Qudsia Arif

Dramatic Antitumor Effects of the Dual MET/RON Small-Molecule Inhibitor LY2801653 in Non–Small Cell Lung Cancer

Lung cancer is a heterogeneous disease encompassing a wide array of genetic abnormalities. The MET receptor tyrosine kinase is altered in many lung cancers, especially non-small cell lung cancer (NSCLC), and clinical trials of MET inhibitors that are under way are documenting cases of acquired resistance. On the basis of the evidence that the RON tyrosine kinase receptor can also be overexpressed in NSCLC, we evaluated the potent MET/RON dual kinase inhibitor LY2801653 in this setting. LY2801653 was more efficacious than the MET/ALK/RON/ROS inhibitor crizotinib with a distinct pattern of downstream signaling effects. Using the PamGene platform, we found that inhibition of MET and RON was associated with decreased phosphorylation of CBL, PI3K, and STAT3. In classic and orthotopic mouse xenograft models of lung cancer, LY2801653 decreased tumor growth, dramatically inhibiting mitotic events and angiogenesis. Taken together, our results argued that specific targeting of the MET/RON kinases could provide robust inhibition of cell proliferation and tumor outgrowth in multiple in vitro and in vivo models of NSCLC. These findings offer a robust preclinical proof of concept for MET/RON targeting by LY2801653 as a promising small-molecule modality to treat NSCLC.

MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.

Paxillin mutations affect focal adhesions and lead to altered mitochondrial dynamics: Relevance to lung cancer

Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.

Malignant Mesothelioma Diagnosis

CONTEXT Malignant mesothelioma is a relatively rare pleural tumor that may mimic benign mesothelial lesions and various other tumors including carcinomas and sarcomas. This makes the diagnosis challenging for the pathologist. OBJECTIVE To provide a brief but useful update on the immunohistochemical, cytogenetic, and molecular markers that are currently available for the diagnosis of malignant mesothelioma. DATA SOURCES Reference materials including peer-reviewed publications, text books, and consensus opinion reports among pathologists. CONCLUSIONS It is important to correlate histologic findings on adequate biopsy samples with clinical and radiologic features. Useful diagnostic mesothelial markers include calretinin, WT-1, cytokeratin 5/6, and D2-40 (podoplanin). It is recommended that at least 2 mesothelial and 2 carcinoma markers with greater than 80% sensitivity and specificity be used for the diagnosis of mesothelioma when all clinical, radiologic, and histologic features are concordant. p16 deletion is reported in up to 70% of primary epithelioid and 90% to 100% of sarcomatoid pleural mesotheliomas. Presence of this homozygous gene deletion is so far the best indicator of mesothelioma. To date, this deletion has not been reported in any benign mesothelial lesion. The impact of various histologic patterns on the clinical and prognostic aspects of mesothelioma is addressed. The pleomorphic pattern, when present in more than 10% of tumor, translates into a highly aggressive behavior and is associated with poor survival. Recent studies have shown that the high-grade subgroup of deciduoid mesothelioma with pleomorphic histologic pattern also has a more aggressive clinical course. Nuclear grade (combination of nuclear atypia and mitotic count) may also prove to be an independent prognostic factor.

Proteomic characterization of non-small cell lung cancer in a comprehensive translational thoracic oncology database.

