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B.P. Levy
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MINI 21 - Novel Targets (ID 133)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 13
- Moderators:B.P. Levy, D.S. Tan
- Coordinates: 9/08/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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- Abstract
- Presentation
Background:
Purines are well known as intracellular sources for energy but also act as extracellular signaling molecules. In the last decade there has been a growing interest in the therapeutic potential of purinergic signaling for cancer treatment. The effects carried out depend on the concentration, expressed pattern of purinergic receptors and general dynamics of synthesis and degradation. In this study we analyze different purines and purinergic receptors in bronchoalveolar lavage (BAL) of patients with non-small-cell lung cancer (NSCLC) to provide further insight on their relevance in the tumor microenvironment.
Methods:
In this prospective clinical trial we enrolled 27 patients with NSCLC and 16 patients with chronic obstructive pulmonary disease (COPD) without signs of malignancy. The study was approved by our local ethics committee and registered as a clinical trial in the German Registry for Clinical Trials (DRKS-ID: DRKS00005415). BAL was performed using flexible bronchoscopes. The bronchoscope was wedged into a subsegment were the tumor was present and a total of 300 ml sterile saline was instilled. The BAL-fluid (BALF) was recovered by gentle aspiration. Purines (ATP, ADP, AMP, Adenosine and Inosine) were analyzed using fluorescence/luminescence based assays. Expression of purinergic receptors and Ectonucleotidases in NSCLC (P2X1, P2X4, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2Y13, P2Y14, CD39, CD73) were analyzed using qPCR.
Results:
Patients with NSCLC have significantly lower ATP and ADP concentrations in BALF than patients with COPD without signs of malignancy (p=0.006 and p=0.009). Inosine concentrations however are higher in patients with malignant disease (p=0.01). In the subgroup-analysis of metastasized versus non-metastasized tumors receptor-analysis revealed a higher expression of P2X4 (p=0.07), P2X7 (p=0.0008) and P2Y1 (p=0.009) as well as of the ectonucleotidase CD39 (p=0.007). Analysis of the purine metabolites in the respective groups showed no statistically significant differences. Furthermore there is a positive correlation of the proportion of macrophages in differential cell count in BAL with the expression of P2X7 (r=0.53, p=0.02).
Conclusion:
Previous data suggests pro-inflammatory, zytotoxic and thus anti-neoplastic effects of Adenosine-Triphosphate (ATP) and ADP. Also it has been shown that low ATP concentrations in the tumor microenvironment can lead to enhanced proliferation of tumor cells. In this first in vivo study on purinergic signaling in lung cancer we find lower concentrations of ATP and ADP in samples from NSCLC patients compared to COPD without signs of malignancy in accordance with these findings. Furthermore in aggressive, metastasized NSCLC we find a higher expression of the ectonucleotidase CD39. This enzyme degrades ATP and ADP to Adenosin and has previously been shown to hence induce immune escape in malignant disease. Furthermore we demonstrate elevated expression of P2X4, P2X7 and P2Y1 in the tumor microenvironment of metastasized NSCLC compared to non-metastasized tumors. This suggests a role of these receptors in tumor metastasis, however the exact mechanisms remain unclear. To further illustrate these interactions we are currently initiating a study to identify purinergic receptors in NSCLC tumor cells from pathologic specimen. With this knowledge future translational studies can be conducted to potentially provide new therapeutic targets.
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MINI21.02 - CCT68127 Is a Next Generation CDK2/9 Inhibitor with Potent Antineoplastic Activity Against Lung Cancer Cells (ID 554)
16:50 - 16:55 | Author(s): M. Kawakami, L.M. Mustachio, X. Liu, S. Hu, Y. Lu, D. Sekula, S. Freemantle, E. Dmitrovsky
- Abstract
- Presentation
Background:
Lung cancer growth was significantly repressed by the first generation CDK2/9/7 inhibitor seliciclib (R-roscovitine, CYC202, Cyclacel Ltd). This induced anaphase catastrophe and apoptosis to occur. Anaphase catastrophe happens when supernumerary centrosomes attempt mitosis by clustering extra centrosomes. If this clustering is inhibited, cells segregate chromosomes inappropriately and anaphase catastrophe occurs and leads to death of daughter cells. This study explored antineoplastic effects of a next generation CDK2/9 inhibitor: CCT68127 (Cyclacel) against lung cancer cells. CCT68127 inhibits CDK2/9 more potently and selectively than seliciclib (IC50s for CDK2 and CDK9 are 30nM and 110nM, respectively).
Methods:
Antineoplastic CCT68127 effects in murine (transgenic mouse-derived) and human lung cancer cells were compared to seliciclib using luminescent cell viability assays. Cell cycle arrest and apoptosis induction by CCT68127 were detected using fluorescence-based cell imaging after staining with propidium iodide (PI) and double-staining with Annexin V and PI. Multipolar anaphase cells were scored after a tubulin and DNA staining. RPPA (Reverse Phase Protein Assay) analyses were performed in CCT68127 and vehicle-treated lung cancer cells to uncover mechanisms engaged by CDK2/9 antagonism. Expression levels of nearly 200 key growth-regulatory proteins were examined before and after 6, 24, and 48 hours of CCT68127 versus vehicle treatments of murine: ED1 (wild-type KRAS) and LKR13 (mutant KRAS) and human lung cancer cells: H522 (wild-type KRAS) and Hop62 (mutant KRAS).
