title: Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.
abstract: 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.
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
authors.forename: Alison C
authors.initials: AC
authors.lastname: Macleod
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
authors.forename: Lillian R
authors.initials: LR
authors.lastname: Klug
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
authors.forename: Janice
authors.initials: J
authors.lastname: Patterson
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Griffith
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
authors.forename: Carol
authors.initials: C
authors.lastname: Beadling
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon.
authors.forename: Ajia
authors.initials: A
authors.lastname: Town
authors.affiliation: Portland VA Medical Center, Portland, Oregon. OHSU Knight Cancer Institute, Portland, Oregon. heinrich@ohsu.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Heinrich
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D065095:Calcineurin Inhibitors / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000069439:Dasatinib; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004357:Drug Synergism; D055785:Gene Knockdown Techniques; D006801:Humans; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism*; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics* / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic
chemical_list: D065095:Calcineurin Inhibitors; D050778:NFATC Transcription Factors; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D019009:Proto-Oncogene Proteins c-kit; D019703:Calcineurin; D000069439:Dasatinib; D002118:Calcium