Driver Signaling Pathways Research Articles

Abstract 3214: Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor of childhood. While the current standard care for MB leads to long term survival in approximately 75 % of patients; recurrent and refractory MB continues to have dismal outcomes. In addition, long term survivors face significant treatment-related sequelae especially poor neurocognitive outcomes. Drug repositioning for known drugs is a promising potential strategy not only for drug discovery but also for accelerated translation into the clinical setting. To systematically explore thousands of known drugs available, we integrated computational biology and empirical biology methods to find old drugs for new indications in MB. Results: A non-parametric, bootstrapping based simulated annealing (NPBSA) algorithm was employed to identify driver signaling pathways for over 1,800 patients with Group 3 and Group 4 MB through an integrative analysis on their mRNA expression, DNA-copy number, DNA-methylation and DNA-seq profiles. Then, drug functional networks were constructed based on gene expression profiles under drug treatment as well as chemical structures and were clustered into drug modules with potential mechanisms of action. By evaluating targeted effects of 1,309 drugs from connectivity map database within each drug module in driver signaling pathways, we identified a group of known cardiac glycosides that top ranked among the total drug candidates for the Group 3 and 4 MB subtypes. In addition to traditional chemotherapeutic agents, members of the cardiac aminoglycoside family were repeatedly identified as potential therapeutic agents for MB. These findings were validated in multiple MB-derived cell lines which showed high rates of growth inhibition by cardiac aminoglycosides compared to controls. To evaluate if this growth inhibition in vitro correlated to prolonged survival in vivo, an extensively characterized patient-derived orthotopic-xenograft (PDOX) model of Group 4 MB (ICb-1078MB) was treated with digoxin (2 mg/kg i.p.) for 2 cycles of 14 days. Digoxin treatment significantly prolonged survival to 113 days from a median of 92 days in untreated controls (p=0.001). Histological evaluation of recurrent tumors following digoxin treatment demonstrated changes in the pattern of tumor spread, vascularity and necrosis compared to untreated controls. Conclusions: Leveraging big data in the domains of pharmacogenomics and the notion of drug functional networks and driver signaling pathways represents a powerful tool to repurpose known drugs for new indications in pediatric cancers. Using this integrative biology approach, we identified the cardiac aminoglycosides family generally and Digoxin, specifically, as potential novel agents in the treatment of pediatric medulloblastoma Citation Format: Lei Huang, Sarah G. Injac, Hong Zhao, Qi Lin, Mari Kogiso, Chris T. Man, Xiao-Nan Li, Ching C. Lau, Stephen T. Wong. Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3214. doi:10.1158/1538-7445.AM2017-3214

Abstract 351: mTOR-ERK co-targeting strategies for head and neck cancer therapy

Abstract Despite therapeutic advances in the past decade, head and neck squamous cell carcinoma (HNSCC) continues to be a major public health concern. Although recent studies of human cancer genomes identified remarkable complexity of genomic alterations in HNSCC, surprisingly, most aberrations have fallen within key driver signaling pathways. Among them, PI3K/AKT/mTOR pathway is most frequently activated in over 80% of the HNSCC patients. We showed that the majority of HNSCC lesions exhibit high levels of phosphorylated ribosomal S6 protein (pS6), key downstream target of the AKT-mTOR pathway, and that inhibition of mTOR by the use of rapamycin causes a rapid decrease in the level of pS6 and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. Our initial studies identified the AKT-mTOR pathway as a potential therapeutic target for HNSCC and provided strong rational to initiate multiple clinical trials. Indeed, inhibitors of PI3K/AKT/mTOR block mTOR activity and exert a beneficial response in HNSCC patients. However, therapeutic resistance was evidenced in several trials, which might be due to the activation of adaptive survival signaling in tumor cells. To explore the therapeutic option that can overcome the resistance, we performed a synthetic lethality screen in HNSCC cell lines using shRNA libraries. We found that multiple molecules involved in ERK pathway were highly represented. Furthermore, treating HNSCC cell lines harboring activated mTOR with small molecule inhibitors that target PI3K/AKT/mTOR pathway can sequentially induce ERK activation, which is consistent with the findings observed in numerous clinical trials. We demonstrated that co-targeting mTOR and ERK using trametinib, a MEK1/2 inhibitor or cetuximab, an EGFR monoclonal antibody, exhibited the synergistic effect by sensitizing HNSCC to priming with the mTOR inhibitor. Specifically, mTOR-ERK co-targeting prevented the growth of HNSCC tumor xenografts by decreasing cell proliferation, reducing lymphangiogenesis and activating apoptosis. In addition, we have recently used a phosphoproteomics approach to identify the molecular mechanisms of ERK feedback activation caused by PI3K/AKT/mTOR inhibition. Overall, our promising findings from these preclinical studies suggest that the use of mTOR-ERK co-targeting strategies may provide the novel alternative approach to achieve durable responses (cure) in HNSCC cancer patients. Citation Format: Zhiyong Wang, Esteban Delgado, Kosuke Yamaguchi, Ramiro Iglesias-Bartolomé, Panomwat Amornphimoltham, Alfredo Molinolo, Qianming Chen, J.Silvio Gutkind. mTOR-ERK co-targeting strategies for head and neck cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 351. doi:10.1158/1538-7445.AM2017-351