Results:
IC50s of CCT68127 in murine lung cancer cells (ED1, LKR13, and 393P) were <1µM while IC50 of seliciclib was >25µM. KRAS mutant murine lung cancer cells (LKR13 and 393P) were more sensitive to CCT68127 than the KRAS wild-type line (ED1). In contrast, growth inhibition in C10 immortalized murine pulmonary epithelial cells was negligible. IC50s in human lung cancer cell lines (Hop62, A549, H2122, H522, and H1703) were comparable to murine lung cancer cell lines. KRAS mutant lung cancer cells (Hop62, A549, and H2122) were more sensitive than KRAS wild-type lung cancer cell lines (H522 and H1703). Immortalized human bronchial epithelial cells (BEAS-2B) were resistant to CCT68127 treatment. CCT68127 triggered apoptosis in a dose-dependent manner in murine lung cancer cell lines and at much lower concentrations than seliciclib. CCT68127 caused G1 arrest. Its growth inhibition was partially reversed in washout experiments. CCT68127 also induced apoptosis in human lung cancer cells (Hop62, A549, H522, and H1703). A mechanism responsible for these effects was found. Anaphase catastrophe was triggered by CCT68127 treatment of murine and human lung cancer cell lines and was independent of KRAS mutation status. RPPA analyses uncovered distinct protein profiles after CCT68127 treatment. These included DNA repair, Hippo and Rab GTPase pathway members that were each markedly down-regulated.
Conclusion:
CCT68127 is a next generation CDK2/9 inhibitor that has more potent antineoplastic activity against KRAS mutant and wild-type lung cancer cells than the prior inhibitor, seliciclib. This occurred via induced anaphase catastrophe and was linked to changes in expressed growth regulatory proteins. Taken together, these findings implicate use of a next generation CDK2/9 inhibitor for human lung cancer cases.
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MINI21.03 - CACNA2D1 Is a Novel Biomarker for Tumor Initiating Cells and Has Therapeutic Effect in Non-Small Cell Lung Cancer (ID 3074)
16:55 - 17:00 | Author(s): Y. Ma
- Abstract
- Presentation
Background:
Tumor initiating cells (TICs) are a small subpopulation within cancer that is thought to be resistant to conventional therapies and capable of reinitiating tumors. However, only a few biomarkers of TICs have been well elucidated.
Methods:
By the methods of QPCR, FACS, western blot, colony formation and statistic analysis, we have investigated whether CACNA2D1 (α2δ1) to enrich TICs of non-small cell lung cancer (NSCLC) and and tharget therapy of its antibody.
Results:
In comparison to α2δ1[-], α2δ1[+] cells demonstrated greater TICs properties with higher potential of self-renewal, differentiation and reconstituting tumors. Following treatment, these cells were enriched in clinical samples. We verified a monoclonal antibody of α2δ1 mAb which targets to α2δ1 had therapeutic treatment to TICs of NSCLC and further α2δ1 mAb combined with the common anti-cancer drug of carboplatin was obtained to suppress the established xenograft tumors. Importantly, the disease free survival and overall survival of NSCLC patients with increased α2δ1 expression was significantly shorter than that of patients with decreased expression. Mechanically, our results showed that a role of α2δ1 in up-regulating stemness of NSCLC cells was associated with NOTCH signaling.
Conclusion:
Collectively, our data indicate that α2δ1 could be used as a marker for identifying TICs of NSCLC and targeting these cells might provide a way to treat this disease.
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- Abstract
- Presentation
Background:
Heparan sulfate D-glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1) participates in the biosynthetic steps of heparan sulfate (HS) and found to target VEGF in acute myeloid leukemia(AML) cells thus contributing the angiogenesis and proliferation of AML cells . However, the role of HS3ST3B1 in NSCLC has never been reported. In this study, we aim to investigate the role of HS3ST3B1 in NSCLC epithelial-to-mesenchymal transition.
Methods:
Expression of HS3ST3B1 was investigated by qRT-PCR in specimens of tumor and matched normal tissues of NSCLC patients and also in epithelial and mesenchymal NSCLC cell lines. A549 and HCC827 cell lines was induced to mesenchymal phenotype by TGF-β, and expression of HS3ST3B1, CDH1, and VIM were studied by PCR. HS3ST3B1 was knockdown by siRNA to analyze the effect of HS3ST3B1 on EMT. Computational predicting software was used to predict potential regulators of HS3ST3B1 and dual luciferase report system demonstrated that miR-218 may target HS3ST3B1 in cells. MiR-218 was tranfected into cells to analyze the association of miR-218 and HS3ST3B1 in cells.
