Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

<p>Figure and table legends for supplemental figures 1-4 and supplemental tables 1-3.</p>

<div>Abstract<p>Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of <i>KIT</i>-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of <i>KIT</i>-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct <i>KIT</i>-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each <i>KIT</i>-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of <i>KIT</i>-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease. <i>Mol Cancer Ther; 13(12); 2840–51. ©2014 AACR</i>.</p></div>

<p>Figure S1: HMC1.2 cell line has constitutively actived NFAT2, NFAT2 and NFAT4 species; Figure S2: RT-PCR of transcripts modulated by 24 hour treatment with CSA; Figure S3: CNPIs do not modulate KIT signaling and KIT inhibitors do not affect NFAT localization; Figure S4: Combination therapy does not enhance the effect of KIT inhibitors on KIT or downstream signaling; Table S1: Summary of KIT-mutant mast cell characteristics; Table S2: Summary of antibodies used in immunoblotting experiments; Table S3: qRT-PCR analysis of NFAT expression in KIT-mutant mast cell lines.</p>

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