Journal: bioRxiv

Article Title: Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

doi: 10.64898/2026.01.07.698128

Figure Lengend Snippet: A ) CRISPR screening strategy to identify regulators of KRAS protein stability. B ) Volcano plot of average KRAS stability scores (n=3). Significant hits for genes that either decrease (pink) or increase (purple) KRAS expression are indicated. Callouts represent genes identified as RAS interaction partners by mass spectrometry . C ) Venn diagram of overlapping genes from KRAS stability screen (≤-0.8 KRAS stability score and P≤0.05), RAS BioID2 proteomics (≥1.0 log2 enrichment vs. control, from ref: ), and genes essential in RAS-dependent MM cell lines (≤-1.0 CSS, from ref: ). D ) Western blot analysis of RAS, PPP1R2, and GAPDH 3 days after transduction with control shRNA (shCTRL) or PPP1R2-targeting shRNAs in XG7, RPMI 8226, and MM.1S MM lines, n=3. E ) PPP1R2-BioID2 enrichment over empty vector in RPMI 8226 cells. F ) Western blot analysis of RAS, PPP1R2, PP1C, and GAPDH 3 days after transduction with shCTRL, shPPP1R2.1, and/or ectopic expression of DN PP1C, n=3. G ) Comparison of protein expression levels between KRAS (x-axis) and average PP1C (PPP1CA, PPP1CB, PPP1CC, and PPP1CC;PPP1CB) in 115 MM patient tumors. Display line is simple linear regression; R 2 =0.1593, P<0.0001. I ) Model of PPP1R2 and PP1C regulation of KRAS protein expression. Under a normal state, PPP1R2 inhibits PP1C activity. Following PPP1R2 disruption, PP1C activity reduces KRAS levels. In contrast, PP1C disruption increases KRAS expression.

Article Snippet: Briefly, 150 ng of KRAS gene fragment was combined with 1 μL of SnaBI-linearized BioID-2 vector and 4 μL of 2× NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and incubated for 1 h at 50 °C.

Techniques: CRISPR, Expressing, Mass Spectrometry, Control, Western Blot, Transduction, shRNA, Plasmid Preparation, Comparison, Activity Assay, Disruption

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: MRTX1133 treatment of PDAC KRAS G12D–mutant cells induces M1C expression in an NF-κB autoinductive pathway. A, AsPC-1 cells treated with 50-nmol/L MRTX1133 for the indicated days were analyzed for M1C transcripts by qRT-PCR using primers listed in Supplementary Table S1. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). B, Lysates from AsPC-1 cells treated with the indicated MRTX1133 concentrations for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. C, PANC-1 cells treated with 1-μmol/L MRTX1133 for the indicated days were analyzed for M1C transcripts by qRT-PCR. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). D, Lysates from PANC-1 cells treated with 1-μmol/L MRTX1133 for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. E, AsPC-1 cells treated with 50-nmol/L MRTX1133 for the indicated days were analyzed for NF-κB p65/RELA transcripts by qRT-PCR. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). F, Lysates from AsPC-1 cells treated with the indicated MRTX1133 concentrations for 6 days were immunoblotted with antibodies against the indicated proteins. G, PANC-1 cells treated with 1-μmol/L MRTX1133 for the indicated days were analyzed for NF-κB p65/RELA transcripts by qRT-PCR. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). H, Lysates from PANC-1 cells treated with 1-μmol/L MRTX1133 for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. I and J, Lysates from AsPC-1 ( I ) and PANC-1 ( J ) cells expressing a control scrambled shRNA (CshRNA) or NF-κBshRNA and treated with 50-nmol/L and 1-μmol/L MRTX1133, respectively, for 2 days were immunoblotted with antibodies against the indicated proteins. K, Lysates from AsPC-1 cells treated with MRTX1133 at the indicated concentrations for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. L, Lysates from AsPC-1 cells expressing the indicated vectors and treated with doxycycline (DOX) for 7 days were immunoblotted with antibodies against the indicated proteins.

Article Snippet: HPAF-II KRAS G12D mutant SC cells (ATCC) were cultured in Eagle Minimum Essential Medium (ATCC) supplemented with 10% FBS.

