Fig 2: Development of LYMTAC as a modular tool for lysosomal degradation of membrane proteins.A Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Quantified data are representative of two independent experiments. C Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand) and PROTAC TL-12-186. D Quantitative proteomics analysis of LYMTAC-1. HEK293 cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. Differential expression was assessed with the limma moderated t-test (empirical-Bayes framework, two-sided). Reported P values are unadjusted; no correction for multiple comparisons was applied. E Quantitative proteomics analysis of PROTAC (TL-12-186). HEK293 cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM PROTAC for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. Differential expression was assessed with the limma moderated t-test (empirical-Bayes framework, two-sided). Reported P values are unadjusted; no correction for multiple comparisons was applied. F LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293 cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Quantified data are representative of three independent experiments. G LYMTAC-1 induced dose-dependent degradation of EPHA2. Quantified data are representative of three independent experiments. H, I HEK293 cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Quantified data are representative of two independent experiments.

Fig 3: KRAS relocalization and lysosomal degradation using LYMTAC lead to deeper pathway suppression and potent cell killing compared to SMI.A Schematic of LYMTAC-induced KRAS relocalization from plasma membrane to lysosome. B KRAS localization analyzed by confocal microscopy. FLAG-MTH1-RNF152-stably expressing HEK293 mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 for 30 min and then co-treated with DMSO or 1 µM LYMTAC-4 for 4 h. Cells were fixed, permeabilized, and immunostained with FLAG and LAMP1 antibodies, followed by appropriate secondary fluorophore-conjugated antibodies. Nuclei were stained with Hoechst dye. Data are representative of two independent experiments. C Kinetics of LYMTAC-4 induced KRAS relocalization. FLAG-MTH1-RNF152-stably expressing HEK293 mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 for 30 min and then co-treated with 1 µM LYMTAC-4. Live cell images were acquired every 30 min up to 4 h. Puncta formation in each cell is highlighted with orange, green and pink arrows at each time point. Data are representative of two independent experiments. D Washout experiment in AsPC-1 cells stably expressing FLAG-MTH1-RNF152. Cells were treated with 100 nM pan-KRASi and 100 nM LYMTAC-4 for 4 h, washed three times with PBS and replaced with fresh media in the absence or presence of 5 µM MTH1-ligand for 48 h. Quantified data are representative of two independent experiments. E pan-KRASi, PROTAC, and LYMTAC-4 activity in a 5-day cell proliferation assay in AsPC-1 cells stably expressing MTH1-RNF152. Data are representative of three independent experiments, reported as the mean ± S.E.

Fig 4: LYMTACs induce ubiquitylation and lysosomal degradation of KRASG12D.A Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12F36V-KRASG12D, and the effector is MTH1-RNF152. B Structure of LYMTAC-2 (FKBP12F36V ligand-PEG2-MTH1 ligand). C HiBiT cellular degradation assay in knock-in HCT116 (HiBiT- FKBP12F36V-KRASG12D: (HF-KRAS)) cells in the absence or presence of stably expressing MTH1-RNF152. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h, and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Quantified data are representative of two independent experiments. E KRASG12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as the input. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.