Abstract 640: Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs

Abstract

Abstract Ion channels are important portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates “leak” currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung tumors and KCNK9 has been found to promote tumor survival and growth, by increasing resistance to hypoxia and apoptosis. However, KCNK9's underlying pathophysiologic mechanisms have been elusive due to the absence of specific inhibitors. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their molecular/cellular effects on KCNK9-expressing cells. Murine monoclonal IgG1 antibodies were raised against recombinant KCNK9 extracellular domains expressed in a mammalian system. 40 monoclonal antibodies were generated and demonstrated channel subtype-specific recognition of human KCNK9. Among them, 1 antibody designated Y4-mAb displayed sub-nanomolar binding affinity and inhibited KCNK9 channel conductance by up to 30% when tested in a fluorescent ion-influx assay. Through confocal microscopic analysis, channel inhibition was found to result from antibody-induced internalization and endocytosis of cell-surface channels. Y4-mAb induced up to 30∼40% reduction in cell viability and increased cell death in BEN non-small cell lung carcinoma cells and BT-549 and 410.4 breast cancer cells. In addition to “direct cell killing”, this antibody activated complement, resulting in complement-dependent cancer cell cytotoxicity in vitro. Initial experiments in vivo showed that systemic Y4-mAb inhibited the capacity of BEN non-small cell lung carcinoma cells to propagate subcutaneous tumor xenografts in nude mice (p<0.05). In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that perturbs KCNK9 function and inhibits tumor cell survival and xenograft formation. Anti-KCNK9 antibodies have the potential to be valuable research tools and may lead to novel therapeutics. Moreover, our strategies should be generally applicable to studying the functions of other ion channels in cancer. Citation Format: Han Sun, Liqun Luo, Bachchu Lal, John Laterra, Min Li. Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2015-640