Results:
HS3ST3B1 was significantly up-regulated in tumors compared with matched normal tissues(P=0.002). Its expression was also up-regulated in mesenchymal phenotype NSCLC cells lines compared with epithelial phenotype(P＜0.05). When epithelial phenotype NSCLC cells transformed to mesenchymal phenotype induced by TGF-β, HS3ST3B1 was also significantly up-regulated. Moreover, when HS3ST3B1 was knockdown by siRNA in mesenchymal phenotype NSCLC cell lines, cells were reversed to epithelial phenotype morphologically. With Targetscan, we found that HS3ST3B1 was one potential targets of miR-218 and dual luciferase report system demonstrated that HS3ST3B1 was direct target of miR-218 in cells. When miR-218 was transfected into cells, we found that HS3ST3B1 was down-regulated. Figure 1 Figure 2
Conclusion:
HS3ST3B1 may regulate EMT and it can be regulated by miR-218 in NSCLC.
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MINI21.05 - Discussant for MINI21.01, MINI21.02, MINI21.03, MINI21.04 (ID 3421)
17:05 - 17:15 | Author(s): S. Popat
- Abstract
- Presentation
Abstract not provided
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MINI21.06 - Role of the Focal Adhesion Protein Paxillin in Lung Cancer - From Genetic Alterations to Novel Mitochondrial Functionality (ID 2188)
17:15 - 17:20 | Author(s): R. Hasina, A. Rodriguez, S. Tumuluru, F. Lennon, Y.C. Tan, I. Kawada, R. Kanteti, R. Salgia
- Abstract
- Presentation
Background:
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer including lung cancer, which is attributed to a greater number of deaths than prostate, breast and colorectal cancers combined. Paxillin is a 68 kDa protein that is an integral part of the focal adhesion and acts as an adaptor molecule. We initially cloned the gene for paxillin, and localized it to chromosome 12q24. We have previously reported that paxillin can be mutated (approximately 8%), amplified (5-7%), and/or overexpressed in almost 80% of lung cancer patient samples. Paxillin protein is upregulated in more advanced stages of lung cancer compared with earlier stages and is a prognostic factor for non-small cell lung cancer (NSCLC). Paxillin gene is amplified in some pre-neoplastic lung lesions as well as neoplastic lesions. We identified 22 different variants of paxillin mutation in our initial investigation especially between the LD and the LIM domains (Jagadeeswaran et al. 2008). There are mutations that have been validated in the TCGA set. We selected six mutants to perform further studies ((P52L, A127T, P233L, T255I, D399N, and P487L as well as wild-type as control). Our investigations focused on an effort to understand the contribution of molecular abnormalities found in paxillin and their relationship to mitochondrial functionality.
Methods:
HEK293 cells as well as a paxillin null NSCLC cell line H522 was used to overexpress the above paxillin mutants and wild-type paxillin. Live cell confocal microscopy was performed to evaluate cell motility, immunoprecipitation to determine interaction with other proteins, and gene expression analysis was performed to evaluate effects on gene expression.
Results:
Among the mutations we investigated, we found that the most common paxillin mutant A127T in lung cancer cells enhanced cell proliferation, focal adhesion formation and co-localized with the anti-apoptotic protein B cell CLL/Lymphoma 2 (BCL-2), which among other sites also localizes to the mitochondria. We further found that when these variant clones of activating mutations were expressed in HEK293 cells, they conferred phenotypic changes resembling neoplastic cells. In gene chip microarrays assay investigating gene expression modulation conferred by these mutations in these same HEK293 cells, we found that P52L, A127T, T255I, P233L and D399N mutations, compared to wild-type paxillin, indeed modulated the expression of a significant number of genes. In particular, there were a number of mitochondrial signature proteins that were altered in the various mutants. Analyzing mitochondrial functions by measuring the interaction of these mutants with mitochondrial proteins MFN2, and DRP1, we identified that they alter mitochondrial dynamics, with significant fission rather than fusion. Paxillin also translocated from the focal adhesion to the mitochondrial membrane. In relationship to cisplatin responsiveness, PXN and mutant overexpression lead to cisplatin resistance.
Conclusion:
These data suggest that wild-type and mutant paxillin variants play a prominent role in neoplastic changes with direct implications in lung cancer progression and hence, its potential as a therapeutic target needs to be explored further.
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MINI21.07 - Oncogenic EZH2 Is an Actionable Target in Patients with Adenocarcinoma of the Lung (LUAD) (ID 3169)
17:20 - 17:25 | Author(s): B. Shi, C. Behrens, V. Vaghani, E. Riquelme, H. Lin, J.R. Canales, H. Lui, H. Kadara, I.I. Wistuba, G.R. Simon
- Abstract
- Presentation
Background:
The methyltransferase enhancer of zeste homolog 2 (EZH2) belongs to the polycomb repressive 2 complex (PRC2). EZH2 is upregulated in several malignancies including prostate, breast and lung cancer. The EZH2 protein forms one of the critical protein complexes of PRC2 by partnering with EED (embryonic ectoderm development) protein. This EED/EZH2 complex has been shown to interact with histone deacytelase (HDAC). This interaction is highly specific and HDAC does not interact with any other PRC2 protein complexes. In the present study, we investigated the link between EZH2 and HDAC in lung cancer cell lines and in human tumor tissue microarrays (TMAs). We also further investigated EZH2 as a marker for response to HDAC inhibitors.