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Control, shRNA

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: Resistance of PDAC KRAS G12D–mutant cells to MRTX1133 is M1C-dependent. A, AsPC-1/control scrambled shRNA (CshRNA) and AsPC-1/MUC1shRNA#2 cells treated with 50-nmol/L MRTX1133 for 6 days were analyzed for colonyformation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). B, AsPC-1 cells were treated with the indicated concentrations of GO-203 and MRTX1133 for 3 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). Shown are the combination indices determined using Bliss scores. C, PANC-1/CshRNA and PANC-1/MUC1shRNA#2 cells treated with 1-μmol/L MRTX1133 for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). D, PANC-1 was treated with the indicated concentrations of GO-203 and MRTX1133 for 3 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). Shown are the combination indices determined using Bliss scores. E and F, Gene set enrichment analysis of RNA-seq data from AsPC-1/MR cells with M1C silencing using the HALLMARK KRAS SIGNALING UP ( E ) and DN ( F ) gene signatures. G and H, Lysates from (i) AsPC-1/MR/CshRNA and AsPC-1/MR/MUC1shRNA#2 cells ( G ) and (ii) AsPC-1/MR cells treated with the indicated concentrations of GO-203 for 4 days ( H ) were immunoblotted with antibodies against the indicated proteins. I and J, Lysates from (i) PANC-1/MR/CshRNA and PANC-1/MR/MUC1shRNA#2 cells ( I ) and (ii) PANC-1/MR cells treated with the indicated concentrations of GO-203 for 4 days ( J ) were immunoblotted with antibodies against the indicated proteins.

Article Snippet: HPAF-II KRAS G12D mutant SC cells (ATCC) were cultured in Eagle Minimum Essential Medium (ATCC) supplemented with 10% FBS.

Techniques: Mutagenesis, Control, shRNA, Staining, RNA Sequencing

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: PDAC KRAS G12D–mutant cells are sensitive to killing with an M1C ADC. A, AsPC-1 and PANC-1 cells treated with the indicated concentrations of M1C ADC for 6 days were analyzed for cell viability by alamarBlue staining. The results (mean ± SD of three determinations) are expressed as relative cell number (percentage of control) compared with that for untreated cells. Shown are the M1C ADC IC 50 values. B, AsPC-1 cells left untreated or treated with 50-nmol/L MRTX1133 for 2 days were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot shows the mean fluorescence intensity, presented as the mean ± SD of three independent determinations (right). C, AsPC-1 cells treated with 50-nmol/L MRTX1133 and 20-nmol/L M1C ADC for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). D, PANC-1 cells left untreated or treated with 1-μmol/L MRTX1133 for 6 days were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot shows the MFI, presented as the mean ± SD of three independent determinations (right). E, PANC-1 cells treated with 1-μmol/L MRTX1133 and 20-nmol/L M1C ADC for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). F, AsPC-1/MR and AsPC-1 cells were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot depicts MFI fold change (anti-M1C/IgG). The results (mean ± SD of three determinations) are expressed as relative levels compared with those obtained for AsPC-1 cells (assigned a value of 1; right). G, AsPC-1/MR and AsPC-1 cells treated with the indicated concentrations of M1C ADC for 6 days were analyzed for cell viability by alamarBlue staining. The results (mean ± SD of three determinations) are expressed as relative cell number (percentage of control) compared with that for untreated cells. The M1C ADC IC 50 values are shown. H, AsPC-1/MR were treated with the indicated concentrations of M1C-ADC and MRTX1133 for 6 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). The combination indices determined using Bliss scores are shown. I, PANC-1/MR and PANC-1 cells were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot depicts MFI fold change (anti-M1C/IgG). The results (mean ± SD of three determinations) are expressed as relative levels compared with those obtained for PANC-1 cells (assigned a value of 1; right). J, PANC-1/MR and PANC-1 cells treated with the indicated concentrations of M1C ADC for 6 days were analyzed for cell viability by alamarBlue staining. The results (mean ± SD of three determinations) are expressed as relative cell number (percentage of control) compared with that for untreated cells. The M1C ADC IC 50 values are shown. K, PANC-1/MR cells were treated with the indicated concentrations of M1C-ADC and MRTX1133 for 6 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). The combination indices determined using Bliss scores are shown.

Article Snippet: HPAF-II KRAS G12D mutant SC cells (ATCC) were cultured in Eagle Minimum Essential Medium (ATCC) supplemented with 10% FBS.

