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The clinical success of combined androgen deprivation therapy (ADT) and radiation therapy (RT) in prostate cancer (PCa) created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in PCa cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at steady state in asynchronously growing PCa cells. Our data indicate that the clinical benefit of ADT and RT is not due to the direct regulation of DDR gene transcription by AR.

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Recent data show that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways, suggesting that the inhibition of a single component of a canonical pathway is insufficient for the treatment of cancer. The biologic outcome of signaling through a network is inherently more robust and resistant to inhibition of a single network component. In this study, we conducted a functional chemical genetic screen to identify novel interactions between signaling inhibitors that would not be predicted on the basis of our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. The synergistic cytotoxicities identified did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. Among the most robust results was synergy between sorafenib, a multikinase inhibitor with activity against RAF, and diclofenac, a nonsteroidal anti-inflammatory drug (NSAID). Drug substitution experiments using the NSAIDs celecoxib and ibuprofen or the MAP–ERK kinase inhibitor PD325901 and the RAF inhibitor RAF265 suggest that inhibition of COX and mitogen-activated protein kinase signaling are targets for the synergistic cytotoxicity of sorafenib and diclofenac. Cotreatment with sorafenib and diclofenac interrupts a positive feedback signaling loop involving extracellular signal–regulated kinase, cellular phospholipase A2, and COX. Genome-wide expression profiling shows synergy-specific downregulation of survival-related genes. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially, depending on unexplored components of the cell genotype. Mol Cancer Ther; 11(11); 2505–15. ©2012 AACR.

PDF file, 428K, The combination of sorafenib and diclofenac causes cell death.

Prostate cancer (PCa) is a very complex disease that is a major cause of death in men worldwide. Currently, PCa dependence on the androgen receptor (AR) has resulted in use of AR antagonists and anti-androgen therapies that reduce endogenous steroid hormone production. However, within two to three years of receiving first line androgen deprivation therapy, the majority of patients diagnosed with PCa progress to castration resistant prostate cancer (CRPC). There is an urgent need for therapies that are more durable than antagonism of the AR axis. Studies of Runt-related transcription factors (RUNX) and their heterodimerization partner, Core-Binding Factor Subunit b (CBFβ), are revealing that the RUNX family are drivers of CRPC. In this review, we describe what is presently understood about RUNX members in PCa, including what regulates and is regulated by RUNX proteins, and the role of RUNX proteins in the tumor microenvironment and AR signaling. We discuss the implications for therapeutically targeting RUNX, the potential for RUNX as PCa biomarkers, and the current pressing questions in the field.

Lung cancer rates are rising globally and non-small cell lung cancer (NSCLC) has a five year survival rate of only 24%. Unfortunately, the development of drugs to treat cancer is severely hampered by the inefficiency of translating pre-clinical studies into clinical benefit. Thus, we sought to apply a tumor microenvironment system (TMES) to NSCLC. Using microvascular endothelial cells, lung cancer derived fibroblasts, and NSCLC tumor cells in the presence of in vivo tumor-derived hemodynamic flow and transport, we demonstrate that the TMES generates an in-vivo like biological state and predicts drug response to EGFR inhibitors. Transcriptomic and proteomic profiling indicate that the TMES recapitulates the in vivo and patient molecular biological state providing a mechanistic rationale for the predictive nature of the TMES. This work further validates the TMES for modeling patient tumor biology and drug response indicating utility of the TMES as a predictive tool for drug discovery and development and potential for use as a system for patient avatars.

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR–CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR–CHK2 interactions. The destabilization of AR – CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR–CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR–CHK2 interactions. The destabilization of AR–CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.