Methods:
We analyzed EZH2 and HDAC1 mRNA expression in two lung adenocarcinoma datasets (MDACC n=152, and TCGA n=308), and correlated the gene expression with tumors’ clinico-pathological characteristics and patients’ outcome. To study the association of EZH2 and HDAC1 expression with response to the HDAC1 inhibitor suberanilohydroxamic acid (SAHA), we examined mRNA and protein expression by RT-PCR and Western blot, respectively, in twelve lung adenocarcinoma (LUAD) cell lines at baseline and after overexpression or knock-down of EZH2 or HDAC1 gene expression using siRNA. Response to (SAHA) in cell lines was measured by MTT assay and correlated with protein and mRNA expression levels of EZH2 and HDAC1.
Results:
Direct and positive correlation was found between EZH2 and HDAC1 expression NSCLC cell lines (P <0.0001). This correlation was confirmed in NSCLC specimens from MDACC (Spearman’s correlation r=0.416; p < 0.0001) and TCGA datasets (r=0.221; p <0.0001).Patients with high EZH2 and high HDAC1 expression in stage I NSCLC specimens of MDACC and TCGA datasets had lowest survival compared to the patients who had either or both low expressions. Overall survival in the univariate analysis (MDACC dataset; Hazard Ratio (HR)=2.97; p=0.031 and TCGA dataset; HR=2.6 and p=0.041) and multivariate analysis (MDACC; HR=2.92 and p=0.034 and TCGA; HR=3.17 p=0.016). When EZH2 expression was knock down, there was a significant reduction in HDAC1 expression; conversely, when HDAC1 was knocked down EZH2 expression was also decreased. These concordant change in expression was seen both at the protein and mRNA level. Importantly, while all 8 cell lines with high EZH2 protein expression responded to SAHA treatment with average inhibition rate reaching 73.1%, three out of four cell lines with low EZH2 expression had a significantly lower response rate to SAHA inhibition with average inhibition rate 43.2% (P<0.0001). Additionally, altering the expression of EZH2 concordantly altered the sensitivity to SAHA i. e. forced increased expression of EZH2 increased the response to SAHA and vice versa.
Conclusion:
Our data suggest that EZH2 and HDAC expression are correlated in LUAD cell lines in human tissue microarrays and overexpression of both is a negative prognostic indicator. Additionally we show that increased EZH2 expression predicts for response to HDAC inhibitors and thus could serve as a biomarker for selecting LUAD patients with HDAC inhibitors.
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MINI21.08 - Bombesin Receptor Subtype-3: An Underappreciated Growth Factor in Lung Cancer (ID 722)
17:25 - 17:30 | Author(s): P. Moreno, T.W. Moody, S.H. Lee, S.A. Mantey, R.T. Jensen
- Abstract
- Presentation
Background:
Human bombesin receptors (BnR) are one of the most frequently over-expressed receptor families by human lung-cancers. It is known the activation of the classical members of the BnR family (GRPR, NMBR), causes a marked effect on cell-signaling and growth, often autocrine in nature, on lung-cancer cells. In addition, it has been discovered that the orphan receptor related to the BnR family, BRS-3, is widely distributed in central/peripheral normal tissues, and numerous tumors include lung-cancer cells. However, in contrast to the classical BnRs (GRPR, NMBR), BRS-3 has received little attention in lung-cancer in large part due to the fact its natural ligand is still unknown, and also because, until recently, the lack of specific pharmacological tools to study it. To address this, in this study, we examined the frequency of hBRS-3 expression in lung-cancer cells, and the effects of specific hBRS-3 activation on cell-signaling and cell-function (growth) in different lung-cancer cell lines.
Methods:
17 human lung-cancer (LC) cell lines were studied (9 NSCLC, 8 SCLC), as well as hBRS3 transfected H1299 and Balb 3T3 cells. The BRS-3 selective agonist, MK-5046 and selective BRS-3 antagonist, Bantag-1 were used. BnR expression was assessed by PCR using specific primers for hBRS3, hGRPR or hNMBR. Receptor activation was determined by assessing PLC and MAPK cascade activation using Western Blotting, and cytosolic Ca[2+] release. Proliferation was studied by clonogenic and [[3]H]-Thymidine assays, and EGFR transactivation was assessed using Western blotting.
Results:
Of the 17 LC cell lines, 9 (53%) express hBRS3 [H358, H460, H520, H720, H727, H69, H82, N417, H510], 14 (82%) express hGRPR [H28, H157, H358, H520, A549, H838, H1299, SK-LU-1, H720, H727, H69, H82, H345, H510] and 13 (77%) express hNMBR [H28, H157, H358, A549, H838, H1299, SK-LU-1, H720, H727, H82, H345, N417, H510]. MK-5046 stimulated PLC activation in 6/9 cells which express hBRS3 (H358, H720, H727, H69, H82, N417), and MAPK activation in all 9 hBRS3 cell lines. Cytosolic Ca[2+] increased with MK-5046 addition in all hBRS-3-containing cells, included Balb and H1299 transfected cells, except in H358 cells. Similarly, MK-5046 increased [3]H-Thymidine uptake in 5/9 cells (H460, H520, H720, H727, H82, H510), as well as in Balb and H1299 hBRS-3 transfected cells, and this increase was in a dose-response manner in H727, H69 and N417 cells. MK-5046 stimulated the clonal growth of N417 and H727 cells. MK-5046 addition to H358, H460, H727 (NSCLC) and H69, N417, H510 (SCLC) caused Tyr[1068] phosphorylation of the EGFR.
