PLX4032 has robust activity in BRAF mutated melanoma. The preclinical use of this molecule identifies criteria for its proper clinical application, describes patterns of and reasons for response/resistance, and affords insight into the role of a BRAF mutation in melanoma.

This study reports the identification and characterization of a cAMP-specific phosphodiesterase from the parasitic hemoflagellate Trypanosoma brucei. TbPDE2A is a class I phosphodiesterase. Its catalytic domain exhibits 30 -40% sequence identity with those of all 11 mammalian phosphodiesterase (PDE) families, as well as with PDE2 from Saccharomyces cerevisiae, dunce from Drosophila melanogaster, and regA from Dictyostelium discoideum. The overall structure of TbPDE2A resembles that of human PDE11A in that its N-terminal region contains a single GAF domain. This domain is very similar to those of the mammalian PDE2, -5, -6, -10, and -11, where it constitutes a potential cGMP binding site. TbPDE2A can be expressed in S. cerevisiae, and it complements an S. cerevisiae PDE deletion strain. Recombinant TbPDE2A is specific for cAMP, with a K m of ϳ2 M. It is entirely resistant to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine, but it is sensitive to trequinsin, dipyridamole, sildenafil, and ethaverine with IC 50 values of 5.4, 5.9, 9.4, and 14.2 M, respectively. All four compounds inhibit proliferation of bloodstream form trypanosomes in culture, indicating that TbPDE2A is an essential enzyme.

Proliferation and migration of vascular smooth-muscle cells (VSMCs) are essential events in neointimal hyperplasia. Recent findings that active oxygen species induce pro-oncogene expression, and stimulate VSMC DNA synthesis and cell division, suggest that active oxygen species may play an important role in intimal proliferation after arterial injury. To determine how the redox state of the artery was altered by injury, the levels of thiobarbituric-acid-reactive substances (TBARs, markers of lipid oxidation) and glutathione peroxidase (GSH-PX, the enzyme responsible for the production of glutathione, a major intracellular antioxidant) were measured in the rat aorta after balloon injury. There was an inverse relationship between the level of TBARs, which increased significantly to a maximum 17% greater than normal at 10 days after injury (p < 0.05, n = 7), and the activity of GSH-PX, which decreased significantly to a minimum 36.5% less than normal at 10 days after injury (p < 0.05, n = 7). To determine whether maintaining a more reduced vessel environment would inhibit intimal proliferation, L-cysteine was administered by intraperitoneal injection from 3 days before to 14 days after balloon injury. At 14 days after the arterial injury, the aorta was harvested for histological and morphometric measurements of TBARs and GSH-PX. At 7 and 14 days after balloon injury, the aorta was harvested for [³H]thymidine incorporation studies. Efficacy of therapy was demonstrated by a significant decrease in the level of TBARs and an increase in the activity of GSH-PX (p < 0.05 vs. the control group, n = 7). In the L-cysteine group, all parameters of intimal proliferation were significantly reduced compared to the controls, including the intima:me-dia ratio (0.091 vs. 0.253, p < 0.05); the cross-sectional area of the neointima (0.0563 compared to 0.140 mm², p < 0.05), coverage of the internal elastic lamina by the neointima (23.44 compared to 43.27%, p < 0.05), and [³H]thymidine incorporation (at 7 days 240.58 vs. 350.68 cpm/mg tissue, p < 0.05; at 14 days 181.71 vs. 275.30 cpm/mg tissue, p < 0.05). These results demonstrate dynamic alterations in the vessel redox state after arterial injury, and suggest that maintaining a more reduced environment (e.g. administration of L-cysteine) will reduce intimal proliferation after arterial injury.

