Treatment Of Late Stage Melanoma Research Articles

Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC50) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.

Abstract IA20: Immune checkpoint blockade in cancer therapy: New insights and opportunities, and prospects for cures

Abstract The existence of multiple nonredundant inhibitory pathways that limit T-cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T-cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen-presenting cells. Antibodies to CTLA-4 have proved effective against multiple tumor types in both preclinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long-term (>4 years) survival benefit in about 20% of patients in a randomized, placebo-controlled trial in late-stage melanoma. In 2011 it was approved by the FDA for treatment of late-stage melanoma, and it remains part of the standard of care for that disease. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T-cell antigen receptor-mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. Combination of anti-PD-1 and anti-CTLA-4 in melanoma provides objective responses in about 50% of late-stage melanoma patients. The FDA has now approved 6 different checkpoint antibodies for a variety of cancers including urothelial, renal, and lung cancers. A major focus of current efforts is the rational identification of combinatorial treatments that improve the response rate and expand the range of immune checkpoint inhibitors. We used high-parameter flow cytometry to identify the mostly nonoverlapping cellular mechanisms of CTLA-4 and PD-1 blockade, which may partially explain the enhanced effect of their combination. Further work has shown that combined CTLA-4 and PD-1 blockade, which provides higher response rates against a wider range of cancers than either monotherapy, acts through a population of T cells not enriched in either single treatment. T-cell responses are largely directed toward neoantigens arising as a result of mutational events associated with the carcinogenesis. While all tumors with antigens recognizable by the immune system should be targets for checkpoint blockade, tumors with lower burdens of mutations (e.g., prostate, breast, and kidney cancer) present special challenges for immune therapy. Recent evidence suggests that even the few neoantigens carried by these can provide targets for immune attack. Strategies for effective treatment of such tumors will be discussed. Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights and opportunities, and prospects for cures [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA20.

Abstract IA30: Immune checkpoint blockade in cancer therapy: New insights and opportunities, and prospects for cures

Abstract The existence of multiple nonredundant inhibitory pathways that limit T-cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen-presenting cells. Antibodies to CTLA-4 have proved effective against multiple tumor types in both preclinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long-term (>4 years) survival benefit in about 20% of patients in a randomized, placebo-controlled trial in late-stage melanoma. In 2011 it was approved by the FDA for treatment of late-stage melanoma and is now a standard of care for that disease. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T-cell antigen receptor-mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. A recent phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late-stage melanoma patients. Altogether, the FDA has approved 17 checkpoint antibodies/indications for melanoma and lung cancer. Emerging data suggest that T-cell responses are largely directed toward neoantigens arising as a result of mutational events associated with the carcinogenesis. While all tumors with antigens recognizable by the immune system should be targets for checkpoint blockade, it is becoming clear that certain types of tumors with lower burdens of mutations (e.g., prostate, breast, and kidney cancer) present special challenges for immune therapy. Strategies for effective treatment of such tumors will be discussed. We have also used high-parameter flow cytometry to identify the cellular mechanisms of CTLA-4 and PD-1 blockade. These are mostly nonoverlapping, which may be a partial explanation for the enhanced effect of the combination. Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights and opportunities, and prospects for cures [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA30.

Abstract IA08: Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for cures

Abstract The existence of multiple non-redundant inhibitory pathways that limit T cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen presenting cells. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>4 years) survival benefit in about 20% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease. The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. A recent phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients. Emerging data suggest that T cell responses are largely directed toward neoantigens arising as a result of mutational events associated with the carcinogenesis. While all tumors with antigens recognizable by the immune system should be targets for checkpoint blockade, it is becoming clear that certain types of tumors with lower burdens of mutations (e.g. prostate, breast, and kidney cancer) present special challenges for immune therapy. Strategies for effective treatment of such tumors will be discussed. Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for cures. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA08.

Abstract 967: Vemurafenib regulates miRNA-211 expression in melanoma - effects on extracellular vesicle RNA cargo and function