// Devin G. Roller 1 , Brian Capaldo 2 , Stefan Bekiranov 2 , Aaron J. Mackey 3 , Mark R. Conaway 3 , Emanuel F. Petricoin 4 , Daniel Gioeli 1 , Michael J. Weber 1 1 Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, 22908 USA 2 Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, 22908 USA 3 Department of Public Health Sciences, University of Virginia, Charlottesville, VA, 22908 USA 4 Center for Applied Proteomics and Molecular Medicine, School of Systems Biology, College of Science, George Mason University, Manassas, VA 20110, USA Correspondence to: Michael J. Weber, e-mail: mjw@virginia.edu Keywords: melanoma, therapeutic resistance, cell signaling, BRAF, MAP Kinase Received: August 20, 2015 Accepted: November 21, 2015 Published: December 10, 2015 ABSTRACT Over half of BRAFV600E melanomas display intrinsic resistance to BRAF inhibitors, in part due to adaptive signaling responses. In this communication we ask whether BRAFV600E melanomas share common adaptive responses to BRAF inhibition that can provide clinically relevant targets for drug combinations. We screened a panel of 12 treatment-naive BRAFV600E melanoma cell lines with MAP Kinase pathway inhibitors in pairwise combination with 58 signaling inhibitors, assaying for synergistic cytotoxicity. We found enormous diversity in the drug combinations that showed synergy, with no two cell lines having an identical profile. Although the 6 lines most resistant to BRAF inhibition showed synergistic benefit from combination with lapatinib, the signaling mechanisms by which this combination generated synergistic cytotoxicity differed between the cell lines. We conclude that adaptive responses to inhibition of the primary oncogenic driver (BRAFV600E) are determined not only by the primary oncogenic driver but also by diverse secondary genetic and epigenetic changes (“back-seat drivers”) and hence optimal drug combinations will be variable. Because upregulation of receptor tyrosine kinases is a major source of drug resistance arising from diverse adaptive responses, we propose that inhibitors of these receptors may have substantial clinical utility in combination with inhibitors of the MAP Kinase pathway.

Recent data show that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways, suggesting that the inhibition of a single component of a canonical pathway is insufficient for the treatment of cancer. The biologic outcome of signaling through a network is inherently more robust and resistant to inhibition of a single network component. In this study, we conducted a functional chemical genetic screen to identify novel interactions between signaling inhibitors that would not be predicted on the basis of our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. The synergistic cytotoxicities identified did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. Among the most robust results was synergy between sorafenib, a multikinase inhibitor with activity against RAF, and diclofenac, a nonsteroidal anti-inflammatory drug (NSAID). Drug substitution experiments using the NSAIDs celecoxib and ibuprofen or the MAP–ERK kinase inhibitor PD325901 and the RAF inhibitor RAF265 suggest that inhibition of COX and mitogen-activated protein kinase signaling are targets for the synergistic cytotoxicity of sorafenib and diclofenac. Cotreatment with sorafenib and diclofenac interrupts a positive feedback signaling loop involving extracellular signal–regulated kinase, cellular phospholipase A2, and COX. Genome-wide expression profiling shows synergy-specific downregulation of survival-related genes. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially, depending on unexplored components of the cell genotype. Mol Cancer Ther; 11(11); 2505–15. ©2012 AACR.

Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with emphasis on the cellular receptors for HSV, nectin-1, nectin-2 and HVEM. The ability of an HSV strain with FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral entry by that receptor. A FFWO form of gB was not sufficient to switch the entry of HSV from a pH-dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, the results indicate that the roles of gB in FFWO and in virus–cell fusion during entry are related but not identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity.

Herpes simplex virus (HSV) entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in HSV entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on HSV entry. HSV can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, HSV successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. We propose that proteasomal degradation of virion and/or host proteins is required for efficient delivery of incoming HSV capsids to the nucleus.

