006, ~90% at day 30; PC9, 89% at day 25), although regrowth of the tumors occurred with all three xenografts. AZD4547 yielded little or no growth inhibition as a monotherapy, but significantly enhanced the degree of tumor shrinkage and delayed the time to tumor progression in H1650 and HCC4006 tumors, but not PC9 tumors.
Conclusion:
Combination of the FGFR inhibitor AZD4547 with AZD9291 affords greater growth suppression relative to AZD9291 alone in HCC4006 and H1650 cells that undergo EMT and induction of an FGF2-FGFR1 pathway. Predictably, this combination was not more effective compared to AZD9291 alone in PC9 cells that fail to undergo EMT in response to EGFR TKI treatment. The studies support the efficacy of combined AZD9291 and AZD4547 treatment of a subset of lung tumors driven by mutated EGFR, although the features of these particular lung tumors that predict this response is unknown at this time.
