Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion.

During tumor development, constant interplay occurs between tumor cells and surrounding stromal cells. We report evidence that gastrointestinal stromal tumor (GIST) cells invade the interstitial stroma through the release of the oncogenic protein tyrosine kinase (KIT)-containing exosomes, which triggers the phenotypic conversion of progenitor smooth muscle cells to tumor-promoting cells. These recipient cells display morphologic changes and acquire tumor-associated phenotypes, including enhanced adhesion to extracellular matrix proteins, activation of intracellular pathways downstream of KIT, expression of Interstitial Cell of Cajal-like markers, and release of various matrix metalloproteinases (MMPs), particularly MMP1. This report shows stimulation of MMP1 production by stromal cells via uptake of tumor-derived exosomes, which leads to tumor cell invasion. Exosomes derived from GIST patients but not healthy donors show enhanced MMP1 secretion by smooth muscle cells and tumor cell invasion, whereas selective blocking of exosome-mediated MMP1 secretion decreases tumor invasiveness. Our study indicates that exosome release and subsequent MMP1 induction creates a positive feedback mechanism established between tumor and stromal cells that drives GIST development and offers unique insights for potential therapeutic strategies to block GIST progression and metastatic spread.

PTN signaling: Components and mechanistic insights in human ovarian cancer.

Molecular vulnerabilities represent promising candidates for the development of targeted therapies that hold the promise to overcome the challenges encountered with non-targeted chemotherapy for the treatment of ovarian cancer. Through a synthetic lethality screen, we previously identified pleiotrophin (PTN) as a molecular vulnerability in ovarian cancer and showed that siRNA-mediated PTN knockdown induced apoptotic cell death in epithelial ovarian cancer (EOC) cells. Although, it is well known that PTN elicits its pro-tumorigenic effects through its receptor, protein tyrosine phosphatase receptor Z1 (PTPRZ1), little is known about the potential importance of this pathway in the pathogenesis of ovarian cancer. In this study, we show that PTN is expressed, produced, and secreted in a panel of EOC cell lines. PTN levels in serous ovarian tumor tissues are on average 3.5-fold higher relative to normal tissue and PTN is detectable in serum samples of patients with EOC. PTPRZ1 is also expressed and produced by EOC cells and is found to be up-regulated in serous ovarian tumor tissue relative to normal ovarian surface epithelial tissue (P < 0.05). Gene silencing of PTPRZ1 in EOC cell lines using siRNA-mediated knockdown shows that PTPRZ1 is essential for viability and results in significant apoptosis with no effect on the cell cycle phase distribution. In order to determine how PTN mediates survival, we silenced the gene using siRNA mediated knockdown and performed expression profiling of 36 survival-related genes. Through computational mapping of the differentially expressed genes, members of the MAPK (mitogen-activated protein kinase) family were found to be likely effectors of PTN signaling in EOC cells. Our results provide the first experimental evidence that PTN and its signaling components may be of significance in the pathogenesis of epithelial ovarian cancer and provide a rationale for clinical evaluation of MAPK inhibitors in PTN and/or PTPRZ1 expressing ovarian tumors.

The Integrated Genomic Landscape of Thymic Epithelial Tumors.

Thymic epithelial tumors (TETs) are one of the rarest adult malignancies. Among TETs, thymoma is the most predominant, characterized by a unique association with autoimmune diseases, followed by thymic carcinoma, which is less common but more clinically aggressive. Using multi-platform omics analyses on 117 TETs, we define four subtypes of these tumors defined by genomic hallmarks and an association with survival and World Health Organization histological subtype. We further demonstrate a marked prevalence of a thymoma-specific mutated oncogene, GTF2I, and explore its biological effects on multi-platform analysis. We further observe enrichment of mutations in HRAS, NRAS, and TP53. Last, we identify a molecular link between thymoma and the autoimmune disease myasthenia gravis, characterized by tumoral overexpression of muscle autoantigens, and increased aneuploidy.

Multiscale modelling of relationships between protein classes and drug behavior across all diseases using the CANDO platform.

We have examined the effect of eight different protein classes (channels, GPCRs, kinases, ligases, nuclear receptors, proteases, phosphatases, transporters) on the benchmarking performance of the CANDO drug discovery and repurposing platform (http://protinfo.org/cando). The first version of the CANDO platform utilizes a matrix of predicted interactions between 48278 proteins and 3733 human ingestible compounds (including FDA approved drugs and supplements) that map to 2030 indications/diseases using a hierarchical chem and bio-informatic fragment based docking with dynamics protocol (> one billion predicted interactions considered). The platform uses similarity of compound-proteome interaction signatures as indicative of similar functional behavior and benchmarking accuracy is calculated across 1439 indications/diseases with more than one approved drug. The CANDO platform yields a significant correlation (0.99, p-value < 0.0001) between the number of proteins considered and benchmarking accuracy obtained indicating the importance of multitargeting for drug discovery. Average benchmarking accuracies range from 6.2 % to 7.6 % for the eight classes when the top 10 ranked compounds are considered, in contrast to a range of 5.5 % to 11.7 % obtained for the comparison/control sets consisting of 10, 100, 1000, and 10000 single best performing proteins. These results are generally two orders of magnitude better than the average accuracy of 0.2% obtained when randomly generated (fully scrambled) matrices are used. Different indications perform well when different classes are used but the best accuracies (up to 11.7% for the top 10 ranked compounds) are achieved when a combination of classes are used containing the broadest distribution of protein folds. Our results illustrate the utility of the CANDO approach and the consideration of different protein classes for devising indication specific protocols for drug repurposing as well as drug discovery.

