Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Principle of the CANDDY technology. Each CANDDY molecule includes a target interactor (burgundy hexagon) and a CANDDY tag (yellow oval). The target protein is bound by the CANDDY molecule via the target interactor and is directly degraded by proteasome via the CANDDY tag. b, The CANDDY tag was synthesized by chemically inactivating the inhibitor site of MLM2238 (MLN). c, Structures of the RAS-SOS inhibitor (ref. 5), RAS-SOS-NH 2 , and TUS-007. d, KRAS G12D was mixed with DMSO, RAS-SOS inhibitor (4 µM) or TUS-007 (4 µM) and incubated under heating from 25 °C to 99 °C. The denature of KRAS G12D was monitored by the fluorescence. The typical curve of each group was shown. e, The correlation between relative amount of KRAS G12D and concentration of TUS-007 were approximated with Rodbard using the means of 3 independent examinations. The DC50 was estimated about 4 µM. f, TUS-007 induced KRAS G12D chemical knockdown by 26S proteasome in a cell free assay. KRAS G12D was incubated with 26S proteasome and agents as indicated for 3 h.

Article Snippet: Human KRAS (G12D) and the corresponding His tag (12259-H07E1) were purchased from Sino Biological (Beijing, China).

Techniques: Synthesized, Incubation, Fluorescence, Concentration Assay, Cell-Free Assay

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, TUS-007 induced chemical knockdown was independent of ubiquitination. GFP fused KRAS G12D and DsRed were transiently expressed in HeLa cells. The cells were treated with TUS-007 in the presence of a proteasome inhibitor (MLN), a ubiquitination inhibitor (NAEi) and a molecular chaperon inhibitor (HSP70 inhibitor: VER-155008) for 24 h. b & c, RAS-less MEFs expressing KRAS G12D, G12V or G12C were incubated with TUS-007 for 72 h. Representative blots of KRAS are shown (b). The relative amounts of KRAS mutants normalized to GAPDH are shown in the bar graph (mean ± SEM; n = 3–4). The cell viability was also measured (mean ± SEM; n = 3–4) (g). *P < 0.05 and ** P < 0.01 vs. DMSO. TUS-007 induced chemical knockdown did not cause non-specific cytotoxicity.

Article Snippet: Human KRAS (G12D) and the corresponding His tag (12259-H07E1) were purchased from Sino Biological (Beijing, China).

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, KRAS G12D chemical knockdown induced by TUS-007 in SW1990 cells. SW1990 cells were treated with TUS-007 for 72 h and analyzed by immunoblotting. The KRAS G12D band intensity was normalized to that of GAPDH (mean ± SEM; n = 4). *P < 0.05 vs. DMSO. b, The proportion of annexin V-positive apoptotic SW1990 cells treated with DMSO, RAS-SOS-NH 2 or TUS-007 for 72 h (mean ± SEM; n = 3). *P < 0.05 vs. DMSO or RAS-SOS-NH 2 . c, TUS-007 increased the caspase 3/7 activity in SW1990 cells after treatment for 96 h (mean ± SEM; n = 3). *P < 0.05 and **P < 0.01 vs. DMSO. d, Caspase 3/7 activation by TUS-007 was proteasome dependent and ubiquitination independent. SW1990 cells were treated with the agents for 96 h (mean ± SEM; n = 3). **P < 0.01 vs. DMSO, ## P < 0.01 vs. TUS-007 (100 µM) only, and † no statistically significant difference compared with TUS-007 (100 µM) only. e, Tumor volume in mice with SW1990 cells transplanted subcutaneously and treated with p.o. administered TUS-007 or vehicle (mean ± SEM; n = 6–9). The agents were administered every 3 days. **P < 0.01 vs. vehicle alone. f, Body weight changes in mice with SW1990 cells transplanted subcutaneously (mean ± SEM; n = 6–9).

Article Snippet: Human KRAS (G12D) and the corresponding His tag (12259-H07E1) were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Images showing the luciferase activity and total flux in orthotopic tumors transplanted SW1990-Luc cells in vivo . b, Tumor growth (bioluminescence) in identical mice treated with vehicle or TUS-007 administered p.o. every 3 days. (mean ± SEM; n = 6). *P < 0.05 vs. vehicle alone. c, Representative histochemical staining of KRAS and TUNEL in orthotopic tumor sections from the same mice used in a . The scale bar is 60 µm. d, Immunoblotting of KRAS G12D in tumor lysates from the same mice used in a . The bar graph shows quantification of KRAS G12D normalized to GAPDH (mean ± SEM; n = 6). **P < 0.01 vs. vehicle alone.

Article Snippet: Human KRAS (G12D) and the corresponding His tag (12259-H07E1) were purchased from Sino Biological (Beijing, China).

