Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) Calu1 and its KRAS-G12Ci–resistant cells (Calu1 R ) and MIA PaCa-2 and its resistant cells (MIA PaCa-2 R ) were treated with MRTX849 at the indicated concentrations. The percentage of viable cells is shown relative to untreated controls by CCK-8 assay ( n = 3 biologically independent samples; *** P < 0.001; two-way ANOVA; data are presented as means ± SD). ( B to D ) Cell clonogenic assay. The cells were treated with MRTX849 (5 μM) for 24 hours and observed for 7 to 14 days. The cell colonies were stained with 0.1% crystal violet for evaluation. Quantification was performed by ImageJ ( n = 3 biologically independent samples; ** P < 0.01; Student’s t test; data are presented as means ± SD). ( E ) Number of differentially expressed genes identified by iDEP96 using an FDR cutoff of 0.1 and a minimum fold change of 2 between the indicated groups. ( F ) KEGG pathway analysis of differentially down-regulated genes in MIA PaCa-2 cells treated with MRTX849 compared to the control. ( G ) KEGG pathway analysis of differentially down-regulated genes in MIA PaCa-2–resistant cells (MIA PaCa-2 R ) treated with MRTX849 compared to the control. ( H ) KEGG pathway analysis of differentially up-regulated genes in MIA PaCa-2–resistant cells (MIA PaCa-2 R ) compared to MIA PaCa-2 cells. ( I ) GSEA showing that the MAPK signaling pathway was statistically up-regulated between MIA PaCa-2 R and MIA PaCa-2 cells (nominal P value of <0.01). ( J ) Immunoblot analysis of the indicated proteins of cells treated with 1 μM MRTX849 at the indicated time. The blots shown are representative of three repeats. h, hours; KEGG, Kyoto Encyclopedia of Genes and Genomes; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: CCK-8 Assay, Clonogenic Assay, Staining, Control, Western Blot

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) GO enrichment of RNA-seq analysis. ( B ) GSEA was performed using a clinical tissue sample from an AMG510-resistant patient, available in a published database. A filtering step was applied to select gene sets associated with plasma membrane functions. ( C ) Heatmap showing the expression of ABC transporters in cells treated with MRTX849 (4 hours, 1 μM), based on RNA-seq. ( D and E ) Expression of ABCC1 and ABCG2 was examined by immunoblot analyses. Quantification was performed by the Image Lab software. ( F ) Partial volcano plot of 45 ABC transporters analyzed from genome-wide CRISPR-Cas9 screening. The x axis represents log 2 fold changes of each gene. The y axis represents the negative logarithm of the P value. ( G to I ) MIA PaCa-2 R or Calu1 R and their ABCC1 knockout (KO) cells were treated with MRTX849 followed by CCK-8 assays. ( J and K ) Small interfering RNA targeting ABCB1 , ABCC3 , ABCG1 , and ABCG2 was used for gene knockdown, followed by MRTX849 treatment and cell viability assays. The gene expressions in MIA PaCa-2 R cells were measured by quantitative PCR (Student’s t test). ( L ) Cells were treated with the ABCC1 inhibitor MK-571 and MRTX849 for 3 days, followed by CCK-8 assays. ( M ) Cells were transfected with an empty vector or ABCC1, treated with MRTX849 for 3 days, and subjected to CCK-8 assays. ( N ) Cells were treated with 1 μM MRTX849 for 6 hours or with 10 μM MK-571 for 24 hours, followed by 1 μM MRTX849 for 6 hours. Intracellular MRTX849 accumulation was quantified using LC-MS/MS (Student’s t test). The blots shown are representative of three independent experiments. Statistical analysis was performed using two-way ANOVA unless otherwise noted (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). Data are presented as means ± SD, with n = 3 biologically independent samples.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: RNA Sequencing, Clinical Proteomics, Membrane, Expressing, Western Blot, Software, Genome Wide, CRISPR, Knock-Out, CCK-8 Assay, Small Interfering RNA, Knockdown, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) Heatmap of the phosphorylation of kinases in MIA PaCa-2 and MIA PaCa-2 R by human phosphokinase array. ( B and C ) GO terms of gene enrichment in the MF (B) and CC (C) categories of the 246 differentially KRAS-G12Ci resistance-associated genes from an analysis of a CRISPR screening. CC, cellular component; MF, molecular function. ( D ) Heatmap of the expression pattern of 25 transcription factors in MIA PaCa-2 and MIA PaCa-2 R in response to MRTX849 by next-generation sequencing. ( E ) Immunoprecipitation (IP) assay with anti-JUN antibodies using chromatin fraction in MIA PaCa-2 and MIA PaCa-2 R cells. ( F ) The ABCC promoter region is divided into a, b, c, and d fractions. ChIP assays were performed using digested chromatin from Calu1/Calu1 R , MIA PaCa-2/MIA PaCa-2 R , and anti-JUN antibodies. The binding DNA was purified. The ABCC1 promoter fractions a to d were measured by quantitative real-time PCR. TSS, Transcription Start Site. ( G ) Immunoblots of the indicated protein levels in MIA PaCa-2, MIA PaCa-2 R , Calu1, and Calu1 R cells. ( H and I ) MIA PaCa-2 and Calu1 cells were transfected with pCDH-Flag-JUN and vector plasmid for 24 hours and then treated with MRTX849 for 3 days. The expression of the indicated proteins was shown by Western blot analysis. Cell viability was assessed. ( J ) Calu1 cells were infected with lentivirus expressing WT JUN followed by CRISPR-Cas9–mediated ABCC1 knockout. The expression of ABCC1 and FLAG-JUN in Calu1 was measured by Western blot. Calu1 cells with JUN overexpression in the absence or presence of ABCC1 expression were exposed to the indicated drugs at the indicated concentrations for 72 hours, and cell viability was assessed. The blots shown are representative of three repeats. Statistical analysis was performed using two-way ANOVA (** P < 0.01; **** P < 0.0001). Data are presented as means ± SD, with n = 3 biologically independent samples.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: Phospho-proteomics, CRISPR, Expressing, Next-Generation Sequencing, Immunoprecipitation, Binding Assay, Purification, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Infection, Knock-Out, Over Expression

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) Procedures of the US Food and Drug Administration (FDA)-approved drug screen. Pie chart of drugs with color codes corresponding to hit target type is shown. The calculating score is determined by taking log 2 of the ratio of cell viability between drug-treated MIA PaCa-2 cells and drug-treated MIA PaCa-2 R cells. ( B ) ZIP synergy score was calculated by SynergyFinder between dasatinib/palbociclib and MRTX849. ( C and D ) MIA PaCa-2/Calu1 and MIA PaCa-2 R /Calu1 R cells were treated with MRTX849 and dasatinib for 3 days. Cell viability was measured by CCK-8 assay. ( E ) Cell clonogenic assay. MIA PaCa-2 and MIA PaCa-2 R cells were treated with the MRTX849 (1 μM) and dasatinib for 24 hours. Calu1 and Calu1 R cells were treated with the MRTX849 (10 μM) and dasatinib for 24 hours. All cells were observed for 10 days, followed by evaluation using crystal violet staining. Quantification was performed by ImageJ. ( F ) Heatmap showing the expression of the cyclin proteins in MIA PaCa-2 and MIA PaCa-2 R in response to MRTX849 by next-generation sequencing. ( G ) Volcano plot of differential gene expression between MRTX849 and vehicle in MIA PaCa-2 (absolute fold change of ≥2; q value <0.05). CCND1 was substantially down-regulated. ( H ) GSEA showing that the JNK signaling pathway is up-regulated between MIA PaCa-2 R and MIA PaCa-2 cells (nominal P value of <0.0001). ( I ) Immunoblot analysis of the indicated proteins of cells treated with 1 μM dasatinib and 1 μM MRTX849 for 24 hours. The blots shown are representative of three independent experiments. Statistical analysis was performed using two-way ANOVA unless otherwise noted (* P < 0.05; ** P < 0.01; *** P < 0.001). Data are presented as means ± SD, with n = 3 biologically independent samples.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: CCK-8 Assay, Clonogenic Assay, Staining, Expressing, Next-Generation Sequencing, Gene Expression, Western Blot

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) MIA PaCa-2 and MIA PaCa-2 R cells were treated with dasatinib (1 μM, 24 hours) and MRTX849 (1 μM, 48 hours), followed by human phosphokinase array and quantification. ( B ) Cells were treated with 1 μM dasatinib and 1 μM MRTX849 for 24 hours, followed by RAS-GTP pull-down assays, Western blotting, and quantification. ( C ) Model of phosphorylated RAF1 bound to the SH2. (a) Structure of the computer-generated model: RAF1 (based on AlphaFold AF-P04049-F1) and SRC (based on the PDB). (b) SH2 (PDB: 1FMK) complexed with phosphorylated tyrosine pentapeptide Y341 (RAF1), interacting with Arg 178 and Thr 182 of SRC. (c) Human SH2 (PDB: 1HCS, dark gray) complexed with Y341 (yellow). ( D ) Immunoblot analysis of gene knockdown in Calu1 R and MIA PaCa-2 R cells. ( E ) Cells were treated with MRTX849 for 3 days and then assessed by CCK-8 assays. ( F ) Cells were transfected with an empty vector, SRC-WT, SRC-K298M (kinase dead), or SRC-Y530F (active form), treated with drugs for 3 days, and assessed by CCK-8 assays. ( G ) Cells were transfected with the indicated plasmids, treated with MRTX849 (1 μM) for 3 days and analyzed by immunoblotting. ( H ) HOP62 and H2030 cells were treated with MRTX849 (1 μM) for 1 and 7 days, followed by immunoblots. ( I and J ) ABCC1 WT and knockout cells of HOP62 and H2030 cells were transfected with an empty vector or SRC-WT-Flag and treated with MRTX849 for 3 days, followed by CCK-8 assays. The blots shown are representative of three independent experiments. Statistical analysis was performed using two-way ANOVA unless otherwise noted (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). Data are presented as means ± SD, with n = 3 biologically independent samples.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: Western Blot, Generated, Knockdown, CCK-8 Assay, Transfection, Plasmid Preparation, Knock-Out

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) Schematic diagram of the therapeutic strategy of the combination of MRTX849 (35 mg/kg, p.o., oral gavage) and dasatinib (35 mg/kg, oral gavage) in MIA PaCa-2/MIA PaCa-2 R xenograft models. sc, subcutaneously. ( B ) The tumor growth was monitored twice per week ( n = 8 to 10 tumor; ** P < 0.01; *** P < 0.001, two-way ANOVA). ( C and D ) Body weight was monitored after the administration of MRTX849 and dasatinib in nude mice for the indicated weeks after MIA PaCa-2/MIA PaCa-2 R inoculation. ( E ) Survival curve of each group [ n = 5 mice per group; ** P < 0.01 compared with the vehicle; ## P < 0.01 compared with MRTX849 treatment, log-rank (Mantel-Cox) test]. ( F ) Schematic diagram of the therapeutic strategy of the combination of MRTX849 (25 mg/kg, p.o., twice a week for 6 weeks) and dasatinib (35 mg/kg, p.o., twice a week for 6 weeks) in Calu1/Calu1 R xenograft models. ( G ) Tumor growth was monitored twice per week ( n = 8 to 10 tumors). ( H ) Body weight was monitored after the administration of MRTX849 and dasatinib in nude mice for the indicated weeks after Calu1/Calu1 R inoculation. ( I ) Survival curve of each group [ n = 5 mice per group; * P < 0.05, log-rank (Mantel-Cox) test]. ( J ) Staining of ABCC1, p-ERK1/2, and PCNA in MIA PaCa-2/MIA PaCa-2 R xenograft models on day 29. Scale bars, 50 μm. Quantification of immunohistochemistry analysis was performed by calculation using the immunoreactive score ( n = 3; * P < 0.05 or ** P < 0.01 compared with the vehicle; ## P < 0.01 compared with MRTX849 treatment).

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: Staining, Immunohistochemistry

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) Bright-field microscopy images of 3D sphere cultures of MIA PaCa-2 R and mKRC.1 with vehicles, MRTX849, dasatinib, DGY-06-116, and bosutinib as well as combinations, after 7 days of treatment. Scale bars, 500 μm. ( B ) Measurement of cell viability in 3D sphere cultures of MIA PaCa-2 R and mKRC.1 with different treatments ( n = 3 biologically independent samples; **** P < 0.0001, two-way ANOVA). ( C ) Schematic diagram of the therapeutic strategy of the combination of MRTX849 (35 mg/kg, oral gavage, p.o.) and bosutinib (75 mg/kg, oral gavage) in mKRC.1 syngeneic mouse models. The tumor growth was monitored twice per week ( n = 8 to 10 tumors; * P < 0.05; *** P < 0.001; **** P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). ( D ) Photo of tumors on day 27. ( E ) Tumor weight on day 27. n = 8 tumor per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, two-way ANOVA. ( F ) Body weight was monitored after the treatments for the indicated weeks. n = 5 mice per group; two-way ANOVA. ( G ) Organ weight in each group was measured on day 27 ( n = 5; * P < 0.05; *** P < 0.001; **** P < 0.0001, two-way ANOVA). ( H ) Representative images of H&E staining of the liver, lung, pancreas, and spleen from mice bearing mKRC.1 tumors, treated with either vehicle or the combination of MRTX849 and bosutinib. Scale bars, 50 μm.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: Microscopy, Staining

Journal: Science Advances

Article Title: SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition

doi: 10.1126/sciadv.adq4274

Figure Lengend Snippet: ( A ) Patient-derived organoids HCC4300-PDX-ORG were treated with MRTX849 for 1 or 7 days, and the expression of the indicated proteins was measured. The blots shown are representative of three repeats. ( B ) Patient-derived organoids HCC4300-PDX-ORG treated with vehicle, MRTX849, dasatinib, or DGY-06-116, as well as combinations, after 7 days of treatment. Cell viability of the organoids was measured by CCK-8. ( C ) Bright-field microscopy images of HCC4300-PDX-ORG treated with vehicle, MRTX849, or bosutinib, as well as combinations, after 7 days of treatment. Scale bars, 50 μm. ( D ) HCC4300-PDX-ORG was treated with vehicle, MRTX849, or bosutinib, as well as combinations, after 7 days of treatment. Cell viability of the organoids was measured by CCK-8. ( E ) HCC4285-PDX-ORG was treated with vehicle, MRTX849, bosutinib, DGY-06-116, or dasatinib, as well as combinations, after 7 days of treatment. Cell viability of the organoids was measured by CCK-8. ( F ) 3D synergy map displaying ZIP synergy score between MRTX849 and dasatinib, DGY-06-116, or bosutinib in HCC4300-PDX-ORG. ( G ) Proposed mechanisms and strategies to overcome MRTX849-induced multidrug resistance. In G12Ci-resistant cells, SRC binding with RAF1 bypasses the inhibition of KRAS-G12C by MRTX849, resulting in JUN activation and subsequent ABCC1 and CCND1 expression, and confers a multidrug-resistant phenotype and uncontrolled cell proliferation. Dasatinib, bosutinib, or DGY-06-116 suppresses the phosphorylation of SRC on Y416 and thus inhibits JUN activation and the expression of ABCC1 and CCND1, preventing or reversing G12Ci-induced multidrug resistance and inducing G 1 cell cycle arrest. Statistical analysis was performed using two-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). n = 3 biologically independent samples unless otherwise specified.

Article Snippet: MRTX849 (S8884), JNK inhibitor IX (S7508), JNK-IN-8 (S4901), bosutinib (S1014), PP1 (S7060), and PP2 (S7008) were purchased from Selleckchem.

Techniques: Derivative Assay, Expressing, CCK-8 Assay, Microscopy, Binding Assay, Inhibition, Activation Assay, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades

doi: 10.3390/ijms22042057

Figure Lengend Snippet: Effects of UVA on phosphorylation and protein levels of p38/ERK/JNK. HDFs were exposed to UVA at the indicated doses in culture and cell lysates prepared at the indicated times post-irradiation were subjected to western blotting as described in the Materials and Methods section. Representative immunoblots from three independent experiments are shown.

Article Snippet: The p38 inhibitor (SB239063) and the JNK inhibitor (JNK inhibitor II) were from Santa Cruz Biotechnology and Merck KGaA (Darmstadt, Germany), respectively.

Techniques: Phospho-proteomics, Irradiation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades

doi: 10.3390/ijms22042057

Figure Lengend Snippet: Effects of signaling inhibitors on mRNA expression levels of HAS2 and HYBID in HDFs. Effects of inhibitors of p38, JNK, NF-kB, and STAT3 on UVA- or sham-stimulated mRNA expression of HAS2 in HDFs. Signaling inhibitors were added at the indicated concentrations immediately after UVA irradiation at 10 J/cm 2 or sham irradiation. Cell lysates prepared at the indicated times post-irradiation were subjected to RT-qPCR analysis. ( a ) p38 inhibitor, 12 h, n = 4, ( b ) JNK inhibitor, 12 h, n = 4, ( c ) NF-kB inhibitor, 3 h, n = 4, ( d ) STAT3 inhibitor, 3 h, n = 4, *: p < 0.05, **: p < 0.01.

Article Snippet: The p38 inhibitor (SB239063) and the JNK inhibitor (JNK inhibitor II) were from Santa Cruz Biotechnology and Merck KGaA (Darmstadt, Germany), respectively.

Techniques: Expressing, Irradiation, Quantitative RT-PCR