A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer.

Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity.

CANDO and the infinite drug discovery frontier.

The Computational Analysis of Novel Drug Opportunities (CANDO) platform (http://protinfo.org/cando) uses similarity of compound-proteome interaction signatures to infer homology of compound/drug behavior. We constructed interaction signatures for 3733 human ingestible compounds covering 48,278 protein structures mapping to 2030 indications based on basic science methodologies to predict and analyze protein structure, function, and interactions developed by us and others. Our signature comparison and ranking approach yielded benchmarking accuracies of 12-25% for 1439 indications with at least two approved compounds. We prospectively validated 49/82 'high value' predictions from nine studies covering seven indications, with comparable or better activity to existing drugs, which serve as novel repurposed therapeutics. Our approach may be generalized to compounds beyond those approved by the FDA, and can also consider mutations in protein structures to enable personalization. Our platform provides a holistic multiscale modeling framework of complex atomic, molecular, and physiological systems with broader applications in medicine and engineering.

The prognostic value of BRCA1 promoter methylation in early stage triple negative breast cancer.

INTRODUCTION: Methylation of the BRCA1 promoter is frequent in triple negative breast cancers (TNBC) and results in a tumor phenotype similar to BRCA1-mutated tumors. BRCA1 mutation-associated cancers are more sensitive to DNA damaging agents as compared to conventional chemotherapy agents. It is not known if there is an interaction between the presence of BRCA1 promoter methylation (PM) and response to chemotherapy agents in sporadic TNBC. We sought to investigate the prognostic significance of BRCA1 PM in TNBC patients receiving standard chemotherapy. METHODS: Subjects with stage I-III TNBC treated with chemotherapy were identified and their formalin-fixed paraffin-embedded (FFPE) tumor specimens retrieved. Genomic DNA was isolated and subjected to methylation-specific PCR (MSPCR). RESULTS: DNA was isolated from primary tumor of 39 subjects. BRCA1 PM was detected in 30% of patients. Presence of BRCA1 PM was associated with lower BRCA1 transcript levels, suggesting epigenetic BRCA1 silencing. All patients received chemotherapy (anthracycline:90%, taxane:69%). At a median follow-up of 64 months, 46% of patients have recurred and 36% have died. On univariate analysis, African-American race, node positivity, stage, and BRCA1 PM were associated with worse RFS and OS. Five year OS was 36% for patients with BRCA1 PM vs. 77% for patients without BRCA1 PM (p=0.004). On multivariable analysis, BRCA1 PM was associated with significantly worse RFS and OS. CONCLUSIONS: We show that BRCA1 PM is common in TNBC and has the potential to identify a significant fraction of TNBC patients who have suboptimal outcomes with standard chemotherapy.

Drug repurposing for gastrointestinal stromal tumor.

Despite significant treatment advances over the past decade, metastatic gastrointestinal stromal tumor (GIST) remains largely incurable. Rare diseases, such as GIST, individually affect small groups of patients but collectively are estimated to affect 25 to 30 million people in the United States alone. Given the costs associated with the discovery, development, and registration of new drugs, orphan diseases such as GIST are often not pursued by mainstream pharmaceutical companies. As a result, "drug repurposing" or "repositioning," has emerged as an alternative to the traditional drug development process. In this study, we screened 796 U.S. Food and Drug Administration (FDA)-approved drugs and found that two of these compounds, auranofin (Ridaura) and fludarabine phosphate, effectively and selectively inhibited the proliferation of GISTs, including imatinib-resistant cells. One of the most notable drug hits, auranofin, an oral, gold-containing agent approved by the FDA in 1985 for the treatment of rheumatoid arthritis, was found to inhibit thioredoxin reductase activity and induce reactive oxygen species (ROS) production, leading to dramatic inhibition of GIST cell growth and viability. Importantly, the anticancer activity associated with auranofin was independent of imatinib-resistant status, but was closely related to the endogenous and inducible levels of ROS. Coupled with the fact that auranofin has an established safety profile in patients, these findings suggest for the first time that auranofin may have clinical benefit for patients with GIST, particularly in those suffering from imatinib-resistant and recurrent forms of this disease.

An RNA interference lethality screen of the human druggable genome to identify molecular vulnerabilities in epithelial ovarian cancer.

Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 "hits" affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer.