FDW028, a novel FUT8 inhibitor, impels lysosomal proteolysis of B7-H3 via chaperone-mediated autophagy pathway and exhibits potent efficacy against metastatic colorectal cancer

Abstract

Metastatic colorectal cancer (mCRC) is a major cause of cancer-related mortality due to the absence of effective therapeutics. Thus, it is urgent to discover new drugs for mCRC. Fucosyltransferase 8 (FUT8) is a potential therapeutic target with high level in most malignant cancers including CRC. FUT8 mediates the core fucosylation of CD276 (B7-H3), a key immune checkpoint molecule (ICM), in CRC. FUT8-silence-induced defucosylation at N104 on B7-H3 attracts heat shock protein family A member 8 (HSPA8, also known as HSC70) to bind with 106-110 SLRLQ motif and consequently propels lysosomal proteolysis of B7-H3 through the chaperone-mediated autophagy (CMA) pathway. Then we report the development and characterization of a potent and highly selective small-molecule inhibitor of FUT8, named FDW028, which evidently prolongs the survival of mice with CRC pulmonary metastases (CRPM). FDW028 exhibits potent anti-tumor activity by defucosylation and impelling lysosomal degradation of B7-H3 through the CMA pathway. Taken together, FUT8 inhibition destabilizes B7-H3 through CMA-mediated lysosomal proteolysis, and FDW028 acts as a potent therapeutic candidate against mCRC by targeting FUT8. FDW028, an inhibitor specifically targeted FUT8, promotes defucosylation and consequent HSC70/LAMP2A-mediated lysosomal degradation of B7-H3, and exhibits potent anti-mCRC activities.

FDW028, an inhibitor specifically targeted FUT8, promotes defucosylation and consequent HSC70/LAMP2A-mediated lysosomal degradation of B7-H3, and exhibits potent anti-mCRC activities.

Fig. 1. B7-H3 is N-glycosylated and core fucosylated in CRC cells.

A Immunoblots of B7-H3 treated with or without PNGase F at 37 °C for 3 h. B Immunoblots of truncated B7-H3 containing the first IgV-IgC domain (aa1-258) treated with or without PNGase F at 37 °C for 3 h. C Immunoblots of truncated B7-H3 containing N91Q, N104Q, N189Q or N215Q mutation. D AOL blot of Flag-B7-H3 immunoprecipitated by anti-Flag beads. E IHC stains of AOL and B7-H3 in CRC tissues. F Immunoblots of B7-H3 in SW480 and HCT-8 cells upon knockdown of each fucosyltransferase. G Immunoblots of membranous AOL and B7-H3 in SW480 and HCT-8 cells upon FUT8 knockdown. H Immunoblots of B7-H3 and its downstream AKT/MTOR pathway molecules in SW480 and HCT-8 cells upon FUT8 knockdown. All experiments were performed in technical triplicates and are displayed as mean ± s.d.; ns no significance; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Defucosylation-promoted lysosomal degradation of B7-H3.

A Immunoblots of B7-H3 protein in SW480 and HCT-8 cells that were treated with 100 μM CHX for the indicated time after FUT8 knockdown or not. B Immunoblots of B7-H3 protein in SW480 and HCT-8 cells that were treated with MG-132 (20 μM), PS-341 (50 nM), CHQ (50 μM), and AC (100 μM), respectively. C Immunoblots of B7-H3 protein in SW480 and HCT-8 cells that were treated with CHQ (50 μM) or AC (100 μM) after FUT8 knockdown. D Immunofluorescence shows the colocalization of B7-H3 and LAMP1 upon FUT8 knockdown. The intensity profiles of B7-H3 and LAMP1 are plotted in the middle panel. The statistical results of colocalization factor (Pearson’s R value) are shown on the right panel. All experiments were performed in technical triplicates and are displayed as mean ± s.d.; ns no significance; **P < 0.01, ***P < 0.001.

Fig. 3. Defucosylation-promoted CMA degradation of B7-H3.