Conclusion:
These results show the orphan human BnR, hBRS-3 is present in more than one-half of human lung cancer cells. Furthermore, these receptors are functional with their activation effecting cell signaling (MAPK, PLC, Ca[2+]) and cell growth. Transactivation of EGFR is likely an important transduction cascade. These results suggest this orphan BnR, similar to classical BNRs, will be important to assess for growth effects and expression in human lung tumors, and its pharmacological inhibition may be a useful therapeutic approach.
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MINI21.09 - Discussant for MINI21.06, MINI21.07, MINI21.08 (ID 3422)
17:30 - 17:40 | Author(s): J.S. Lee
- Abstract
- Presentation
Abstract not provided
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MINI21.10 - The TORK/DNA-PK Inhibitor CC-115 Shows Combination Anti-Proliferative Effects with Erlotinib in NSCLC Cells Resistant to EGFR Inhibition (ID 641)
17:40 - 17:45 | Author(s): S. Ekman, D. Chan, M. Wynes, Z. Zhang, K. Hege, E. Filvaroff, H. Raymon, R. Hassan, L. Rozeboom, F.R. Hirsch
- Abstract
Background:
In non-small cell lung cancer (NSCLC), activation of the phosphoinositide-3-kinase (PI3K)/mTOR pathway is common in tumors resistant to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). CC-115 (Celgene Corporation), an mTOR kinase inhibitor that targets both mTORC1 and mTORC2 as well as DNA-dependent protein kinase (DNA-PK), is currently under early clinical development. We evaluated CC-115 in combination with Erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.
Methods:
In the present study we investigated whether CC-115 is able to increase the therapeutic effect of the EGFR TKI Erlotinib in several different NSCLC cell lines which exhibit intermediate or high resistance to EGFR TKIs: A549, H1975, H1650, HCC95, H2122 and H23. Mechanisms of inhibition were analyzed with assays for proliferation, apoptosis, and cell cycle progression. Cell signaling activity was analyzed using phospho-specific antibodies in Western blotting. Xenograft mice studies were performed to confirm the results in vivo.
Results:
CC-115 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.07 up to 6.9 mM. The anti-proliferative efficacy of Erlotinib was increased in the NSCLC cells synergistically by combination treatment with CC-115 with combination indices down to 0.04-0.2, indicating strong synergy. The synergistic, anti-proliferative effect of the combination treatment could be explained by increased cell cycle arrest and inhibition of signaling components in the mTOR pathway, especially 4E-BP1. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of Erlotinib and CC-115.
Conclusion:
We demonstrate that the therapeutic effect of the EGFR tyrosine kinase inhibitor Erlotinib can be increased by simultaneous treatment with the mTOR kinase/DNA-PK inhibitor CC-115, justifying further clinical studies in lung cancer patients with primary or acquired resistance to EGFR TKIs.
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MINI21.11 - A Novel Cell Line Model of EGFR Exon 20 Insertion Mutations (ID 2828)
17:45 - 17:50 | Author(s): A. Estrada-Bernal, A.T. Le, H. Zhou, S.A. Noonan, D.L. Aisner, R. Camidge, R.C. Doebele
- Abstract
- Presentation
Background:
In-frame insertions in exon 20 of EGFR are infrequent activating mutations in the tyrosine kinase domain that have decreased sensitivity to EGFR inhibitors and currently have no available targeted therapies. In vitro studies ectopically expressing some of the common insertions (3 to 21 bp between codons 762 and 770) show reduced sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Non-small cell lung cancer (NSCLC) patients whose tumors harbor these mutations do not respond to EGFR kinase inhibitors. To date, there are no known patient-derived cell lines that harbor the EGFR exon 20 insertions that recapitulate patient insensitivity to EGFR TKIs. Here we report the isolation and characterization of a patient derived cell line with an EGFR exon20 insertion.
Methods:
The CUTO-14 cell line was derived from a malignant pleural effusion of a lung adenocarcinoma patient harboring the EGFR exon 20 insertion p.A767_V767dupASV after obtaining IRB-approved informed consent. PCR amplification of EGFR exon 20 and subsequent Sanger sequencing was performed on genomic DNA isolated from CUTO-14. H3255 (L858R) and HCC827 (exon 19 del) cell lines were used as controls because they harbor sensitizing EGFR mutations. Cell viability was evaluated by MTS proliferation assay. Phosphorylation status and signaling was analyzed by western blot and an EGFR phosphorylation array. For tumor xenograft studies, nude mice were injected with 1.5 x 10[6] cells in matrigel and evaluated weekly for tumor growth.
Results:
Genomic sequencing of CUTO-14 demonstrated that the cell line maintains the pA767_V767dupASV EGFR exon 20 insertion. CUTO-14 showed relative resistance to gefitinib inhibition compared to HCC827 and H3255 in ERK1/2 phosphorylation assays. CUTO-14 also demonstrated reduced sensitivity to gefitinib compared to HCC827 and H3255 in cell proliferation assays. Tumor formation was observed in mice after injection in nude mice.