Abstract In melanoma, more than 50% of the patients harbor BRAF somatic missense mutations, most affecting the V600 region. Vemurafenib, a potent BRAF inhibitor used for the treatment of late stage melanoma, has shown promising results by causing programmed cell death in melanoma cells. Recently, extracellular vesicles including apoptotic bodies, microvesicles and exosomes has been shown to contain genetic information especially RNA with distinct repertoire of RNA, which could be transferred from one cell to another. Here we investigate the effects of Vemurafenib on melanoma cells and the RNA content in their subsets of extracellular vesicles. Upon vemurafenib treatment, we found that the melanoma cells harboring the BRAF mutation significantly increases the RNA and protein cargo in the different subsets of extracellular vesicles. The RNA profile also changes substantially in apoptotic bodies and microvesicles with significant changes in the ribosomal RNA ratio. Further, using small RNA sequencing, we found that extracellular vesicles from treated cells harbor unique miRNAs. The significantly different miRNA detected with sequencing analysis was validated in vitro with PCR and confirmed that miR-211 is upregulated in cells as well as subsets of extracellular vesicles after treatment. Furthermore, miR-211 was validated to be significantly upregulated in tumors tissues harvested from patient derived xenograft (PDX) as well as cell line derived xenograft and its vesicular subsets. This shows that vemurafenib induces miR-211 upregulation in BRAF mutant cells and PDX especially in the subsets of extracellular vesicles. miR-211 induction upon oncogene treatment could be potentially used as a biomarker in melanoma patients with BRAF mutation. Citation Format: Taral R. Lunavat, Lesley Cheng, Berglind Einarsdottir, Cecilia Lässer, Jonas A. Nilsson, Andrew F. Hill, Jan Lötvall. Vemurafenib regulates miRNA-211 expression in melanoma - effects on extracellular vesicle RNA cargo and function. [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 967.

Abstract IA01: Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for cures

Abstract The existence of multiple non-redundant inhibitory pathways that limit T cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen presenting cells. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>4 years) survival benefit in about 20% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease. The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. A recent phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients. Emerging data suggest that T cell responses are largely directed toward neoantigens arising as a result of mutational events associated with the carcinogenesis. While all tumors with antigens recognizable by the immune system should be targets for checkpoint blockade, it is becoming clear that certain types of tumors with lower burdens of mutations (eg. prostate, breast, and kidney cancer) present special challenges for immune therapy. Strategies for effective treatment of such tumors will be discussed. Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for cures. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA01.

Cytotoxic T-lymphocyte-associated Protein 4 and Cancer Therapy

The existence of multiple non-redundant l inhibitory pathways that limit T cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits CD28 mediated costimulation. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>4.5 years) survival benefit in about 23% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease. A recent retrospective study of almost 5,000 patients showed an inflection point at about 2.5 years with essentially no deaths of about 20% of patients for 10 years following treatment.The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. We have recently uncovered a mechanism whereby anti-CTLA-4 antibodies expand Treg in lymph nodes but cause their depletion in the tumor microenvironment. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms. We have also shown that CTLA-4 blockade results in a 2-5 fold increase in the frequency of CD4 T cells expression ICOS (inducible costimulator) in both tumor tissues and blood. This population contains that vast majority of tumor antigen specific cells that produce IFNg and TNFa. The appearance of the ICOS+ CD4 cells serves as a pharmacodynamic marker of a biological effect of anti-CTLA-4 activity. Using mouse models, we have shown that the ICOS/ICOSL pathway is critical for optimal anti-tumor activity of anti-CTLA-4. Furthermore, we have shown that agonist stimulation of ICOS coupled with CTLA-4 blockade results in enhanced anti-tumor efficacy in mouse models, suggest that ICOS is a compelling molecule to develop as a target for agonistic targeting of costimulatory checkpoints.PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25% . A recent phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients. Our studies indicate that the mechanisms of anti-PD-1 mediated tumor immunity are distinct from those of anti-CTLA-4, at least as for the role of ICOS+ CD4 T cells.These studies and their implications for cancer therapy will be discussed. DisclosuresAllison:Jounce Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: Licensed patent.; Bristol Meyers-Squibb: Patents & Royalties: Licensed patent owned by the University of California. Previously received royalties.

Abstract IA19: Immune checkpoint blockade in cancer therapy: New insights and opportunities

Abstract The existence of multiple non-redundant inhibitory pathways that limit T cell responses offers novel strategies for unleasing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen presenting cells. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>10 years) survival benefit in about 20% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease. The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25% . A recent phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients. These studies and their implications for cancer therapy will be discussed. Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights and opportunities. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr IA19.

Abstract B175: A novel plant sesquiterpene lactone derivative DETD suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice

