M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

Abstract

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.

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

“…In this study, blockade of the ER export of KIT with BFA/M-COPA decreased KIT’s autophosphorylation in leukemia cells. Together with the results previous reports [25, 27, 76, 77], our findings may offer a trafficking blockade of receptor mutants as a third strategy for inhibition of oncogenic signaling.…”

“…Recently, we reported that in MCL, AL-mut, such as KIT D816V (human) and KIT D814Y (mouse), accumulates in EL (Table 1) [24, 25]. Unlike JM-mut, AL-mut in MCL activates AKT and STAT5 on EL and the ER, respectively.…”

“…Previously, we reported that in MCL, KIT D816V (human) and KIT D814Y (mouse) activate AKT and the signal transducer and activator of transcription 5 (STAT5) in EL and on the ER, respectively (Table 1) [24, 25]. Furthermore, previous studies showed that in cells other than those of MCL, such as GIST and NIH3T3 cells, JM-mut or AL-mut accumulates on the Golgi apparatus, where it initiates oncogenic signals (Table 1) [26–29].…”

“…The major activating KIT mutations are found at D816 and N822 (26 cases and 14 cases in 63 KIT mutation-positive patients, respectively) [33]. Although spatio-temporal analyses of KIT D816V signals have been performed [24, 25, 28], it is unclear whether the N822K mutation in leukemia affects KIT localization and the signal platform.…”

Drugging the Small GTPase Pathways in Cancer Treatment: Promises and Challenges

“…In fact, AMF-26 can induce complete tumor regression in breast cancer xenografts [54], reduce the proliferation of 39 different cancers in a variety of human organs (such as breast, colon, kidney, skin, central nervous system, lung, ovary, and stomach) [53,55], as well as diminish angiogenesis through suppressing the activation of the vascular endothelial growth factor receptor 1/2 (VEGFR1/2) and the nuclear factor-κB (NF-κB) pathways [56]. In addition, AMF-26 deactivates a mutant form of the endolysosomal Kit, leading to sensitizing carcinogenic mast cell to imatinib [57]. Finally, AMF-26 also prevents Shiga toxin-dependent apoptosis by decreasing its translocation into the Golgi apparatus [58].…”