BackgroundIn recent years, there has been tremendous growth and interest in translational research, particularly in cancer biology. This area of study clearly establishes the connection between laboratory experimentation and practical human application. Though it is common for laboratory and clinical data regarding patient specimens to be maintained separately, the storage of such heterogeneous data in one database offers many benefits as it may facilitate more rapid accession of data and provide researchers access to greater numbers of tissue samples.DescriptionThe Thoracic Oncology Program Database Project was developed to serve as a repository for well-annotated cancer specimen, clinical, genomic, and proteomic data obtained from tumor tissue studies. The TOPDP is not merely a library--it is a dynamic tool that may be used for data mining and exploratory analysis. Using the example of non-small cell lung cancer cases within the database, this study will demonstrate how clinical data may be combined with proteomic analyses of patient tissue samples in determining the functional relevance of protein over and under expression in this disease.Clinical data for 1323 patients with non-small cell lung cancer has been captured to date. Proteomic studies have been performed on tissue samples from 105 of these patients. These tissues have been analyzed for the expression of 33 different protein biomarkers using tissue microarrays. The expression of 15 potential biomarkers was found to be significantly higher in tumor versus matched normal tissue. Proteins belonging to the receptor tyrosine kinase family were particularly likely to be over expressed in tumor tissues. There was no difference in protein expression across various histologies or stages of non-small cell lung cancer. Though not differentially expressed between tumor and non-tumor tissues, the over expression of the glucocorticoid receptor (GR) was associated improved overall survival. However, this finding is preliminary and warrants further investigation.ConclusionThough the database project is still under development, the application of such a database has the potential to enhance our understanding of cancer biology and will help researchers to identify targets to modify the course of thoracic malignancies.

Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma.

Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.

Combined MET Inhibition and Topoisomerase I Inhibition Block Cell Growth of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)–induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC. Mol Cancer Ther; 13(3); 576–84. ©2013 AACR.

O-6-methylguanine-deoxyribonucleic acid methyltransferase methylation enhances response to temozolomide treatment in esophageal cancer

Background: World-wide, esophageal cancer is a growing epidemic and patients frequently present with advanced disease that is surgically inoperable. Hence, chemotherapy is the predominate treatment. Cytotoxic platinum compounds are mostly used, but their efficacy is only moderate. Newer alkylating agents have shown promise in other tumor types, but little is known about their utility in esophageal cancer. Methods: We utilized archived human esophageal cancer samples and esophageal cancer cell lines to evaluate O-6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT) hypermethylation status and determined sensitivity to the alkylating drug temozolomide (TMZ). Immunoblot analysis was performed to determine MGMT protein expression in cell lines. To assess and confirm the effect of TMZ treatment in a methylated esophageal cancer cell line in vivo, a mouse flank xenograft tumor model was utilized. Results: Nearly 71% (12/17) of adenocarcinoma and 38% (3/8) of squamous cell carcinoma (SCC) patient samples were MGMT hypermethylated. Out of four adenocarcinoma and nine SCC cell lines tested, one of each histology was hypermethylated. Immunoblot analyses confirmed that hypermethylated cell lines did not express the MGMT protein. In vitro cell viability assays showed the methylated Kyse-140 and FLO cells to be sensitive to TMZ at an IC50 of 52-420 μM, whereas unmethylated cells Kyse-410 and SKGT-4 did not respond. In an in vivo xenograft tumor model with Kyse-140 cells, which are MGMT hypermethylated, TMZ treatment abrogated tumor growth by more than 60%. Conclusion: MGMT methylation may be an important biomarker in subsets of esophageal cancers and targeting by TMZ may be utilized to successfully treat these patients.