P11.08 Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor of childhood. While the current standard care for MB leads to long term survival in approximately 75 % of patients; recurrent and refractory MB continues to have dismal outcomes. In addition, long term survivors face significant treatment-related sequelae especially poor neurocognitive outcomes. Drug repositioning for known drugs is a promising potential strategy not only for drug discovery but also for accelerated translation into the clinical setting. To systematically explore thousands of known drugs available, we integrated computational biology and empirical biology methods to find old drugs for new indications in MB. Results: A non-parametric, bootstrapping based simulated annealing (NPBSA) algorithm was employed to identify driver signaling pathways for over 1,800 patients with Group 3 and Group 4 MB through an integrative analysis on their mRNA expression, DNA-copy number, DNA-methylation and DNA-seq profiles. Then, drug functional networks were constructed based on gene expression profiles under drug treatment as well as chemical structures and were clustered into drug modules with potential mechanisms of action. By evaluating targeted effects of 1,309 drugs from connectivity map database within each drug module in driver signaling pathways, we identified a group of known cardiac glycosides that top ranked among the total drug candidates for the Group 3 and 4 MB subtypes. In addition to traditional chemotherapeutic agents, members of the cardiac aminoglycoside family were repeatedly identified as potential therapeutic agents for MB. These findings were validated in multiple MB-derived cell lines which showed high rates of growth inhibition by cardiac aminoglycosides compared to controls. To evaluate if this growth inhibition in vitro correlated to prolonged survival in vivo, an extensively characterized patient-derived orthotopic-xenograft (PDOX) model of Group 4 MB (ICb-1078MB) was treated with digoxin (2 mg/kg i.p.) for 2 cycles of 14 days. Digoxin treatment significantly prolonged survival to 113 days from a median of 92 days in untreated controls (p=0.001). Histological evaluation of recurrent tumors following digoxin treatment demonstrated changes in the pattern of tumor spread, vascularity and necrosis compared to untreated controls. Conclusions: Leveraging big data in the domains of pharmacogenomics and the notion of drug functional networks and driver signaling pathways represents a powerful tool to repurpose known drugs for new indications in pediatric cancers. Using this integrative biology approach, we identified the cardiac aminoglycosides family generally and Digoxin, specifically, as potential novel agents in the treatment of pediatric medulloblastoma.

A comprehensive analysis of cancer-driving mutations and genes in kidney cancer.

An accumulation of driver mutations is important for cancer formation and progression, and leads to the disruption of genes and signaling pathways. The identification of driver mutations and genes has been the subject of numerous previous studies. The present study was performed to identify cancer-driving mutations and genes in renal cell carcinoma (RCC), prioritizing noncoding variants with a high functional impact, in order to analyze the most informative features. Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping version 2 (Polyphen2) and MutationAssessor were applied to predict deleterious mutations in the coding genome. OncodriveFM and OncodriveCLUST were used to detect potential driver genes and signaling pathways. The functional impact of noncoding variants was evaluated using Combined Annotation Dependent Depletion, FunSeq2 and Genome-Wide Annotation of Variants. Noncoding features were analyzed with respect to their enrichment of high-scoring variants. A total of 1,327 coding mutations in clear cell RCC, 258 in chromophobe RCC and 1,186 in papillary RCC were predicted to be deleterious by all three of MutationAssessor, Polyphen2 and SIFT. In total, 77 genes were positively selected by OncodriveFM and 1 by OncodriveCLUST, 45 of which were recurrently mutated genes. In addition, 10 signaling pathways were recurrently mutated and had a high functional impact bias (FM bias), and 31 novel signaling pathways with high FM bias were identified. Furthermore, noncoding regulatory features and conserved regions contained numerous high-scoring variants, and expression, replication time, GC content and recombination rate were positively correlated with the densities of high-scoring variants. In conclusion, the present study identified a list of cancer-driving genes and signaling pathways, features like regulatory elements, conserved regions, replication time, expression, GC content and recombination rate are major factors that affect the distribution of high-scoring non-coding mutations in kidney cancer.

Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 632.

Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

Par6 family proteins in cancer.