Techniques: Mutagenesis, Staining, Control, Flow Cytometry, Expressing, Fluorescence

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: Targeting MRTX1133-resistant PDAC G12D-mutant cells with the M1C ADC inhibits self-renewal capacity and tumorigenicity. A and B, AsPC-1 and PANC-1 ( A ) and AsPC-1/MR and PANC-1/MR ( B ) cells left untreated or treated with vehicle or 50-nmol/L M1C ADC for 7 days were analyzed for tumorsphere formation. Shown are representative photomicrographs of tumorspheres (left). The results (mean ± SD of three determinations) are expressed as relative tumorsphere formation compared with that for vehicle-treated cells (assigned a value of 1; right). C and D, Nude mice were injected subcutaneously with 5 × 10 6 AsPC-1/MR ( C ) or PANC-1/MR ( D ) cells. Mice bearing established AsPC-1/MR tumors were randomized into two groups when the mean tumor volume reached 100 mm 3 and treated with vehicle ( n = 6) or 5 mg/kg M1C ADC weekly × 3 every 28 days for two cycles ( n = 6). Mice bearing established PANC-1/MR tumors were randomized into two groups when the mean tumor volume reached 150 mm 3 and treated with vehicle ( n = 5) or 5-mg/kg M1C ADC weekly × 3 and then 7.5 mg/kg weekly × 3 ( n = 5). Tumor volumes are expressed as the mean ± SEM. E, Lysates from ST-00013312-T cells treated with the indicated MRTX1133 concentrations for 2 days were immunoblotted with antibodies against the indicated proteins. F, ST-00013312-T cells treated with vehicle, 5-μmol/L GO-203, and 1-μmol/L MRTX1133 for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony numbers compared with those for untreated cells (assigned a value of 1). G, ST-00013312-T cells were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression. H, ST-00013312-T cells treated with vehicle, 50-nmol/L M1C ADC, and 1-μmol/L MRTX1133 for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). I, ST-00013312-T cells treated with vehicle or 50-nmol/L M1C ADC for 7 days were analyzed for tumorsphere formation. Shown are representative photomicrographs of tumorspheres (left). The results (mean ± SD of three determinations) are expressed as relative tumorsphere formation compared with that for vehicle-treated cells (assigned a value of 1; right). J, ST-00013312-T cells were implanted subcutaneously in NOD/SCID gamma mice. Mice randomized into two groups when the mean tumor volume reached 100 mm 3 were treated with vehicle ( n = 7) or 7.5-mg/kg M1C ADC weekly × 3 every 28 days for two cycles ( n = 7). Tumor volumes are expressed as the mean ± SEM.

Article Snippet: HPAF-II KRAS G12D mutant SC cells (ATCC) were cultured in Eagle Minimum Essential Medium (ATCC) supplemented with 10% FBS.

Techniques: Mutagenesis, Injection, Flow Cytometry, Control, Expressing

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: M1C is a potential target for the treatment of PDAC tumors. A and B, Analysis of The Cancer Genome Atlas–PDAC/PAAD dataset for associations of MUC1-high and MUC1-low expression with relapse-free survival (RFS; A ) and overall survival (OS; B ) using the Kaplan–Meier plotter. C, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) representation of total cells analyzed in the aggregated PDAC reference atlas. Cells are colored by annotated cell type (above) and by MUC1 expression (below) within normal donor, adjacent normal tissue, primary tumor, and metastatic lesion–derived samples. D, Ductal compartment pseudobulk expression of MUC1 across samples. Significance determined by the DESeq2 method. E, Representative examples of IHC staining for M1C expression in normal pancreas and PDAC tissues. Bar represents 250 microns. F, IHC staining for M1C of primary PDAC tissues. Bar represents 250 microns. G, The indicated PDO cells were analyzed for (i) MUC1 expression by RNA-seq , (ii) sensitivity to MRTX1133 and M1C ADC for 6 days as determined by IC 50 values, and (iii) effects of the MRTX1133 + M1C ADC combination as determined by Bliss scores. H, PF405 cells were treated with the indicated concentrations of M1C-ADC and MRTX1133 for 6 days. Cell viability was assessed using the CellTiter-Glo 3D Cell Viability Assay (mean of five determinations). The combination indices determined using Bliss scores are determined. I, The present results support a model in which MRTX1133 induces M1C expression in PDAC KRAS G12D–mutant cells by activating an M1C/NF-κB autoinductive pathway. In turn, M1C induces STAT1 and the IFN type I pathway, conferring MRTX1133 resistance. Targeting M1C genetically and pharmacologically thus reverses the MRTX1133-resistant phenotype. In support of these results, we found that treatment with the M1C ADC is effective against MRTX1133-resistant PDAC cell lines, patient-derived organoids, and patient-derived xenograft models. Patients with refractory PDAC KRAS G12D–mutant tumors have limited therapeutic options. The present findings identify M1C as a potential target for ADC treatment of patients with PDAC who are refractory to MRTX1133 treatment. TNM, tumor–node–metastasis. (Adapted from an image created with BioRender.com . Ozawa, H. [2025] https://app.biorender.com/illustrations/69132633069d50fee858e910 ).