Abstract LB-239: Inhibition of MET/RON in lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Lung cancer can be a heterogeneous group of disorders. We have identified that the MET receptor tyrosine kinase is overexpressed, mutated, and/or amplified in various subsets of lung cancer. Several clinical trials of MET inhibitors that are underway have documented cases of acquired resistance. Based on evidence that the RON tyrosine kinase receptor (family member of MET receptor) can also be overexpressed in NSCLC, we determined the effect of sequential and concurrent inhibition of MET and RON with gene silencing. We identified that both MET and RON gene silencing led to inhibition of signal transduction, but that the combination was more effective. We evaluated the potent MET/RON dual kinase inhibitor LY2801653 in this setting and found it to be more efficacious than the MET/ALK/RON/ROS inhibitor crizotinib with a distinct pattern of changes in downstream signaling. Using the protein phosphorylation detection platform PamGene®, we found that inhibition of MET and RON was associated with decreased phosphorylation of CBL, PI3K and STAT3. A systematic analysis of the PamGene® data allowed us to define abnormalities of the signal transduction pathways. We further report that LY2801653 also has biological and tumor inhibitory effect both in vitro and in vivo. In mouse flank and orthotopic lung xenografts of NSCLC, LY2801653 decreased tumor growth, dramatically inhibiting mitotic events and angiogenesis.. Taken together, our results argued that specific targeting of the MET/RON kinases could provide robust inhibition of cell proliferation and tumor growth in multiple in vitro and in vivo models of NSCLC. These findings offer a robust preclinical proof of concept for MET/RON targeting by LY2801653 as a promising small molecule treatment modality. Methods: LY2801653 was provided by Eli Lilly and Company (Indianapolis, IN). NSCLC cell lines purchased from ATCC were utilized to conduct in vitro and in vivo studies. Cell proliferation assays were performed using MTT, standard immunoblot assays and the high-throughput protein phosphorylation detection platform PamGene® was used to demonstrate phosphorylation events. To determine drug effects in vivo, the mouse flank xenograft model and an orthotopic lung tumor model was used. In the orthotopic model, tumor progression was monitored by bioluminescence using Xenogen IVIS®. Results: We have reported that LY2801653 specifically inhibited proliferation of NSCLC cell lines at IC50 as low as 6-600 nM with inhibition of phosphorylation in downstream signaling of MET/RON. The PamGene® platform also showed a network of downstream signaling molecules affected by MET/RON inhibition, such as CBL, PI3K and STAT3. We further report that LY2801653 also has tumor growth inhibitory effects in mouse xenografts and dramatic effects on mitosis and angiogenesis. Conclusions: Specific inhibition of MET/RON by a small molecule inhibitor LY2801653 was able to abrogate cell proliferation, tumor growth, as well as affect downstream signal transduction of MET/RON in NSCLC. These results demonstrate the promising therapeutic potential of LY2801653 in treating lung cancer. Citation Format: Rifat Hasina, Ichiro Kawada, Qudsia Arif, Jeffrey Mueller, Aliya N. Husain, Everett E. Vokes, Ravi Salgia. Inhibition of MET/RON in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-239. doi:10.1158/1538-7445.AM2014-LB-239

Abstract 1861: EPHB4 as a potential therapeutic target for esophageal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Despite substantial improvements in screening, diagnosis, and treatment of esophageal cancer, the prognosis of this disease remains bleak. Survival at 5 years for all esophageal cancer patients taken together, with amenable treatments and with or without surgery ranges from 5-20% (Cancer Facts and Figures. American Cancer Society 2011). Although a multidisciplinary approach to include surgery, radiation, and chemotherapy, alone or in combination, attempts to improve the survival of this aggressive disease, these statistics underscore the continued need for attention to this disease and for identification of new targets in its treatment. We have been studying the role of receptor tyrosine kinases (RTKs) in cancer, particularly the EphB4 receptor, which has become increasingly associated with the pathobiology of adult cancers over the past several years. It has been shown to be aberrantly expressed and/or to play an oncogenic role in cancers of the breast, bladder, ovary, uterus, colon, head and neck, and prostate through its effects on cellular motility, growth, and migration, as well as on tumors’ ability to induce neoangiogenesis. To date, its role and potential as a target for therapy in different cancers have not been thoroughly investigated and remain poorly understood. Targeted inactivation of EphB4 and its ligand Ephrin-B2 have demonstrated that both are essential for angiogenic remodeling and embryonic survival (Adams and Klein 2000; Kim, Hu et al. 2008). In this report, we determine that EphB4 is overexpressed, has increased gene copy number and is involved in enhanced motility and migration in esophageal cancer. Archival patient samples consisting of 93 squamous cell carcinoma, 100 adenocarcinoma and 25 adjacent normal control samples as well as four adenocarcinoma and nine squamous cell carcinoma cell lines were used for the studies. Extensive mouse modeling of esophageal cancer was also used. We have reported that there is consistently higher expression of EphB4 in both squamous and adenocarcinoma compared to adjacent normal tissue with a statistically significant correlation between EphB4 expression and higher grades of squamous cell carcinoma. In a chemically induced esophageal squamous cell carcinoma model in mice, EphB4 was found overexpressed compared to normal controls. This study identifies EphB4 to be an important pathway in esophageal neoplastic lesions. It would now be useful to bring this to clinical fruition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1861. doi:1538-7445.AM2012-1861