The regulatory networks of cell polarization and polarity effector proteins have been subjects of feverish interest in the field of cell and developmental biology but many advances in the polarity research may have flown unnoticed past the radar of mainstream cancer biologist. However, recent findings suggesting important cell cycle gatekeeping functions for polarity proteins may change that. For example, PAR6 proteins interact with classical cancer driver signaling pathways, including MAPK and PI3K and moreover, PAR6 (PARD6) genes are frequently altered in various cancers. Mammalian genomes harbor three different PARD6 genes. Recent studies in breast cancer have suggested that different PARD6 genes are not only important players but may play even opposite roles during tumorigenesis.

DrGaP: A Powerful Tool for Identifying Driver Genes and Pathways in Cancer Sequencing Studies

Cancers are caused by the accumulation of genomic alterations. Driver mutations are required for the cancer phenotype, whereas passenger mutations are irrelevant to tumor development and accumulate through DNA replication. A major challenge facing the field of cancer genome sequencing is to identify cancer-associated genes with mutations that drive the cancer phenotype. Here, we describe a powerful and flexible statistical framework for identifying driver genes and driver signaling pathways in cancer genome-sequencing studies. Biological knowledge of the mutational process in tumors is fully integrated into our statistical models and includes such variables as the length of protein-coding regions, transcript isoforms, variation in mutation types, differences in background mutation rates, the redundancy of genetic code, and multiple mutations in one gene. This framework provides several significant features that are not addressed or naively obtained by previous methods. In particular, on the observation of low prevalence of somatic mutations in individual tumors, we propose a heuristic strategy to estimate the mixture proportion of chi-square distribution of likelihood ratio test (LRT) statistics. This provides significantly increased statistical power compared to regular LRT. Through a combination of simulation and analysis of TCGA cancer sequencing study data, we demonstrate high accuracy and sensitivity in our methods. Our statistical methods and several auxiliary bioinformatics tools have been incorporated into a computational tool, DrGaP. The newly developed tool is immediately applicable to cancer genome-sequencing studies and will lead to a more complete identification of altered driver genes and driver signaling pathways in cancer.

Abstract 2689: Drivers of cancer cell invasion and metastasis.

Abstract Metastatic spread of cancer cells from their primary site requires invasion into the vasculature, evasion at the distant organ site and colonization of the distant organ. Here we studied mechanisms of attachment and invasion into endothelial monolayers by cancer cells to identify driver molecules and signaling pathways that are crucial for the invasive and metastatic phenotype. Different human and mouse cancer cell lines were transduced with lentiviral-based, genome-wide or kinome-targeted shRNA libraries. Cancer cell populations of different invasive and metastatic phenotype were then selected for their loss- or gain-of-function respectively in endothelial invasion in vitro or by tumor growth and metastasis selection in vivo. After several rounds of selection, the respective shRNA targeted genes were identified from the selected cancer cell populations or from clonal cell lines. Eighteen different genes targeted by two to four distinct shRNAs each were identified in the in vitro selection for endothelial attachment and invasion. The function of the genes as well as the clinical significance appear to explain their phenotypic selection. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Louis M. Weiner, Zhang-Zhi Hu, Anna T. Riegel, Anton Wellstein. Drivers of cancer cell invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2013-2689

Abstract B63: Cancer cell invasion determined by cell density-dependent, cytokine-mediated crosstalk

Abstract Metastatic spread of cancer cells from their primary site requires invasion into the vasculature, evasion at the distant organ site and colonization of the distant organ. Here we studied mechanisms of attachment and invasion into endothelial monolayers by cancer cells to identify driver molecules and signaling pathways that are crucial for the invasive and metastatic phenotype. In vitro studies were complemented by studies in zebrafish embryos and mouse models of cancer cell invasion and metastasis. In one approach we compared gene expression profiles across isogenic cell lines with distinct invasive behavior to identify candidate drivers and pathways. These were then subjected to functional validation. This comparison of gene expression profiles of isogenic cancer cell lines showed an upregulation of immune response cytokines associated with an endothelial invasive phenotype in human breast cancer cells. The role of the respective cytokine pathways was validated by blocking the respective receptors in in vitro assays and in a zebrafish metastasis model. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Zhang-Zhi Hu, Eric Glasgow, Anna T. Riegel, Anton Wellstein. Cancer cell invasion determined by cell density-dependent, cytokine-mediated crosstalk. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B63.

