Oncogenic Kit signalling on the Golgi is suppressed by blocking secretory trafficking with M-COPA in gastrointestinal stromal tumours

Abstract

Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as "AMF-26"), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs.

N822K- or V560G-mutated KIT activation preferentially occurs in lipid rafts of the Golgi apparatus in leukemia cells

“…We next examined the site of KIT activation in leukemia cells. To determine the signal platform for KIT, we immuno-stained for phospho-tyrosine residues in the kinase domain which would indicate KIT activation [7–9, 26, 27]. In Kasumi-1 cells, phospho-KIT Y703 (pKIT [Y703]) was clearly detected (Fig.…”

“…We then examined whether KIT activated downstream molecules on the Golgi in leukemia cells. To resolve this question, we used inhibitors of intracellular trafficking, such as brefeldin A (BFA), 2-methylcoprophilinamide (M-COPA) (inhibitors of ER export to the Golgi) [27, 34, 35, 41], monensin (an inhibitor of intra -Golgi trafficking) [26, 42], and bafilomycin A1 (BafA1, an inhibitor of endosome-to-lysosome trafficking) [24, 43]. In Kasumi-1 and HMC-1.1, treatment with BFA or M-COPA significantly increased colocalization of KIT with an ER marker, calnexin (Fig.…”

“…Recently, we showed that in GISTs, KIT on the ER is dephosphorylated by protein tyrosine phosphatases (PTPs) [27]. We then considered the role of PTPs in KIT inactivation in the ER in leukemia cells.…”

“…Finally, we asked whether lipid rafts play a role in oncogenic signaling by all KIT mutants. GIST-T1 cells ( KIT ⊿560–578 ) grow in a manner dependent on KIT signaling on the Golgi, whereas HMC-1.2 (mast cell leukemia, KIT V560G/D816V ) requires pAKT on EL and pSTAT5 on the ER [24–27] (Additional file 1: Figure S6A & Table 1). In both cell types, TKIs increased PM distribution of KIT mutants (Additional file 1: Figure S6B), supporting our data obtained with Kasumi-1 that mutant KIT localizes to intracellular compartments in a manner dependent on its kinase activity.…”

Identification of 10 Hub genes related to the progression of colorectal cancer by co-expression analysis

“…Subpopulations of naive and memory B cells also express GZMB in mammary gland draining lymph nodes (Arabpour et al, 2019). Oncogenic signaling of Kit tyrosine kinase selectively occurs in the Golgi apparatus in the gastrointestinal stromal tumors and causing inhibition of the carcinogenesis by blocking the secretory transport of M-COPA in the gastrointestinal stromal tumors (Obata et al, 2018;Obata et al, 2017). Multiple reports have identified genes directly or indirectly involved in different biological pathways associated with CRC.…”