Article Snippet: HPAF-II KRAS G12D mutant SC cells (ATCC) were cultured in Eagle Minimum Essential Medium (ATCC) supplemented with 10% FBS.

Techniques: Expressing, Derivative Assay, Immunohistochemistry, RNA Sequencing, Viability Assay, Mutagenesis

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: MRTX1133 treatment of PDAC KRAS G12D–mutant cells induces M1C expression in an NF-κB autoinductive pathway. A, AsPC-1 cells treated with 50-nmol/L MRTX1133 for the indicated days were analyzed for M1C transcripts by qRT-PCR using primers listed in Supplementary Table S1. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). B, Lysates from AsPC-1 cells treated with the indicated MRTX1133 concentrations for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. C, PANC-1 cells treated with 1-μmol/L MRTX1133 for the indicated days were analyzed for M1C transcripts by qRT-PCR. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). D, Lysates from PANC-1 cells treated with 1-μmol/L MRTX1133 for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. E, AsPC-1 cells treated with 50-nmol/L MRTX1133 for the indicated days were analyzed for NF-κB p65/RELA transcripts by qRT-PCR. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). F, Lysates from AsPC-1 cells treated with the indicated MRTX1133 concentrations for 6 days were immunoblotted with antibodies against the indicated proteins. G, PANC-1 cells treated with 1-μmol/L MRTX1133 for the indicated days were analyzed for NF-κB p65/RELA transcripts by qRT-PCR. The results (mean ± SD of four determinations) are expressed as relative levels compared with those obtained for control cells (assigned a value of 1). H, Lysates from PANC-1 cells treated with 1-μmol/L MRTX1133 for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. I and J, Lysates from AsPC-1 ( I ) and PANC-1 ( J ) cells expressing a control scrambled shRNA (CshRNA) or NF-κBshRNA and treated with 50-nmol/L and 1-μmol/L MRTX1133, respectively, for 2 days were immunoblotted with antibodies against the indicated proteins. K, Lysates from AsPC-1 cells treated with MRTX1133 at the indicated concentrations for 2 and 6 days were immunoblotted with antibodies against the indicated proteins. L, Lysates from AsPC-1 cells expressing the indicated vectors and treated with doxycycline (DOX) for 7 days were immunoblotted with antibodies against the indicated proteins.

Article Snippet: PANC-1 KRAS G12D mutant, TP53 R273H mutant, SB (ATCC) cells were cultured in DMEM with 10% FBS.

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Control, shRNA

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: Resistance of PDAC KRAS G12D–mutant cells to MRTX1133 is M1C-dependent. A, AsPC-1/control scrambled shRNA (CshRNA) and AsPC-1/MUC1shRNA#2 cells treated with 50-nmol/L MRTX1133 for 6 days were analyzed for colonyformation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). B, AsPC-1 cells were treated with the indicated concentrations of GO-203 and MRTX1133 for 3 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). Shown are the combination indices determined using Bliss scores. C, PANC-1/CshRNA and PANC-1/MUC1shRNA#2 cells treated with 1-μmol/L MRTX1133 for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). D, PANC-1 was treated with the indicated concentrations of GO-203 and MRTX1133 for 3 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). Shown are the combination indices determined using Bliss scores. E and F, Gene set enrichment analysis of RNA-seq data from AsPC-1/MR cells with M1C silencing using the HALLMARK KRAS SIGNALING UP ( E ) and DN ( F ) gene signatures. G and H, Lysates from (i) AsPC-1/MR/CshRNA and AsPC-1/MR/MUC1shRNA#2 cells ( G ) and (ii) AsPC-1/MR cells treated with the indicated concentrations of GO-203 for 4 days ( H ) were immunoblotted with antibodies against the indicated proteins. I and J, Lysates from (i) PANC-1/MR/CshRNA and PANC-1/MR/MUC1shRNA#2 cells ( I ) and (ii) PANC-1/MR cells treated with the indicated concentrations of GO-203 for 4 days ( J ) were immunoblotted with antibodies against the indicated proteins.