Diacylglycerol kinases (DGKs) are a family of highly conserved enzymes that are promiscuously responsive to growth factors and other mitogenic signaling cascades. DGKs are activated by binding of Ca2+ and phosphoserine, and catalyze conversion of diacylglycerol (DAG) to phosphatidic acid (PA). PA has multiple effects on downstream cellular signaling, while activated DGKs can associate with scaffolding proteins, or undergo degradation or translocation to the nucleus through atypical localization signals and thereby promote cell division and survival. A repurposed small-molecule inhibitor of DGKα activity is shown here to antagonize tumor growth in xenografts of primary and metastatic brain tumors and syngeneic mouse tumor grafts. Based on recent prior research and in this work in xenograft models, DGKα inhibition may arrest or kill tumors through cancer addiction to DGKα activity. Here, in syngeneic tumor models of primary and metastatic brain tumors, an additional anti-tumor effect of DGKα inhibition via up-regulation of normal immune function is possibly present. This work uses two syngeneic in vivo tumor models to compare DGKα inhibition with the monoclonal antibody immunotherapy anti-CTLA-4, and also tests the effect of combination of the two treatments on survival, tumor size, and immunologic activity. The anti-tumor activity of the DGKα inhibitor and the contribution of other DGK family members is shown using cell-based toxicity and flow cytometry assays. Direct interaction between the inhibitor and DGKα is shown with biochemical reconstitution of inhibitor-enzyme interactions These early results suggest that DGKα inhibition may be a promising strategy for patients with glioblastoma and melanoma brain metastasis.

Herpesviruses commandeer distinct cellular pathways to enter target cells. The mechanism by which herpes simplex virus (HSV) selects a pH-dependent, endocytic route or a pH-independent route remains to be elucidated. We investigated the role of the non-glycosylated viral envelope protein UL45 in HSV entry via endocytosis. UL45 plays a role in mediating cell–cell fusion and has been proposed to functionally interact with gB to regulate membrane fusion. Thus, we also probed the impact of UL45 on the structure and function of gB present in virions. A UL45 deletion virus successfully entered cells via low pH, endocytic pathway with wild type kinetics. In the absence or presence of UL45, the antigenic conformation of virion gB appeared unaltered. Antibodies to gB neutralized infection of the UL45-deletion virus and wild type virus to a similar extent, regardless of whether the target cells supported low pH endocytic or non-endocytic entry routes. Lastly, HSV virions were inactivated by low pH regardless of the presence of UL45. The results, together with previous studies, suggest that UL45 plays distinct roles in cell–cell fusion and virus–cell fusion during acid-dependent entry.

Fifty percent of cutaneous melanomas are driven by activated BRAF V600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAF V600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.

Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies Adaptive survival responses to targeted therapy reduce therapeutic benefit, and thus there is a need to identify drug combinations that co-target both oncogenic drivers and critical adaptive responses. We screened a panel of twelve V600E BRAF melanoma cell lines with a library of targeted drugs, and identified the combination of lapatinib (a HER family inhibitor) plus PLX4720 (a RAF inhibitor) as synergistically cytotoxic in the six lines that displayed complete or partial resistance to PLX4720 alone. To understand the mechanism(s) of drug resistance and synergy, and to identify potential biomarkers for utilization of this combination, we have performed an extensive molecular analysis of the cell lines and their responses to treatment with the drugs, singly and in combination. Alamar blue was used to calculate the cytotoxic effects of the drugs, and the cells were profiled by gene expression microarrays and reverse phase protein microarrays, before and after drug treatment. DNA methylation microarrays, and exome sequencing were used to identify the epigenetic and genetic context of the lines. Unsupervised hierarchical clustering analysis of the cytotoxic effects of the single drug and combinatorial treatments revealed four well defined groups: synergistic to combination treatment, PLX4720 sensitive, lapatinib resistant, and insensitive to either drug. The cell lines were analyzed using principal component analysis on the basal gene expression profiles. This analysis revealed four major clusters whose membership was identical to the clusters observed in the cytotoxicity analysis, indicating a strong genotype-drug phenotype relationship (i.e. cell lines that showed similar basal transcriptional programs also demonstrated similar responses to treatment. Differential gene expression analysis was performed between the synergistic group and each of the other groups. The most prominent gene expression differences were between the synergistic and PLX4720 sensitive lines, which correlated with differences in MITF expression and transcriptional output. Changes in gene expression and phosphoproteome levels in response to PLX4720 and lapatinib treatment between the four groups were used to identify genes and pathways associated with drug resistance and synergy. Genes involved in apoptosis and cell cycle regulation were found that could play a role in conferring resistance and synergistic response to PLX4720 and lapatinib treatment. These differing patterns of adaptive responses provide insights into molecular mechanisms of drug resistance and synergy and a pathway to rational construction of drug combinations. Citation Format: Brian J. Capaldo, Devin G. Roller, Mark Axelrod, Alex Koeppel, Michael J. Weber, Aaron Mackey, Dan Gioeli, Stefan Bekiranov. Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies. [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 4166. doi:10.1158/1538-7445.AM2014-4166

The effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling, which generates adaptive resistance mechanisms and reduces therapeutic responses. We have performed high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that confer resistance to the cytotoxic effects of targeted therapy. We screened subsets of over 1,000 drug combinations in 14 different epithelial cell lines representing three distinct cancer lineages, and 19 melanoma cell lines, and assessed the ability of each combination to cause synergistic cytotoxicity. We focused on synergistic combinations because they point to mechanistic linkages between the signaling pathways, and also because of the possibility of improved therapeutic index in vivo. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple epithelial cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern with respect to lineage specificity or mutational status of commonly altered oncogenes in the observed synergies. Within the melanoma panel, BRAF mutational status predicted response to single-agent BRAF inhibition, but did not predict synergistic drug combinations, which were different for each cell line. These results reflect the heterogeneity of genetic alterations and the plasticity of cell signaling networks even among cell lines of the same tissue of origin that contain the same predominant driver mutations. We suggest that there is not a sharp dichotomy between “driver” and “passenger” mutations, and that the biological responses to combination therapies are determined by functionally important modifier mutations that we term “back-seat drivers.” This hypothesis is supported by analysis of the transcriptomes and phosphoproteomes of cells treated with drugs singly and in combination, and by exome sequencing. Such analyses also can reveal critical nodes with the potential to function as effective single targets. We found that co-inhibition of EGFR and PI3 Kinase causes synergistic cytotoxicity in some epithelial cancer cell lines, and that phosphoproteomic analysis of signaling pathway responses revealed concordant synergistic inhibition of p70S6 Kinase in KU-7 bladder cancer cells. Using an epistasis paradigm, restoration of p70S6 Kinase signaling by expression of mutationally activated p70S6 Kinase resulted in protection from cytotoxicity, indicating that p70S6 Kinase is a critical node for enhanced cytotoxicity due to combination treatment. AT7867, a potent inhibitor of p70S6 Kinase, was able to inhibit phosphorylation of ribosomal protein S6 and induce cytotoxicity as effectively as the combination drug treatment. We suggest that p70S6 Kinase acts as a functionally important node within the EGFR/PI3 Kinase signaling network and is an attractive target for therapeutic intervention. Citation Format: Mark J. Axelrod, Devin Roller, Brian Capaldo, Aaron J. Mackey, Mark Conaway, Daniel Gioeli, Michael J. Weber. Combinatorial screens with targeted inhibitors reveal diverse compensatory responses and mechanisms of adaptive resistance to therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B29.