Conclusion:
CUTO-14 cells represent a novel model for the investigation of therapeutic strategies for EGRF exon 20 insertions mutations. The cell line has the ability to develop tumors in vivo and importantly shows reduced sensitivity to EGFR TKIs mimicking the lack of response in patients with these mutations.
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MINI21.12 - Identification of a First in Class TWIST1 Inhibitor with Activity in KRAS Mutant NSCLC (ID 1616)
17:50 - 17:55 | Author(s): L. Mazzacurati, J. Cades, S. Chatley, Z. Yochum, K. Nugent, A. Somasundarum, Y. Cho, C.M. Rudin, P. Tran, T.F. Burns
- Abstract
- Presentation
Background:
Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that the basic helix-loop-helix transcription factor, Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models, and that inhibition of Twist1 in murine models and KRAS mutant NSCLC cell lines led to oncogene-induced senescence (OIS) and in some cases, apoptosis. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact.
Methods:
We used gene expression profiles from KRAS mutant human NSCLC cell lines following shRNA-mediated TWIST1 knockdown to perform connectivity map (CMAP) analysis to identify pharmacologic inhibitors of TWIST1. Growth inhibition was determined through the colony formation and MTS assays. Apoptosis (cl-PARP, active anti-C3) and OIS (SA-β-Gal) was assessed. Genetic (shRNA) and pharmacologic inhibition of the TWIST1-E2A pathway was performed. Lung tumor burden as well as levels of TWIST1 protein, apoptosis and proliferation were measured after treatment with harmine in the CCSP-rtTA/tetO-KrasG12D/Twist1-tetO7-luc(CRT) mice.
Results:
We found that several of our CMAP compounds had significant growth inhibitory effects in NSCLC cell lines. Interestingly, a family of related harmala alkaloids including harmine ranked highly in our CMAP analysis. We observed that harmine could inhibit growth in KRAS mutant NSCLC cell lines through the induction of OIS or apoptosis and phenocopied genetic inhibition of TWIST1. Remarkably, harmine treatment led to TWIST1 protein degradation as well as degradation of its binding partners, the E2A proteins, E12/E47. Furthermore, the growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1 and were independent of its ability to inhibit the DYRK kinases. In addition, we demonstrated that heterodimer formation of TWIST1/E12/E47 resulted in a reciprocal stabilization of each binding partner and that E12/E47 are required for TWIST1 mediated suppression of OIS and apoptosis. Importantly, we found that harmine preferential targets the TWIST1-E12 heterodimer for degradation and the growth inhibitory effects of harmine are in due in at least part to the ability to inhibit the TWIST1/E12/E47 heterodimer as overexpression of the E2A proteins can suppress harmine induced cytotoxicity. Finally, we have demonstrated that harmine treatment lead to Twist1 protein degradation and tumor growth inhibition in our Kras[G12D]/Twist1 murine model of lung adenocarcinoma. We are currently testing and designing structure analogs of the initial candidate agents to develop more specific and potent inhibitors of TWIST1.
Conclusion:
We have identified a novel TWIST1 inhibitor harmine that induces degradation of TWIST1 and its binding partners, E12/E47 and inhibits the growth of KRAS mutant NSCLC both in vitro and in vivo. Therefore, we believe that targeting the TWIST1-E2A pathway would be an effective therapeutic strategy. Since TWIST1 is essential not only for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC, the development of this novel class of TWIST1 inhibitors could have a significant clinical impact.