Identification of druggable targets along with new therapies in metastatic melanoma is a main concern given the bad prognosis of this disease in the metastatic setting. The development of targeted therapies for cancer has fundamentally changed the ways in which we develop new drugs, select therapy for patients, design and conduct clinical trials and assess treatment outcomes. The problems can be most effectively attacked by an integrated approach that leads from discovery through preclinical and clinical development. Our therapeutic focus is the development of targeted therapeutics and associated predictive biomarkers that can be used to treat melanoma. Here we present data indicating that the IGF-1R and ErbB3 growth factor pathways are co-active in preclinical models of melanoma and propose that MM-141, a novel bispecific antibody co-inhibitor of IGF-1R and ErbB3, has the potential to provide therapeutic benefit to melanoma patients. Our approach is based on a set of 48 melanoma cell lines that have already been extensively characterized both in terms of genetic background and pharmacologic response. We have quantitatively measured basal expression and activation of 40 total and phospho receptor tyrosine kinases expressed by melanoma cell lines. This characterization was placed in the context of the genetic and pharmacologic data that has already been generated in order to develop pathophysiological hypotheses and points of intervention in melanoma. We have also determined anti-proliferative response and mechanism of action for the MM-141 activity in this cell line panel. Proteomic data analyses showed that melanoma cells have lower basal phospho-protein levels relative to other solid tumor cell line panels. The data showed that EGFR and ErbB2 were not overexpressed. ErbB3 and IGF-1R were the most widely expressed RTKs on melanoma cells, and c-MET and FGFR1 were also prevalent. Genomic and proteomic analyses indicated that ErbB3 and IGF-1R mRNA and protein expression were positively correlated. Upon this preliminary analysis of the data we have tested the activity of MM-141 on 48 human melanoma cell lines with well-established genotypes. Taking 15% growth inhibition as a cutoff for response in cell-based assays after 100μg/ml (500nM) MM-141 treatment, 24 out of 48 cell lines resulted in inhibition of cell proliferation 5 days post-treatment. The anti-proliferative response to MM-141 was independent of genotype. We chose 6 cell lines, 3 from the responder population and 3 from the non-responder population to further investigate the mechanism of action of MM-141. No evidence of apoptosis was found after 5 days of MM-141 treatment. All the sensitive cell lines showed a G0/G1 cell cycle arrest in response to MM-141. These combined data provide the rationale for further preclinical and clinical evaluation of MM-141 as a melanoma therapeutic. Citation Format: Erika M. Von Euw, Emily Pace, Karina Covarrubias, Abhi Jairam, Diana Chai, Veerauo Konkankit, Ke-Wei Gong, Bryan Johnson, Birgit Schoeberl, Alexey Lugovskoy, Finn Richard, Dennis Slamon. MM141, a novel bispecific antibody co-inhibitor of IGF-1R and ErbB3, inhibits the proliferation of melanoma cells. [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 2077A. doi:10.1158/1538-7445.AM2013-2077A

Background: Melanoma, a malignancy originating in pigment-producing melanocytes, is generally resistant to conventional treatments such as radiotherapy or chemotherapy. Molecular targeted therapeutics against BRAF and MEK have shown marked efficacy for patients with metastatic melanoma. However these targeted therapies have a limited duration of response. Palbociclib is a highly selective inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6) that has been shown to inhibit growth of malignant cell lines in vitro and in vivo by preventing the phosphorylation of Rb and stopping the progression G1/S of cell cycle. Cyclin D-CDK4/6-Rb axis has been shown to be dysregulated in 85-90% of melanomas and may represent a new therapeutic target. Our main goal in this study was to evaluate the therapeutic potential of palbociclib in a large panel of human melanoma cell lines to identify potential biomarkers of sensitivity and resistance. Material and Methods: We evaluated the response to palbociclib treatment in 48 melanoma cell lines. A comprehensive biomarker screen was performed using mutation and gene expression (Agilent) data. The mechanism of action of the drug was investigated by western blot (WB) analysis measuring phospho-Rb and senescence markers. Effects on the cell cycle and apoptosis were study by flow cytometry. Results: A marked differential response to palbociclib was observed across the melanoma cell lines with IC50s ranging from 28nM to over 1µM. A subset (14/48) of cell lines were classified as sensitive at clinically achievable concentrations (IC50&lt;150nM). BRAF and NRAS activating mutation did not correlate with sensitivity in vitro. Increased expression of genes correlating with cyclin D-CDK4/6 activation strongly predicted for palbociclib sensitivity. We also found that genomic and expression markers of activation of the hedgehog/smoothened (SHH) pathway strongly predicted for in vitro resistance to palbociclib. These markers included chromosomal amplification of SHH, and high baseline expression of SMO and GLI2. Protein analysis by WB showed a dramatic decrease in phospho-Rb protein only in sensitive cell lines and also it caused a decrease in FOXM1 protein, indicating palbociclib may partially act by inducing senescence in treated cells. Flow cytometry analysis confirmed that palbociclib induces a strong G1/S arrest, but not significant apoptosis. Conclusion: Nearly 30% of the melanoma cell lines evaluated were found to be sensitive to palbociclib in vitro. Response to palbociclib did not correlate with BRAF and NRAS mutational status. A gene expression signature for CDK 4/6 activation successfully identified those cell lines most sensitive to palbociclib. We also identified markers of hedgehog/smoothened/GLI activation that strongly predicted for resistance to palbociclib. These data support the development of CDK 4/6 inhibitors in melanoma and provide a hypothesis for patient enrichment in clinical trials. Note: This abstract was not presented at the meeting. Citation Format: Erika M. von Euw, Dylan Conklin, Hong-Mei Rong, Ke-Wei Gong, Richard S. Finn, Dennis J. Slamon. Identification of markers of sensitivity and resistance to palbociclib (PD0332991) in melanoma. [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 1321. doi:10.1158/1538-7445.AM2014-1321