Techniques: Luciferase, Activity Assay, In Vivo, Staining, TUNEL Assay, Western Blot

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Tumor growth, as assessed by luciferase signal, in individual mice orthotopically transplanted SW1990-Luc cells treated with TUS-007 (orange) or vehicle alone (blue) (mean ± SEM; n = 6). b, Pancreases from orthotopic xenograft model mice at day 21 after treatment with TUS-007 (lower-left panel) or the vehicle alone (upper-left panel) and comparison of their weights (right graph) (mean ± SEM; n = 6). *P < 0.05 vs. vehicle alone. c, The quantitative analyses of the KRAS G12D-positive area (left) and TUNEL-positive area (right) in (mean ± SEM; n = 6). **P < 0.01 vs. vehicle alone. d, Body weight changes in mice subjected to orthotopic transplantation of SW1990-Luc cells (mean ± SEM; n = 6).

Article Snippet: Human KRAS (G12D) and the corresponding His tag (12259-H07E1) were purchased from Sino Biological (Beijing, China).

Techniques: Luciferase, TUNEL Assay, Transplantation Assay

Journal: Translational Lung Cancer Research

Article Title: Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines

doi: 10.21037/tlcr-2026-0798

Figure Lengend Snippet: MTT cytotoxicity assays of one KRAS wildtype (BH1440), one KRAS G12D (BH1194) and one KRAS G13C (BH1450) NSCLC cell line. Cells were treated with 9 two-fold dilutions starting at 2 µM of (A) pan-KRAS(ON) inhibitor RMC-6236 and (B) KRAS(ON) G12D inhibitor RMC-9805. Data are presented as mean ± standard deviation.

Article Snippet: The KRAS(ON) targeting compounds RMC-6236 (pan-KRAS), RMC-6291 (KRAS G12C), RMC-9805 (KRAS G12D) and RMC-5127 (KRAS G12V) were acquired from Targetmol ( T74698 , T75131 , T78212 , T204085, Wellesley Hills, MA, USA).

Techniques: Standard Deviation

Journal: Translational Lung Cancer Research

Article Title: Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines

doi: 10.21037/tlcr-2026-0798

Figure Lengend Snippet: Heatmap of RMC compound efficacy sorted by cell line origin and KRAS mutation. This heatmap shows the minimum percentage of surviving cells after treatment with 2 µM RMC-6236, RMC-6291, RMC-9805, or RMC-5127. This heatmap was sorted cell line origins and respective KRAS mutations. The BHxx cell lines represent NSCLC cell lines established from malignant pleural effusions. BH1522 is a control cell line of a rhabdomyosarcoma in the lung, which was also established via pleural effusion.

Article Snippet: The KRAS(ON) targeting compounds RMC-6236 (pan-KRAS), RMC-6291 (KRAS G12C), RMC-9805 (KRAS G12D) and RMC-5127 (KRAS G12V) were acquired from Targetmol ( T74698 , T75131 , T78212 , T204085, Wellesley Hills, MA, USA).

Techniques: Mutagenesis, Control

Journal: Translational Lung Cancer Research

Article Title: Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines

doi: 10.21037/tlcr-2026-0798

Figure Lengend Snippet: Relative phosphorylation of selected proteins in BH1467 cells after treatment with RMC-6236, RMC-6291, or RMC-9805. Data are presented as mean ± standard deviation. Asterisks indicate significant differences versus untreated control (* P<0.05, ** P<0.01, *** P<0.001).

Article Snippet: The KRAS(ON) targeting compounds RMC-6236 (pan-KRAS), RMC-6291 (KRAS G12C), RMC-9805 (KRAS G12D) and RMC-5127 (KRAS G12V) were acquired from Targetmol ( T74698 , T75131 , T78212 , T204085, Wellesley Hills, MA, USA).

Techniques: Phospho-proteomics, Standard Deviation, Control

Journal: Translational Lung Cancer Research

Article Title: Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines

doi: 10.21037/tlcr-2026-0798

Figure Lengend Snippet: MTT cytotoxicity assays of combinations of (A) RMC-6236 and RMC-9805 for KRAS G12D-mutated BH1194 and (B) RMC-6236 and RMC-6291 for KRAS G12C-mutated BH1658. Combinations start at 2 µM with 9 two-fold dilutions. Data are presented as mean ± standard deviation. Synergistic values are depicted by an asterisk (*).

Article Snippet: The KRAS(ON) targeting compounds RMC-6236 (pan-KRAS), RMC-6291 (KRAS G12C), RMC-9805 (KRAS G12D) and RMC-5127 (KRAS G12V) were acquired from Targetmol ( T74698 , T75131 , T78212 , T204085, Wellesley Hills, MA, USA).

