Abstract 3281: Combination therapy to target KIT mutant cells.
Authors: Macleod Alison C. , Heinrich Michael C. , Griffith Diana , Town Ajia 
Published: August 2014

Abstract
Purpose: Gastrointestinal stromal tumors (GIST), the most common abdominal sarcoma, arise from the interstitial cells of Cajal in the wall of the gut. Eighty percent of GISTs harbor activating mutations in the receptor tyrosine kinase KIT. Despite progress in medical treatment of GIST, advanced disease remains incurable. In addition to GIST, activating KIT mutations are found in cases of mast cell neoplasms, AML, melanoma and seminoma. Therefore, there is a compelling need to identify new targets whose inhibition is synergistic when combined with biochemical inhibition of KIT. Based on shared oncogenic kinase mechanisms with CML, we hypothesized that combined inhibition of KIT and a novel Wnt/Ca++/Calcineurin/NFAT pathway would synergistically inhibit the proliferation of KIT mutant cells.
Experimental methods: We sequentially combined four different calcineurin inhibitors with five different KIT inhibitors and measured cell proliferation and caspase 3/7activity in nine unique KIT mutant cell lines. Using the Chou and Talalay method we calculated combination indices to quantify drug-drug interactions. Changes in NFAT phosphorylation and localization in KIT mutant cells were assessed by immunoblotting. Finally, we measured NFAT transcriptional activity using an NFAT-responsive reporter cell line.
Results: In contrast to other cellular models, NFAT appeared to be constitutively active and localized in the nucleus in many KIT mutant cell lines. We observed synergy when we combined a calcineurin inhibitor with a KIT inhibitor in all KIT mutant cell lines where NFAT was constitutively active. Combination index (CI) values for the different KIT mutant cell lines ranged from 0.3-0.5. To investigate whether NFAT was required for this synergy, we combined NFAT specific inhibitors with KIT inhibitors and found synergistic inhibition of cell proliferation. We created a KIT-mutant reporter cell line where NFAT binding drives the expression of luciferase. As expected, NFAT transcriptional activity decreased when our promoter-reporter cell line was treated with calcineurin inhibitors and increased with TPA and ionomycin treatment. Unexpectedly, we also found that KIT inhibitors decreased NFAT transcriptional activity in this cell model.
Conclusions: These data indicate that combination therapy using a calcineurin phosphatase inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant cells. Additionally, the constitutive activation of NFAT in these cell lines indicate aberrant signaling in the Wnt/Ca2+/calcineurin/NFAT pathway, which has not been previously reported in KIT mutant cell lines. The observed synergy between NFAT inhibitors and KIT inhibitors suggests this effect is mediated by NFAT rather than other calcineurin targets. Finally, the fact that KIT inhibitors modulated NFAT transcriptional activity suggests crosstalk between KIT and NFAT signaling pathways.
Citation Format: Alison C. Macleod, Michael C. Heinrich, Diana Griffith, Ajia Town. Combination therapy to target KIT mutant 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 3281. doi:10.1158/1538-7445.AM2013-3281