Article Snippet: PANC-1 KRAS G12D mutant, TP53 R273H mutant, SB (ATCC) cells were cultured in DMEM with 10% FBS.

Techniques: Mutagenesis, Control, shRNA, Staining, RNA Sequencing

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: PDAC KRAS G12D–mutant cells are sensitive to killing with an M1C ADC. A, AsPC-1 and PANC-1 cells treated with the indicated concentrations of M1C ADC for 6 days were analyzed for cell viability by alamarBlue staining. The results (mean ± SD of three determinations) are expressed as relative cell number (percentage of control) compared with that for untreated cells. Shown are the M1C ADC IC 50 values. B, AsPC-1 cells left untreated or treated with 50-nmol/L MRTX1133 for 2 days were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot shows the mean fluorescence intensity, presented as the mean ± SD of three independent determinations (right). C, AsPC-1 cells treated with 50-nmol/L MRTX1133 and 20-nmol/L M1C ADC for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). D, PANC-1 cells left untreated or treated with 1-μmol/L MRTX1133 for 6 days were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot shows the MFI, presented as the mean ± SD of three independent determinations (right). E, PANC-1 cells treated with 1-μmol/L MRTX1133 and 20-nmol/L M1C ADC for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). F, AsPC-1/MR and AsPC-1 cells were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot depicts MFI fold change (anti-M1C/IgG). The results (mean ± SD of three determinations) are expressed as relative levels compared with those obtained for AsPC-1 cells (assigned a value of 1; right). G, AsPC-1/MR and AsPC-1 cells treated with the indicated concentrations of M1C ADC for 6 days were analyzed for cell viability by alamarBlue staining. The results (mean ± SD of three determinations) are expressed as relative cell number (percentage of control) compared with that for untreated cells. The M1C ADC IC 50 values are shown. H, AsPC-1/MR were treated with the indicated concentrations of M1C-ADC and MRTX1133 for 6 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). The combination indices determined using Bliss scores are shown. I, PANC-1/MR and PANC-1 cells were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression (left). The bar plot depicts MFI fold change (anti-M1C/IgG). The results (mean ± SD of three determinations) are expressed as relative levels compared with those obtained for PANC-1 cells (assigned a value of 1; right). J, PANC-1/MR and PANC-1 cells treated with the indicated concentrations of M1C ADC for 6 days were analyzed for cell viability by alamarBlue staining. The results (mean ± SD of three determinations) are expressed as relative cell number (percentage of control) compared with that for untreated cells. The M1C ADC IC 50 values are shown. K, PANC-1/MR cells were treated with the indicated concentrations of M1C-ADC and MRTX1133 for 6 days. Cell viability was assessed using alamarBlue staining (mean of three determinations). The combination indices determined using Bliss scores are shown.

Article Snippet: PANC-1 KRAS G12D mutant, TP53 R273H mutant, SB (ATCC) cells were cultured in DMEM with 10% FBS.

Techniques: Mutagenesis, Staining, Control, Flow Cytometry, Expressing, Fluorescence

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: Targeting MRTX1133-resistant PDAC G12D-mutant cells with the M1C ADC inhibits self-renewal capacity and tumorigenicity. A and B, AsPC-1 and PANC-1 ( A ) and AsPC-1/MR and PANC-1/MR ( B ) cells left untreated or treated with vehicle or 50-nmol/L M1C ADC for 7 days were analyzed for tumorsphere formation. Shown are representative photomicrographs of tumorspheres (left). The results (mean ± SD of three determinations) are expressed as relative tumorsphere formation compared with that for vehicle-treated cells (assigned a value of 1; right). C and D, Nude mice were injected subcutaneously with 5 × 10 6 AsPC-1/MR ( C ) or PANC-1/MR ( D ) cells. Mice bearing established AsPC-1/MR tumors were randomized into two groups when the mean tumor volume reached 100 mm 3 and treated with vehicle ( n = 6) or 5 mg/kg M1C ADC weekly × 3 every 28 days for two cycles ( n = 6). Mice bearing established PANC-1/MR tumors were randomized into two groups when the mean tumor volume reached 150 mm 3 and treated with vehicle ( n = 5) or 5-mg/kg M1C ADC weekly × 3 and then 7.5 mg/kg weekly × 3 ( n = 5). Tumor volumes are expressed as the mean ± SEM. E, Lysates from ST-00013312-T cells treated with the indicated MRTX1133 concentrations for 2 days were immunoblotted with antibodies against the indicated proteins. F, ST-00013312-T cells treated with vehicle, 5-μmol/L GO-203, and 1-μmol/L MRTX1133 for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony numbers compared with those for untreated cells (assigned a value of 1). G, ST-00013312-T cells were analyzed by flow cytometry with a control IgG and anti-M1C. Shown are histograms for M1C expression. H, ST-00013312-T cells treated with vehicle, 50-nmol/L M1C ADC, and 1-μmol/L MRTX1133 for 6 days were analyzed for colony formation. The results (mean ± SD of three determinations) are expressed as relative colony number compared with that for untreated cells (assigned a value of 1). I, ST-00013312-T cells treated with vehicle or 50-nmol/L M1C ADC for 7 days were analyzed for tumorsphere formation. Shown are representative photomicrographs of tumorspheres (left). The results (mean ± SD of three determinations) are expressed as relative tumorsphere formation compared with that for vehicle-treated cells (assigned a value of 1; right). J, ST-00013312-T cells were implanted subcutaneously in NOD/SCID gamma mice. Mice randomized into two groups when the mean tumor volume reached 100 mm 3 were treated with vehicle ( n = 7) or 7.5-mg/kg M1C ADC weekly × 3 every 28 days for two cycles ( n = 7). Tumor volumes are expressed as the mean ± SEM.

