Hayashi, Yujiro

Abstract Background & Aims: Most GIST harbor oncogenic KIT or PDGFRA mutations, which are in most cases heterozygous. However, most GIST co-express both wild-type KIT and PDGFRA with the mutated KIT or PDGFRA. Signaling mediated by heterodimers of the wild-type receptors contribute to GIST growth even after blocking oncogenic signaling by KIT/PDGFRA inhibitors such as imatinib. Previously we found that crenolanib (CP-868596), a PDGFRA/B tyrosine kinase inhibitor with >100-fold selectivity over other tyrosine kinases including KIT and a potent inhibitor of oncogenic PDGFRA signaling in vitro, inhibited the proliferation of KIT+PDGFRA+PDGFRB− GIST-T1 cells with a potency comparable to that of imatinib (IC50: 5.3 nM; imatinib: IC50: 3.5 nM) but had very little effect in the KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells (maximum inhibition: <50%; IC50>75 nM) and in mouse stem cells for interstitial cells of Cajal (ICC-SC), a presumed cellular source for GIST, which share a phenotype with GIST48B cells (IC50 >600 nM; Hayashi et al., Gastroenterology 2012; 142:S30). Here we investigated whether the antiproliferative effect of crenolanib was dependent on KIT expression, and studied crenolanib's effect on KIT/PDGFRA signaling and on the stability of ETV1, a transcription factor required for GIST growth and survival. Methods: Protein expression and phosphorylation were studied by Western blotting. KITlow/−PDGFRAlow/+PDGFRB+ murine ICC-SC (2xSCS2F10) were retrovirally transduced to express wild-type murine KIT. Cell proliferation was studied by methyl-tetrazolium salt assay. Results: Expression of wild-type KIT in retrovirally transduced ICC-SC dramatically reduced crenolanib's IC50 from 1157 nM to 121 nM. In GIST-T1 cells, crenolanib significantly reduced PDGFRA and KIT protein expression, inhibited PDGFRA phosphorylation (IC50: 5 nM) but had no effect on KIT phosphorylation. Crenolanib applied at 8.3 nM concentration rapidly and profoundly inhibited phosphorylation of ERK1/2 MAP kinases and reduced ETV1 protein levels by >50% in 2 h. The MEK inhibitor PD98059 (50 μM) reproduced crenolanib's effect on ETV1 protein levels with an identical time-course. The inhibitory effects of both crenolanib and PD98059 on ETV1 protein expression could be reversed with the proteasome inhibitor MG132 (10 μM) and accentuated with the protein synthesis inhibitor cycloheximide (10 μg/mL). ETV1 was undetectable in the crenolanib-resistant, KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells. Conclusions: In KIT mutant, KIT+PDGFRA+PDGFRB− GIST cells, crenolanib induced ETV1 protein degradation by suppressing PDGFRA-dependent MAPK phosphorylation. Crenolanib's antiproliferative effect in these cells may depend on KIT-induced ETV1 expression and reflect the destabilization of ETV1. Grant support: NIH DK58185. Citation Format: Yujiro Hayashi, Michael R. Bardsley, Yoshitaka Toyomasu, Takahiro Taguchi, Brian P. Rubin, Monique Carter, Abhijit Ramachandran, Tamas Ordog. Crenolanib, a highly selective platelet-derived growth factor receptor α/β (PDGFRA/B) tyrosine kinase inhibitor, destabilizes ETV1 protein in KIT-mutant gastrointestinal stromal tumor (GIST) 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 1035. doi:10.1158/1538-7445.AM2013-1035

Key points Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice. Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients. Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity. This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract. AbstractInterstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca2+‐activated Cl− channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca2+ transients between myenteric ICC (ICC‐MY) of small intestine. First, we found spontaneous Ca2+ transients in ICC‐MY in both Ano1 WT and knockout (KO) mice. However, Ca2+ transients within the ICC‐MY network in Ano1 KO mice were uncoordinated, while ICC‐MY Ca2+ transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca2+ transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA‐mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca2+ transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca2+ transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non‐rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca2+ transients in ICC‐MY.