Src family kinase activity is elevated in many human tumors, including breast cancer, and is often associated with aggressive disease. We examined the effects of SKI-606 (bosutinib), a selective Src family kinase inhibitor, on human cancer cells derived from breast cancer patients to assess its potential for breast cancer treatment. Our results show that SKI-606 caused a decrease in cell motility and invasion of breast cancer cell lines with an IC 50 of f250 nmol/L, which was also the IC 50 for inhibition of cellular Src kinase activity in intact tumor cells. These changes were accompanied by an increase in cellto-cell adhesion and membrane localization of B-catenin. By contrast, cell proliferation and survival were unaffected by SKI-606 at concentrations sufficient to block cell migration and invasion. Analysis of downstream effectors of Src revealed that SKI-606 inhibits the phosphorylation of focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), and Crk-associated substrate (p130 Cas ), with an IC 50 similar to inhibition of cellular Src kinase. Our findings indicate that SKI-606 inhibits signaling pathways involved in controlling tumor cell motility and invasion, suggesting that SKI-606 is a promising therapeutic for breast cancer.

1690 Poster Board I-716 Background Patients with relapsed or refractory Hodgkin (HL) and Non Hodgkin Lymphoma (NHL) have few options after salvage therapy and transplant, and new agents are thus needed. MK-5108 is a novel aurora kinase inhibitor (AKI) with specificity against aurora kinase A, that produces G2/M phase cell cycle arrest. We show that addition of vorinostat, a histone and protein deacetylase inhibitor, to AKI treatment results in reactivation of proapoptotic genes and enhanced lymphoma cell death. A panel of HL and NHL cell lines was studied with either drug or the combination, using cell growth, apoptosis, and flow cytometry assays, followed by molecular studies. Results MK-5108 alone at 0.1 – 3 mM results in significant growth inhibition and apoptosis in multiple cell lines representing Hodgkin, Burkitt, and Non-Hodgkin lymphoma types, interestingly,DHL-4 and DHL-6 cells were more sensitive to this agent than to the pan-AKI MK-0457. Vorinostat alone at a dose range of 0.5 – 3 mM reduces cell growth by 50% or more in all lines tested. The combination of 1.5 mM vorinostat and 100 nM MK-5108 results in over 85% apoptosis of multiple lymphoma lines tested at 72 hours. Cell cycle analyses by FACS of MK-5108 treated cells show an increased percentage of cells in G2/M with few cells in sub-G1, whereas in combination with vorinostat the G2/M peak decreases and there is a significant increase in the apoptotic sub-G1 population. Real-time PCR analysis and immunoblotting of L540 cells treated with either single agent or in combination revealed that vorinostat treatment leads to alteration in pro-apoptosis, growth arrest, and DNA damage response genes. Myc mRNA and protein levels are reduced by vorinostat, and repression of microRNAs (miRNAs) in the Myc-regulated polycistronic cluster of miRNAs of chromosome 13, such as miR-17.5p, -17.3p, and 18, occurs with vorinostat and TSA. Prosurvival genes such as bcl-XL and hTERT are downregulated five-fold by vorinostat treatment, while the proapoptotic BAK gene is upregulated 1.5 – 2-fold. Vorinostat treatment leads to enhanced acetylation of p53, with a corresponding increase in the p53 target genes p21 and Noxa. To analyze the role of Myc inhibition in the sensitization by vorinostat of lymphoma cells to MK-5108, siRNA-mediated knock-down of Myc expression in L540 cells was performed. The siRNA-Myc transfected L540 cells showed enhanced sensitivity to MK-5108 as compared to control siRNA-null cells, as well as decreased hTERT levels, confirming the role of Myc inhibition by vorinostat as an integral part of the sensitization of lymphoma cells to MK-5108. Conclusions The HDACi vorinostat leads to both transcriptional and post-transcriptional changes that create a pro-apoptotic milieu, sensitizing the cell to centrosome-acting agents such as the aurora kinase A inhibitor MK-5108. These preclinical data support clinical trials of MK-5108 plus vorinostat in patients with relapsed or refractory lymphomas. [We acknowledge Merck Inc for providing Vorinostat, MK-0457, MK-5108, and research support.] Disclosures Kretzner: Merck: Research Funding. Yen:Merck: Research Funding. Kirschbaum:Merck: Research Funding, Speakers Bureau.