Abstract Melanoma is the most lethal form of skin cancer and cases are increasing year by year. BRAFV600E mutation is found approximately in 50% of melanoma patients that drives MAPK (RAF/MEK/ERK) signaling for cancer progression. Vemurafenib, a small molecule that specifically targets BRAFV600E mutation, received FDA approval for treatment of late-stage melanoma in 2011. Unfortunately, patients develop acquired vemurafenib resistance via reactivation of MAPK signaling or bypass mechanisms. Recent combination therapy such as MEK inhibitor combined with vemurafenib shows improvement in major clinical end points but percentage of patients with adverse toxic events are higher compared to vemurafenib monotherapy and most patients relapse ultimately. It is therefore an urgent need to develop new anti-melanoma drug and/or adjuvant agent for vemurafenib therapy. In this study, we created a novel semi-organically modified derivative DETD from deoxyelephantopin (DET), a plant sesquiterpene lactone demonstrated as an anti-inflammatory and anti-mammary tumor agent in our laboratory. DETD suppressed both parental human BRAFV600E mutant melanoma (A375) and vemurafenib resistance melanoma (A375-R) cell proliferation in vitro. Furthermore, through compound drug combination assay, DETD and vemurafenib was found to show synergism in suppressing proliferation, conoly formation and inducing apoptosis of A375 melanoma cell. In A375 xenograft study, DETD suppressed tumor growth and reduced tumor mass by 70.5%, as effective as vemurafenib (71.9%), compared to tumor control group. Moreover, compound-drug combinational treatment with alternate administration of DETD and vemurafenib also showed similar tumor growth inhibition efficacy by 72.3%, suggesting in vivo synergistic or additive effect of DETD and vemurafenib. In A375-R xenograft study, vemurafenib treatment showed little or no anti-tumor activity with similar tumor growth rate and sizes relative to the tumor control group, whereas DETD could reduce A375-R tumor growth and mass with 46.9%. Notably, the combination of DETD and vemurafenib treatment exhibited the most significant effect, with a 65.3% reduction in tumor mass. Our mechanistic studies revealed the activation of non-receptor tyrosine kinase Src and its downstream signaling pathways, including MAPK, Akt and STAT3 pathways in the vemurafenib resistant melanoma A375-R, and DETD could de-regulate the activation of all these multi- kinase signaling pathways. Overall, our results offer strong evidence of DETD in inhibiting BRAFV600E melanoma growth and its acquired vemurafenib resistance in mice that suggest DETD might have great therapeutic or adjuvant potential in management of melanoma patients with BRAFV600E mutation. Citation Format: Jia-Hua Feng, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, Lie-Fen Shyur. A novel plant sesquiterpene lactone derivative DETD suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B175.

Abstract PL07-01: Immune checkpoint blockade in cancer therapy: New insights and opportunities.

Abstract The existence of multiple non-redundant l inhibitory pathways that limit T cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen presenting cells. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>4.5 years) survival benefit in about 23% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease. The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. We have recently uncovered a mechanism whereby anti-CTLA-4 antibodies expand Treg in lymph nodes but cause their depletion in the tumor microenvironment. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms. PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. A recent Phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients. These studies and their implications for cancer therapy will be discussed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-01. Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights and opportunities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL07-01.

Abstract 863: Tyrosine kinase activity profiling of metastatic malignant melanoma: Identification of possible therapeutic targets and markers predicting response to therapy

Abstract Metastatic melanoma is an aggressive form of skin cancer that is highly resistant to conventional chemotherapy. Thus, novel approaches to improve the current treatment of advanced melanoma are urgently needed. Kinases are key regulators of cellular processes, and since nearly half of the tyrosine kinase complement is implicated in cancer, the information of kinase activity in tumors give important tumor-specific information on affected pathways and potential targets for treatment. With this in mind, we performed tyrosine kinase activity profiling of 26 tissue samples obtained from patients suffering from metastatic stage IV melanoma, prior to dacarbazine (DTIC) treatment, using Tyrosine Kinase PamChip® microarrays. Each sample lysate generated an individual phospho-substrate signature, representing the state of the kinase signalling cascades, which allowed us to compare affected targets in responders and non-responders to DTIC treatment. Further, we attempted to evaluate the kinase activity in response to BRAF inhibitor, PLX4032 (Vemurafenib), which has shown dramatic and positive effects in advanced melanoma tumors, and has recently been approved for the treatment of late stage melanoma. In this case, incubation was carried out on all samples with and without PLX4032. Our preliminary results showed that although the compound is mainly directed against a serine/threonine kinase, BRAF, PLX4032 also showed inhibitory effects on the activity of multiple tyrosine kinases. Since the drug is only beneficial for melanoma patients with BRAF V600E mutations, we compared the activated kinase pathways in patients with and without this mutation in order to identify alternative survival pathways in patients with wild type BRAF. The technology used in this study is based on a unique 3D flow-through microarray format that allows monitoring of the reaction in real time, ideal for the characterization of pathways. Each array contained 144 peptide substrates with sites for phosphorylation, representing about 100 different proteins. The use of kinase inhibitors will cause inhibition of corresponding kinases of activated signalling pathways; hence, providing us leads to therapeutic targets and possible prediction markers for response to therapy. The detailed effects of both PLX4032 and sunitinib in the chosen test system are currently prepared for presentation. 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 863. doi:1538-7445.AM2012-863