Techniques: Standard Deviation

Journal: Translational Lung Cancer Research

Article Title: Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines

doi: 10.21037/tlcr-2026-0798

Figure Lengend Snippet: Relative phosphorylation of selected proteins in BH1194 cells after treatment with RMC-6236, RMC-6291, or RMC-9805. Data are presented as mean ± standard deviation. Asterisks indicate significant differences versus untreated control (*, P<0.05; **, P<0.01; ***, P<0.001).

Article Snippet: The KRAS(ON) targeting compounds RMC-6236 (pan-KRAS), RMC-6291 (KRAS G12C), RMC-9805 (KRAS G12D) and RMC-5127 (KRAS G12V) were acquired from Targetmol ( T74698 , T75131 , T78212 , T204085, Wellesley Hills, MA, USA).

Techniques: Phospho-proteomics, Standard Deviation, Control

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: Successful KRAS G12V degradation by TUS-007. KRAS G12V protein was incubated with TUS-007 at the indicated concentrations in the presence of 26S proteasome for 1 h (mean ± SEM; n = 3). *P < 0.05 vs. DMSO.

Article Snippet: Human KRAS (G12V), 2-186, and the corresponding His tag (R06-32BH) were obtained from SignalChem Lifesciences (Richmond, British Columbia, Canada).

Techniques: Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, TUS-007 induced chemical knockdown was independent of ubiquitination. GFP fused KRAS G12D and DsRed were transiently expressed in HeLa cells. The cells were treated with TUS-007 in the presence of a proteasome inhibitor (MLN), a ubiquitination inhibitor (NAEi) and a molecular chaperon inhibitor (HSP70 inhibitor: VER-155008) for 24 h. b & c, RAS-less MEFs expressing KRAS G12D, G12V or G12C were incubated with TUS-007 for 72 h. Representative blots of KRAS are shown (b). The relative amounts of KRAS mutants normalized to GAPDH are shown in the bar graph (mean ± SEM; n = 3–4). The cell viability was also measured (mean ± SEM; n = 3–4) (g). *P < 0.05 and ** P < 0.01 vs. DMSO. TUS-007 induced chemical knockdown did not cause non-specific cytotoxicity.

Article Snippet: Human KRAS (G12V), 2-186, and the corresponding His tag (R06-32BH) were obtained from SignalChem Lifesciences (Richmond, British Columbia, Canada).

Techniques: Knockdown, Ubiquitin Proteomics, Expressing, Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Immunoblotting analysis of KRAS G12V chemical knockdown in SW620-Luc cells treated with TUS-007 for 48 h at the indicated doses. The KRAS band intensity was normalized to that of GAPDH (mean ± SEM; n = 3). *P < 0.05 and **P < 0.01 vs. DMSO. b & c, The ratio of apoptotic cells were analyzed in colon cancer SW620-Luc cells ( b ) and HT29-Luc cells ( c ), a KRAS-independent, BRAF-dependent cell line (Ctx + Vem: 100 mg/ml cetuximab and 40 µM vemurafenib). Cells were treated with each compound for 48 h in terms of the number of annexin V-positive/PI-negative apoptotic cells detected by flow cytometry (mean ± SEM; n = 2–5). **P < 0.01, and ***P < 0.001 vs. DMSO. d, Relative activity of caspase 3/7 in SW620-Luc cells treated with TUS-007 or 5-fluorouracil (5-FU) for 48 h (mean ± SEM; n = 3). *P < 0.05 and **P < 0.01 vs. DMSO. e, Tumor volume in mice with SW620-Luc cells transplanted subcutaneously and treated with i.p. administration every 3 days (mean ± SEM; n = 6– 8). *P < 0.05 vs. vehicle alone. f, Tumors of SW620-Luc cells at day 21 from the same mice shown in e (n = 6–8).

Article Snippet: Human KRAS (G12V), 2-186, and the corresponding His tag (R06-32BH) were obtained from SignalChem Lifesciences (Richmond, British Columbia, Canada).

Techniques: Western Blot, Knockdown, Flow Cytometry, Activity Assay

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Comparison of SW620-Luc tumor weight at 21 days after injection (mean ± SEM; n = 6–8). *P < 0.05 vs. vehicle alone. b, Immunoblotting of KRAS G12V chemical knockdown in tumors from the same mice used in (mean ± SEM; n = 6). **P < 0.01 vs. vehicle alone. c, Body weight changes in mice with SW620-Luc cells transplanted subcutaneously (mean ± SEM; n = 6–8).

Article Snippet: Human KRAS (G12V), 2-186, and the corresponding His tag (R06-32BH) were obtained from SignalChem Lifesciences (Richmond, British Columbia, Canada).

Techniques: Comparison, Injection, Western Blot, Knockdown