A Immunoblots of B7-H3 protein in SW480 and HCT-8 cells after knockdown of FUT8 and / or HSC70. B Immunoblots of B7-H3 protein in SW480 and HCT-8 cells after knockdown of FUT8 and / or LAMP2A. C Co-IP assays showed the effects of FUT8 knockdown on the interaction between B7-H3 and HSC70 or LAMP2A. D Schematics of truncated B7-H3 (aa 1-258) with the N104 glycosylation site and 106-110SLRLQ motif for HSC70 binding, and AOL blots of the co-IP B7-H3 protein with wildtype N104 and N91Q/N189Q/N215Q mutations. E Schematics of truncated B7-H3 (aa 1-258) with N104Q or Q110A mutation, and immunoblots of truncated B7-H3 in SW480 cells that were treated with 100 μM CHX after transfection of B7-H3 expression plasmids containing N104Q or Q110A mutation. F Schematics of truncated B7-H3 (aa 1-258) with N104Q or VRV112-114NAS mutation, and immunoblots of truncated B7-H3 containing N104Q and / or VRV112-114NAS mutation in SW480 cells. G Co-IP assays showed the interaction between the truncated B7-H3 containing N104Q or Q110A mutation and HSC70 or LAMP2A. All experiments were performed in technical triplicates and are displayed as mean ± s.d.; ns no significance; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. Discovery of FUT8 inhibitor FDW028.

A Strategies for [1, 2, 4] triazolo [1,5-a] pyrimidin-7-ol skeleton discovery. B Docking prediction of FDW028 binding to FUT8. C Binding kinetics for FUT8 versus FDW028 obtained from GCI experiments. Sensograms are shown in multiple color with the respective fits in black, and include table summaries of the dissociation constant K_D. D Protein thermal shift assay curves for FUT8 with 200 μM FDW028 at gradient temperatures from 39.0 to 59.0 °C (up), or with the indicated concentration of FDW028 at 51.9 °C [45]. E The immunoblots (up) and LC–MS/MS spectrum [45] of FUT8 that was pulled down by FDW028-biotin. The structure of FDW028-biotin is also shown.

Fig. 5. Anti-CRC activities of FDW028.

A The colony formation abilities of SW480 and HCT-8 cells treated with FDW028 at 50 μM for 14 days. B The viabilities of SW480 and HCT-8 cells upon the treatment of FDW028 at the indicated concentrations for 72 h, with or without FUT8 knockdown. Transwell assays (C) and wound-healing assays (D) for showing the migration abilities of SW480 and HCT-8 cells treated with FDW028 at 50 μM for 72 h. E Growth curves and photos of SW480 xenografts treated with FDW028 (10 or 20 mg/kg) or 5-Fu (10 mg/kg). F The body weights of tumor-bearing mice with the administration of FDW028 or 5-Fu. G The survival of CRPM mice after i.v. injection of FDW028 at 20 mg/kg once the other day. HE stains of the lung of normal and tumor-bearing mice are shown. i.v. intravenously; t.a. tumor-aside; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. FDW028 promoted defucosylation and CMA degradation of B7-H3.

A Immunoblots of AOL, B7-H3, and AKT/MTOR pathway molecules in SW480 and HCT-8 cells treated with FDW028 or 2F-Fuc at 50 μM for 72 h. B Immunoblots of B7-H3 in SW480 and HCT-8 cells treated with FDW028 at the indicated concentrations for 72 h. C Immunoblots of B7-H3 in SW480 and HCT-8 cells after the treatment of FDW028 (50 μM) with or without following CHQ (50 μM) or AC (100 μM). D Immunofluorescence shows the colocalization of B7-H3 and LAMP1 upon the treatment of FDW028. The intensity profiles of B7-H3 and LAMP1 are plotted in the middle panel. The statistical results of colocalization factor (Pearson’s R value) are shown on the right panel. E Co-IP assays showed the effects of FDW028 on the interaction between B7-H3 and HSC70 or LAMP2A. F Immunoblots of B7-H3 in SW480 and HCT-8 cells treated by FDW028 (50 μM) with or without HSC70 siRNA for 72 h. G Immunoblots of B7-H3 in SW480 and HCT-8 cells treated by FDW028 (50 μM) with or without LAMP2A siRNA for 72 h. All experiments were performed in technical triplicates and are displayed as mean ± s.d.; ns no significance; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7. Schematic diagram showing the pharmacological mechanism of FDW028.

FDW028, an inhibitor specifically targeted FUT8, promotes defucosylation and consequent HSC70/LAMP2A-mediated lysosomal degradation of B7-H3, and exhibits potent anti-mCRC activities.