ALK gene rearrangement was observed in 3%–5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI–resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

, one of the four members of the R-spondin family of proteins, has agonistic activity in the Wnt/β-catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C-mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C-C linkage between the tryptophan residue and an α-mannose. Although Rspo2 has two putative C-mannosylation sites at residues Trp 150 and Trp 153 , it had not been reported to date whether these sites are C-mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C-mannosylated at the Trp 150 and Trp 153 residues. Notably, while this C-mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C-mannosylation had consistent effects on the activation of Wnt/β-catenin signaling in PANC1 and MDA-MB-231 cells, as well as HT1080 cells. Furthermore, overexpression of wild-type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C-mannosylation-defective mutant enhanced migration to a lesser degree. These results suggested that C-mannosylation of Rspo2 may promote cancer progression and that the inhibition of C-mannosylation may serve as a potential novel therapeutic approach for cancer therapy.

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C-mannosylation is a unique type of protein glycosylation via C-C linkage between an a-mannose and a tryptophan residue. This modification has been identified in about 30 proteins and regulates several functions, such as protein secretion and intracellular localization, as well as protein stability. About half of C-mannosylated proteins are categorized as proteins containing thrombospondin type 1 repeat domain or type I cytokine receptors. To evaluate whether C-mannosylation broadly affects protein functions regardless of protein domain or family, we have sought to identify other types of C-mannosylated protein and analyse their functions. In this study, we focused on receptor activity modifying protein 1, which neither contains thrombospondin type 1 repeat domain nor belongs to the type I cytokine receptors. Our mass spectrometry analysis demonstrated that RAMP1 is C-mannosylated at Trp 56 . It has been shown that RAMP1 transports to the plasma membrane after dimerization with calcitonin receptor-like receptor and is important for ligand-dependent downstream signalling activation. Our results showed that C-mannosylation has no effect on this transport activity. On the other hand, C-mannosylation did enhance protein stability and cell migration activity. Our data may provide new insight into both C-mannosylation research and novel RAMP1 analysis.