BRAF is mutationally activated in 50% of melanomas with a variety of data indicating its role as a driver of malignancy. However, while patients with BRAF mutations respond to vemurafenib, most responses are not long lasting. Moreover, the 25% of melanomas harboring an NRAS activating mutation are resistant de novo to BRAF inhibitors. In order to identify effective drug combinations that increase the effectiveness of BRAF inhibition, we used combinatorial small molecule biological screens and global analytical approaches to identify compensatory and redundant pathways. We screened a drug library for combinations with BRAF or MEK inhibitors that cause synergistic cytotoxicity in melanoma cell lines. Although BRAF mutational status predicted sensitivity to inhibitors of BRAF, each BRAF mutant line differed in the combination of drugs that induced synergistic cytotoxicity. One robust and clinically relevant pair identified was the BRAF inhibitor PLX4720 and the EGFR/HER2 inhibitor lapatinib. This drug combination was synergistic in half of the BRAF mutant cell lines tested, including a line that was completely resistant to PLX4720. The combination was also effective in the two NRAS mutant lines tested. Xenograft experiments testing the combination of PLX4720 and lapatinib revealed a significant reduction in tumor volume compared to either single agent alone in both PLX4720 sensitive and resistant BRAF mutant tumors, providing preclinical data supporting the efficacy of this drug combination. Synergy of the PLX4720-lapatinib combination did not correlate with EGFR/HER2 expression, activity state, or mutational status. This further indicates that sensitivity of melanoma to drug combinations is not determined by the targeted oncogenic drivers, but by the existence of unique secondary, compensatory survival responses. We hypothesize that mutationally activated BRAF is wired into the constitutive signaling network differently in each of these cells, presumably a consequence of the diverse secondary mutations that characterize melanomas. Consistent with this, whole exome-sequencing data revealed a diversity of somatic variation among the synergistic and non-synergistic melanomas, while gene expression analysis demonstrated both distinct, line-specific basal transcriptional profiles and the lack of common gene expression signatures in melanomas synergistically inhibited by treatment with PLX4720-lapatinib. Collectively, these data point out the extraordinary robustness of cancer cell signaling networks and the challenges of individualizing therapies. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype and cell signaling network. Citation Format: Devin Roller, Brian Capaldo, Aaron J. Mackey, Mark Conaway, Michael J. Weber, Daniel G. Gioeli. Small molecule biological screens and global analytical approaches reveal the complexity of cancer cell signaling networks and the challenge of personalized medicine. [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 3400. doi:10.1158/1538-7445.AM2013-3400

Cellular signals are not necessarily transmitted via linear pathways, but through a dynamic interconnected network. Effective pharmacologic inhibition of cellular signaling in malignant cells will thus require identifying the key nodes, and intervening at more than one site within the network. To systematically identify functional interactions between signaling proteins within the cell signaling network we have screened nine melanoma cell lines of diverse genetic backgrounds for sensitivity to combinations of drugs targeting the major signaling pathways altered in cancer. Seven drugs representative of standard targets were designated as “primary drugs”. These seven primary targeted therapies were screened against each other and an additional 67 signal transduction inhibitors. The screen was performed robotically with a Biomek NX workstation in a 96-well format. Cell viability was measured with alamarBlue on a microplate reader. Various concentrations of the secondary drug were tested against a partial inhibitory concentration of the primary drug. Twelve percent of a total of 4672 drug dose and cell combinations tested displayed superadditivity in vitro. Nearly 10% of those hits demonstrated greater than 50% superadditivity according to first principles analysis; the actual growth inhibition of the combination was more than 50% greater than the sum of the growth inhibition produced by each drug alone. One intriguing drug combination showing superadditivity in most cell lines tested was sorafenib plus diclofenac. The degree of inhibition and concentration in which superadditivity was observed was cell line dependent, and was independent of known mutational status. The MEK inhibitor, PD325901, was able to substitute qualitatively for sorafenib indicating that Raf may be the major target for sorafenib when used in combination with diclofenac. Consistent with this, sorafenib inhibited MEK and ERK activity at doses that achieved superadditivity with diclofenac. However, the combination of PD325901 with diclofenac was less robust at inhibiting growth than sorafenib plus diclofenac suggesting that the alternate sorafenib targets may play a role in the observed superadditivity. Celecoxib, a COX2 inhibitor, or ibuprofen, a preferential COX1 inhibitor, substituted for diclofenac indicating that diclofenac was acting as a COX inhibitor, although the inhibition was considerably less robust, suggesting a role for a balanced inhibition of COX1 and COX2, or a role for off-target effects. Collectively, these results indicate an unexpected functional interaction between Raf and COX signaling in melanoma. Since both sorafenib and diclofenac are in clinical use, the therapeutic potential of these observations is striking and is being further explored in pre-clinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3843.