Article Snippet: PANC-1 KRAS G12D mutant, TP53 R273H mutant, SB (ATCC) cells were cultured in DMEM with 10% FBS.

Techniques: Mutagenesis, Injection, Flow Cytometry, Control, Expressing

Journal: Clinical Cancer Research

Article Title: Targeting KRAS Inhibitor–Resistant Pancreatic Cancer with an MUC1-C Antibody–Drug Conjugate

doi: 10.1158/1078-0432.CCR-25-2333

Figure Lengend Snippet: M1C is a potential target for the treatment of PDAC tumors. A and B, Analysis of The Cancer Genome Atlas–PDAC/PAAD dataset for associations of MUC1-high and MUC1-low expression with relapse-free survival (RFS; A ) and overall survival (OS; B ) using the Kaplan–Meier plotter. C, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) representation of total cells analyzed in the aggregated PDAC reference atlas. Cells are colored by annotated cell type (above) and by MUC1 expression (below) within normal donor, adjacent normal tissue, primary tumor, and metastatic lesion–derived samples. D, Ductal compartment pseudobulk expression of MUC1 across samples. Significance determined by the DESeq2 method. E, Representative examples of IHC staining for M1C expression in normal pancreas and PDAC tissues. Bar represents 250 microns. F, IHC staining for M1C of primary PDAC tissues. Bar represents 250 microns. G, The indicated PDO cells were analyzed for (i) MUC1 expression by RNA-seq , (ii) sensitivity to MRTX1133 and M1C ADC for 6 days as determined by IC 50 values, and (iii) effects of the MRTX1133 + M1C ADC combination as determined by Bliss scores. H, PF405 cells were treated with the indicated concentrations of M1C-ADC and MRTX1133 for 6 days. Cell viability was assessed using the CellTiter-Glo 3D Cell Viability Assay (mean of five determinations). The combination indices determined using Bliss scores are determined. I, The present results support a model in which MRTX1133 induces M1C expression in PDAC KRAS G12D–mutant cells by activating an M1C/NF-κB autoinductive pathway. In turn, M1C induces STAT1 and the IFN type I pathway, conferring MRTX1133 resistance. Targeting M1C genetically and pharmacologically thus reverses the MRTX1133-resistant phenotype. In support of these results, we found that treatment with the M1C ADC is effective against MRTX1133-resistant PDAC cell lines, patient-derived organoids, and patient-derived xenograft models. Patients with refractory PDAC KRAS G12D–mutant tumors have limited therapeutic options. The present findings identify M1C as a potential target for ADC treatment of patients with PDAC who are refractory to MRTX1133 treatment. TNM, tumor–node–metastasis. (Adapted from an image created with BioRender.com . Ozawa, H. [2025] https://app.biorender.com/illustrations/69132633069d50fee858e910 ).

Article Snippet: PANC-1 KRAS G12D mutant, TP53 R273H mutant, SB (ATCC) cells were cultured in DMEM with 10% FBS.

Techniques: Expressing, Derivative Assay, Immunohistochemistry, RNA Sequencing, Viability Assay, Mutagenesis