Patient-derived breast tumor xenografts facilitating personalized cancer therapy

Despite improved detection and reduction of breast cancer-related deaths over the recent decade, breast cancer remains the second leading cause of cancer death for women in the US, with 39,510 women expected to succumb to metastatic disease in 2012 alone (American Cancer Society, Cancer Facts &Figures 2012. Atlanta: American Cancer Society; 2012). Continued efforts in classification of breast cancers based on gene expression profiling and genomic sequencing have revealed an underlying complexity and molecular heterogeneity within the disease that continues to challenge therapeutic interventions. To successfully identify and translate new treatment regimens to the clinic, it is imperative that our preclinical models recapitulate this complexity and heterogeneity. In this review article, we discuss the recent advances in development and classification of patient-derived human breast tumor xenograft models that have the potential to facilitate the next phase of drug discovery for personalized cancer therapy based on the unique driver signaling pathways in breast tumor subtypes.

Guest editorial: Management of malignant lymphoma is continuously improving

This issue of IJH contains four ‘‘Progress in Hematology(PIH)’’ review articles describing the management ofmalignant lymphoma, with a focus on recent clinical trials.Malignant lymphoma, characterized by its marked hetero-geneity, is the most frequent hematologic malignancy inthe world. Among the various subtypes of malignantlymphoma, the following three are clinically important:Hodgkin lymphoma, follicular lymphoma (FL), and diffuselarge B cell lymphoma (DLBCL). The current and futuremanagement of these three major subtypes are discussed byinternationally distinguished lymphoma experts who havecontributed to the establishment of the current standardmanagement of each subtype. In addition, the current andfuture management of NK/T cell lymphoma is discussed byDr. Yamaguchi, based on clinical trials recently publishedby her group.Current issues to be addressed in the management ofmalignant lymphoma differ somewhat from disease todisease. In Hodgkin lymphoma, continuous efforts toestablish more effective chemotherapy with or withoutradiotherapy have yielded high cure rates in patients withlocalized and advanced diseases. Drs. Eichenauer andEngert of the German Hodgkin Study Group (GHSG)prepared a comprehensive review article regarding thecurrent standard management, based mainly on clinicaltrials conducted by GHSG. Treatment strategies forHodgkin lymphoma are scientifically discussed, andupdated information, including that on ongoing clinicaltrials, should help readers to better understand the mostsuccessful history of using non-surgical treatment modali-ties in clinical oncology.In FL, which remains incurable in most patients, theissues are somewhat different from those in Hodgkinlymphoma. Anti-CD20 monoclonal antibodies, such asrituximab, and radioimmunotherapy have markedly pro-longed survival. In the second PIH article, Drs. Salles andGhesquie`res of Groupe d’Etudes des Lymphomes del’Adulte (GELA), which has recently been renamed theLymphoma Study Association (LYSA), summarize recentadvances in the management of patients with FL, based onthe results of recent clinical trials including the PRIMAStudy, which revealed the efficacy of maintenance use ofrituximab [1]. Considering the marked heterogeneity of FL,the prolonged median survival time probably exceeding15 years under the current treatment modalities, and theemergence of several less toxic but highly effective agents,personalized approaches will be more important in thetreatment of FL in the future.Since the establishment of rituximab plus CHOP as astandard therapy for DLBCL [2], progress has been lessremarkable. In the third PIH article in this issue,Drs. Roschewski, Dunleavy, and Wilson of the NationalCancer Institute in the United States present an excellentreview of further progress in the treatment of DLBCL. Animproved understanding of the biology of DLBCL hasrevealed a number of oncogenic driver mutations and sig-naling pathways essential for growth of the lymphoma cell.As many of these signaling pathways can be targeted bysmall molecule inhibitors, treatment of DLBCL may be instore for a paradigm shift.Finally, Dr. Yamaguchi in Japan summarizes newlydevelopedtreatmentstrategiesforNK/Tcelllymphoma,basedon the results of multicenter clinical trials. NK/T cell lym-phoma is a distinct disease subtype with dismal prognosis

Diffuse large B cell lymphoma: molecular targeted therapy

Diffuse large B cell lymphoma (DLBCL) is a biologically heterogeneous disease and the most common subtype of B cell non-Hodgkin's lymphoma in the USA. Even though it is a curable lymphoma in advanced stages, up to 40 % of patients eventually relapse or fail to achieve remission. Improved understanding of the biologic complexity of DLBCL reveals a diverse range of oncogenic driver mutations and signaling pathways that are essential for growth and survival of malignant cells. Since many of these signaling pathways can be targeted by small-molecule inhibitors, the therapy for DLBCL is currently undergoing a paradigm shift away from conventional chemotherapy and toward targeted agents that capitalize on an improved biologic understanding of the subsets with the highest risk of treatment failure. Participation in well-conducted and rationally designed clinical trials will be essential to realize the potential of these targeted agents and realize the goal of improving overall outcomes in the most common B cell lymphoma in the world.