Abstract POSTER-THER-1431: Combined Src and MEK inhibition decreases ovarian cancer (OVCA) cell growth, tumorigenicity and aldehyde dehydrogenase positive tumor-initiating-cells
Authors: Simpkins F. , Hew K. , Azzam D. , Wei Z. , Zhang G. , Sun J. , Zhang D. , Jang K. , Liu W. , Lu Y. , Mills G. , Tan I. , Slingerland J. 
Published: August 2015

Introduction: More effective treatments for recurrent OVCA are needed. Src kinase and the MAPK pathway are activated in OVCAs. Saracatinib, is a potent inhibitor of Abl and Src family kinases and selumetinib, is a potent, selective, ATP-noncompetitive inhibitor of MEK 1/2 kinases. Our earlier work has shown that resistance to saracatinib monotherapy emerges in vitro and in vivo. Since oncogenic activation of both Src and MEK pathways has been observed in OVCAs and because resistance to Src inhibition emerges with monotherapy, we hypothesized that blocking both kinases with combination therapy will more effectively inhibit OVCA cell proliferation than either drug alone.

Experimental Design: Effects of dual Src and MEK blockade on cell cycle, autophagy, apoptosis and xenografts were assayed. Drug effects were confirmed in primary human OVCA tumor cells. Drug effects were evaluated by RPPA and gene expression analysis by microarray. Cells enriched for stem cell marker, aldehyde dehydrogenase 1 (ALDH1+) were sorted by FACS. Drug effects on ALDH1+ cells, and sphere formation were analyzed. Xenografts after treatment were dissociated and transplanted in vivo in a limiting dilution.

Results: Analysis of TCGA high grade serous OVCAs by RPPA showed the majority express pSrc (T419) (n=300/408) and pMAPK (n=310/408) and patients with high pSrc /pMAPK expression had a poorer survival than those with low pSrc /pMAPK (p=0.004). Increased phosphorylation of MAPK was observed with prolonged saracatinib treatment. While each drug alone caused partial growth arrest, treatment with both saracatinib and selumetinib, decreased Src, MAPK, and Akt and caused synergistic cell cycle arrest, which was mediated by p27 binding to cyclin E/cdk2. Gene expression enrichment analysis showed the top 20 gene sets dowregulated by combination treatment involved cell cycle and the gene set most upregulated was autophagy. RPPA confirmed GEA and demonstrated that combination treatment decreased not only Src, MAPK pathways but the PI3K/AKT pathway. Combination treatment induced apoptosis and autophagy. Src and MEK phosphorylation was increased in ALDH1+ sorted populations and combination treatment decreased %ALDH1+cells and % spheres. Combination treatment was more effective in vivo than monotherapy. Transplantation of dissociated cells after treatment in a limiting dilution in vivo resulted in decreased tumor formation and stem cell frequency in the combination treated grp (p=0.01).

Conclusion: MEK inhibition augments the effects of saracatinib by blocking bypass activation of the MAPK and PI3K/AKT pathway, increasing cell cycle arrest, impairing survival and targeting self-renewing subpopulations. These data support further pre-clinical and clinical evaluation of combined saracatinib and MEK inhibition in OVCA.

Citation Format: F. Simpkins, K. Hew, D. Azzam, Z. Wei, G. Zhang, J. Sun, D. Zhang, K. Jang, W. Liu, Y. Lu, G. Mills, I. Tan, J. Slingerland. Combined Src and MEK inhibition decreases ovarian cancer (OVCA) cell growth, tumorigenicity and aldehyde dehydrogenase positive tumor-initiating-cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1431.