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MINI21.13 - Discussant for MINI21.10, MINI21.11, MINI21.12 (ID 3423)
17:55 - 18:05 | Author(s): G. Giaccone
- Abstract
- Presentation
Abstract not provided
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MS 01 - How to Treat Multiple GGO's (ID 19)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Treatment of Localized Disease - NSCLC
- Presentations: 1
- Moderators:J. Donington, M. Tsuboi
- Coordinates: 9/07/2015, 14:15 - 15:45, 102+104+106
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MS01.03 - Is There a Role for Targeted Therapy or Conventional Chemotherapy in Patients with Multiple GGO's? (ID 1850)
15:00 - 15:20 | Author(s): B.P. Levy
- Abstract
- Presentation
Abstract:
Recent advances in diagnostic imaging and the use of low dose screening CT scans for high risk individuals has increased detection of ground-glass opacities (GGOs). These lesions are defined as hazy lung opacities on CT with preservation of bronchial and vascular markings, and are typically classified as pure, without a solid component, or mixed,with a solid component[1, 2]. While GGOs have historically been characterized by slow growth and indolent tumor biology, their pathogenesis is poorly understood, and progression can be variable. It remains unclear whether these lesions represent dissemination of malignant cells from a single primary tumor (intrapulmonary spread) or synchronous development of multifocal independent clones [3]. In addition, depending on their size and solid component, GGOs can exist anywhere along the histological spectrum from adenomatous hyperplasia (AAH) to invasive adenocarcinoma (AC). Histologic heterogeneity can be reflected in uncertain growth rates, making therapeutic decision making challenging. While historically GGOs have been managed surgically or with close surveillance, chemotherapeutic strategies have been employed. In addition, recent identification of relevant, driver mutations within GGOs has allowed for consideration of targeted therapies including tyrosine kinase inhibitors (TKIs). Given that GGOs frequently represent bronchioloaveolar carcinoma (BAC) (recently reclassified as adenocarcinoma in situ, lepidic predominant adenocarcinoma or mucinous adenocarcinoma), an overview of chemotherapeutic and targeted strategies for such lesions would require extrapolation from the BAC literature. Despite longstanding recognition of BAC as a distinct subclass of lung adenocarcinoma, few completed prospective trials are available to inform on therapy decisions. To date, only two, small phase II prospective studies evaluating cytotoxic chemotherapy for treatment naïve patients with multi-focal BAC have been published. Both trials evaluated single agent paclitaxel and resulted in disappointing response rates (RR) of 11% and 15%, respectively [4, 5]. Post hoc analysis from the sentinel ECOG 1594 study demonstrated a response rate of only 6% to platinum chemotherapy in patients with BAC [6]. In contrast, the French IFCT-0401 trial demonstrated a RR of 21% and PFS of 3 months in 43 patients with BAC who received chemotherapy (platinum doublet; N=38) after disease progression on first line gefitnib[7]. Experience with third-generation agents such as pemetrexed or gemcitabine has been described only in case reports or retrospective series; however, these agents have demonstrated acceptable outcomes and may be considered in systemic treatment plans Subgroup analysis of early studies evaluating the role of EGFR TKIs in NSCLC demonstrated disproportionate and often dramatic responses in those tumors formally classified as BAC. This observation led to several trials exploiting gefitinb or erlotinib as initial therapy for patients with BAC. While overall responses rates in these studies were similar to studies evaluating chemotherapy (RR: 15 to 25%) patients with EGFR mutations derived greater benefit. For example, in a study evaluating erlotinib as initial therapy for patients with advanced BAC, the RR for those with EGFR mutations was 87% compared to 7% for those without EGFR mutations [8]. The association of EGFR mutations with GGOs and the non-mucinous subtype of BAC is supported by multiple studies including a recent comprehensive analysis evaluating genetic alterations in 217 resected GGOs from 215 lung cancer patients[9]. In this study, EGFR mutations were detected in 119 (54.8%). Other relevant driver mutations, including ALK mutations (2.8% in the aforementioned study evaluating resected GGOs) and KRAS mutations in mucinous subtype of AIS, have also been identified. This allows for consideration of other targeted therapies including ALK directed therapies (crizotinib, certinib, alectinib) and those targeting KRAS (selumetinib). Despite the well-established paradigm of offering targeted therapy to molecularly characterized subgroups, the clinical scenarios for patients with multiple GGO’s can be unique in several ways. Should these patients, if confirmed to have EGFR mutations, be treated with TKIs if lesions are slow growing or not growing at all? Or, should the indolent biology of such lesions trump the actionable mutation when making a therapeutic decision? In addition, the notion that a mutation discovered on a biopsied or resected GGOs is representative of all GGOs within a patient may be incorrect. A recent analysis evaluating 72 resected GGO lesions from 35 patients, all with more than one GGO, demonstrated a high rate of mutation discrepancy. In this study, 80% of patients (24/30) who harbored at least one genetic alteration had a driver mutation discrepancy within another GGO supporting the hypothesis that multiple GGOs seem to arise from different primary clones [10]. Given that patients with GGOs represent a spectrum of tumor biology and clinical behavior, an individualized approach that affords flexibility should be employed when implementing treatment strategies (Figure 1). Management decisions need to factor in the potential for indolent disease with consideration of a watch and wait approach for stable or slow growing lesions. If the clinician identifies progressive disease, systemic options should be entertained and include both chemotherapy and TKIs for patients who harbor actionable mutations. Further studies are needed to better define the clonal relationship of GGOs in an effort to optimize targeted approaches for such patients.Figure 1 References: 1. Hansell, D.M., et al., Fleischner Society: glossary of terms for thoracic imaging. Radiology, 2008. 246(3): p. 697-722. 2. Godoy, M.C. and D.P. Naidich, Subsolid pulmonary nodules and the spectrum of peripheral adenocarcinomas of the lung: recommended interim guidelines for assessment and management. Radiology, 2009. 253(3): p. 606-22. 3. Chung, J.H., et al., Epidermal growth factor receptor mutation and pathologic-radiologic correlation between multiple lung nodules with ground-glass opacity differentiates multicentric origin from intrapulmonary spread. J Thorac Oncol, 2009. 4(12): p. 1490-5. 4. West, H.L., et al., Advanced bronchioloalveolar carcinoma: a phase II trial of paclitaxel by 96-hour infusion (SWOG 9714): a Southwest Oncology Group study. Ann Oncol, 2005. 16(7): p. 1076-80. 5. Scagliotti, G.V., et al., A phase II study of paclitaxel in advanced bronchioloalveolar carcinoma (EORTC trial 08956). Lung Cancer, 2005. 50(1): p. 91-6. 6. Schiller, J.H., et al., Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med, 2002. 346(2): p. 92-8. 7. Duruisseaux, M., et al., Chemotherapy effectiveness after first-line gefitinib treatment for advanced lepidic predominant adenocarcinoma (formerly advanced bronchioloalveolar carcinoma): exploratory analysis of the IFCT-0401 trial. J Thorac Oncol, 2012. 7(9): p. 1423-31. 8. Miller, V.A., et al., Molecular characteristics of bronchioloalveolar carcinoma and adenocarcinoma, bronchioloalveolar carcinoma subtype, predict response to erlotinib. J Clin Oncol, 2008. 26(9): p. 1472-8. 9. Ko, S.J., et al., Epidermal growth factor receptor mutations and anaplastic lymphoma kinase rearrangements in lung cancer with nodular ground-glass opacity. BMC Cancer, 2014. 14: p. 312. 10. Wu, C., et al., High Discrepancy of Driver Mutations in Patients with NSCLC and Synchronous Multiple Lung Ground-Glass Nodules. J Thorac Oncol, 2015. 10(5): p. 778-83.
