Fig. 3: KRAS LYMTACs induce pathway suppression irrespective of KRAS degradation. | Nature Communications

Fig. 3: KRAS LYMTACs induce pathway suppression irrespective of KRAS degradation.

From: LYMTACs:chimeric small molecules repurpose lysosomal membrane proteins for target protein relocalization and degradation

Fig. 3

A Schematic of complex formation between KRASG12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B Structures of the pan-KRAS inhibitor and PROTAC C Structures of LYMTAC-3 and LYMTAC-4. D HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12F36V-KRASG12D) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as the input. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Quantified data are representative of two independent experiments. G AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Quantified data are representative of two independent experiments. H HEK293 cells stably expressing HiBiT-FKBP12-KRASG12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Quantified data are representative of two independent experiments.

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