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ORAL 22 - Moving Beyond a Smoking Related-Cancer to the Young, Never-smokers and Inherited Disease (ID 117)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:K. Suda, H.A. Wakelee
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL22.08 - Discussant for ORAL22.05, ORAL22.06, ORAL22.07 (ID 3562)
12:01 - 12:11 | Author(s): B.P. Levy
- Abstract
- Presentation
Abstract not provided
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P1.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 206)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.01-082 - A Phase III Study of MEDI4736 (M) an Anti-PD-L1 Antibody ± Tremelimumab (T), vs Standard of Care (SoC), in Patients with Advanced NSCLC (ARCTIC) (ID 1237)
09:30 - 09:30 | Author(s): B.P. Levy
- Abstract
Background:
M is a human IgG1 mAb that blocks programmed cell death ligand-1 (PD-L1) binding to programmed cell death-1 and CD-80 with high affinity and selectivity, and T is a selective human IgG2 mAb inhibitor of cytotoxic T-lymphocyte antigen-4 (CTLA-4). Both PD-L1 and CTLA-4 are regulators, or checkpoints, of T-cell activation. PD-L1 expression may be associated with greater clinical benefit of anti-PD-1/PD-L1 agents. Thus, the subset of patients with PD-L1-negative tumors represent a cohort with limited therapeutic options, and may benefit from the combination of M+T. Preclinical data, including mouse models of transplantable solid tumors, suggest that targeting both pathways may have synergistic antitumor activity. Emerging pharmacokinetics, pharmacodynamics, safety and efficacy data from a phase Ib study of M+T in advanced NSCLC (NCT02000947) has determined the appropriate dose for this combination.
Methods:
This randomized, open label, multi-center, phase III study (NCT02352948) is designed to evaluate the efficacy and safety of M (10mg/kg once every 2 weeks [Q2W] for up to 12 months) vs SoC (gemcitabine 1000 mg/m[2] iv Days 1, 8, and 15, vinorelbine 30 mg/m[2] iv on Days 1, 8, 15 and 22 or erlotinib 150 mg once daily, on a 4-weekly schedule until PD at the investigator’s discretion) in NSCLC patients with PD-L1-positive tumors (based on archival tumor sample or recent biopsy) (Sub-study A), and the combination of M+T (M 20mg/kg + T 1mg/kg Q4W for 12 weeks then M alone 10mg/kg Q2W for 34 weeks) vs M or T (10mg/kg Q4W for 24 weeks then Q12W for 24 weeks) vs SoC in NSCLC patients with PD-L1-negative tumors (Sub-study B). PD-L1-positive is defined as ≥25% of tumor cells with membrane staining based on central assessment. Approximately 300 patients will be randomized 1:1 in Sub-study A and approximately 600 patients in a 3:2:2:1 ratio (M+T or SoC or M or T) in Sub-study B. Retreatment with immune-therapy is allowed within the setting of PD. For both sub-studies, an interim analysis for OS (and also PFS for Sub-study B) will be performed. Eligible patients include patients (PS of 0-1) with locally advanced or metastatic NSCLC, who have received at least 2 prior treatment regimens including 1 platinum-based chemotherapy. Patients with brain metastases or spinal cord compression are excluded unless asymptomatic, treated and stable off steroids. Patients with known EGFR activating mutations or ALK rearrangements are not eligible, nor patients previously exposed to any anti-PD-1 or anti-PD-L1 antibody. The primary objective is to assess PFS (per RECIST 1.1 as assessed by the Blinded Independent Central Review) and OS of M (PD-L1-positive) and M+T (PD-L1-negative), compared with SoC, in sub-study A and B, respectively. Secondary objectives include proportion of patients alive at 12 months, objective response rate, duration of response, PFS at 6 and 12 months, safety, tolerability, pharmacokinetics, immunogenicity and health-related QoL. Tumor assessments are performed every 8 weeks (first 48 weeks) then every 12 weeks. A confirmatory scan is required following the initial demonstration of PD. Recruitment in the study is ongoing since January 2015.
Results:
Not applicable
Conclusion:
Not applicable
