Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: MUC1-C-dependent upregulation of inflammatory pathways in osimertinib-resistant H1975-OR vs parental H1975 cells. a H1975 cells were treated with increasing osimertinib concentrations for 12 weeks to select for osimertinib-resistant H1975-OR cells. b , c RNA-seq was performed on biological triplicates of H1975-OR/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days. GSEA of the datasets using the indicated HALLMARK gene signatures. d Venn diagram of 2838 shared genes upregulated in H1975-OR vs H1975 cells and downregulated in H1975-OR cells with MUC1-C silencing. Analysis of the 2838 shared genes identifies interferon signaling and cell junction organization pathways. e Heatmap of selected genes upregulated in H1975-OR vs H1975 cells and downregulated by silencing MUC1-C in H1975-OR cells. f H1975-OR cells grown in the absence of osimertinib for the indicated weeks were analyzed for osimertinib sensitivity by Alamar Blue staining (Supplementary Fig. ). The results (mean±SD of 4 determinations) are expressed as the osimertinib IC50 μM values. g Chromatin from H1975, H1975-OR, and H1975-RT cells was immunoblotted with antibodies against the indicated proteins. h . PC9 cells transfected with an empty control vector (PC9/vector) or one expressing MUC1-C (PC9/MUC1-C) were analyzed for MUC1-C mRNA levels by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for untreated cells (assigned a value of 1). i PC9/vector and PC9/MUC1-C cells were treated with the indicated concentrations of osimertinib for 3 days and analyzed for cell viability by Alamar Blue staining. The results (mean±SD of 4 determinations) are expressed as relative cell number (% control) compared to that for PC9/vector cells

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: RNA Sequencing, Staining, Transfection, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: H1975-RT cells are dependent on MUC1-C for activating the memory response of osimertinib resistance. a , b RNA-seq was performed on biologic triplicates of H1975-OR and H1975-RT cells. GSEA of the H1975-OR vs H1975-RT cell transcriptomes using the indicated HALLMARK gene signatures. c Heatmaps of genes downregulated in H1975-RT vs H1975-OR cells. d H1975-OR and H1975-RT cells were analyzed for the indicated transcripts by qRT-PCR using primers listed in Supplemental Table . The results (mean±SD of 4 determinations) are expressed as relative levels compared to those obtained for H1975-OR cells (assigned a value of 1). e H1975-RT cells exposed to osimertinib for 1-3 weeks were analyzed for osimertinib sensitivity by Alamar Blue staining (Supplementary Fig. ). The results (mean±SD of 4 determinations) are expressed as the osimertinib IC50 μM values. f Chromatin from H1975-RT and H1975-RT-OR cells was immunoblotted with antibodies against the indicated proteins. g H1975-RT and H1975-RT-OR cells were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for H1975-RT cells (assigned a value of 1). h H1975-RT/CsgRNA and H1975-RT/MUC1sgRNA cells exposed to osimertinib for 2 weeks were analyzed for osimertinib sensitivity by Alamar Blue staining (Supplementary Fig. ). The results (mean±SD of four determinations) are expressed as the osimertinib IC50 μM values

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: RNA Sequencing, Quantitative RT-PCR, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: MUC1 is induced by a STAT1-mediated pathway in the memory response to osimertinib treatment. a H1975 and H1975-RT cells treated with 1 μM osimertinib for 1-3 days were analyzed for MUC1-C (left) and STAT1 (right) mRNA levels by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for untreated H1975 cells (assigned a value of 1). b Lysates from H1975 and H1975-RT cells treated with 1 μM osimertinib for 2 days were immunoblotted with antibodies against the indicated proteins. c Schematic of the MUC1 gene highlighting a proximal enhancer-like signature-1 (pELS-1) with a STAT1 binding motif. Soluble chromatin from H1975 and H1975-RT cells treated with vehicle or 1 μM osimertinib for 2 days was precipitated with anti-MUC1-C, anti-STAT1 or a control IgG. The DNA samples were amplified by qPCR with primer set-1 encompassing +386 to -746 bp to the TSS (Supplementary Table ). The results (mean ± SD of 3 determinations) are expressed as percent input. d , e H1975-RT/tet-MUC1shRNA cells were treated with vehicle or DOX for 7 days. Chromatin was immunoblotted with antibodies against the indicated proteins ( d ) Soluble chromatin was precipitated with anti-MUC1-C, anti-STAT1 or a control IgG ( e ). The DNA samples were amplified by qPCR for pELS-1. The results (mean ± SD of 3 determinations) are expressed as percent input. f , g H1975-RT cells were treated with vehicle or 3 μM GO-203 for 3 days. Chromatin was immunoblotted with antibodies against the indicated proteins ( f ). Soluble chromatin was precipitated with anti-MUC1-C, anti-STAT1 or IgG ( g ). The DNA samples were amplified by qPCR for pELS-1. The results (mean ± SD of 3 determinations) are expressed as percent input. h H1975-RT/CshRNA and H1975-RT/STAT1shRNA cells treated with 1 μM osimertinib for 2 days were analyzed for STAT1 and MUC1-C mRNA levels by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for H1975-RT/CshRNA cells (assigned a value of 1). i H1975-RT/CshRNA and H1975-RT/STAT1shRNA cells exposed to 1 μM osimertinib for 2 weeks were analyzed for osimertinib sensitivity by Alamar Blue staining (Supplementary Fig. ). The results (mean±SD of 4 determinations) are expressed as osimertinib IC50 values

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: Quantitative RT-PCR, Binding Assay, Control, Amplification, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: Induction of MUC1-C in the inflammatory memory response to osimertinib treatment is JUN/AP-1 dependent. a Schematic of the MUC1 gene highlighting a FOS:JUN binding motif in pELS-2. b Soluble chromatin from H1975-RT cells was precipitated with anti-MUC1-C, anti-JUN, anti-FOS or a control IgG. The DNA samples were amplified by qPCR with primer set-2 encompassing −1010 to −2098 bp to the TSS (Supplemental Table ). The results (mean ± SD of 3 determinations) are expressed as percent input. c Soluble chromatin from H1975-RT/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days and then with 1 μM osimertinib for 2 days was precipitated with antibodies against the indicated proteins and a control IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. d Soluble chromatin from H1975-RT cells treated with 1 μM osimertinib and vehicle or 3 μM GO-203 for 2 days was precipitated with antibodies against the indicated proteins and a control IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. e Soluble chromatin from H1975-RT/tet-AFOS cells treated with vehicle or DOX for 7 days and then with 1 μM osimertinib for 2 days was precipitated with antibodies against the indicated proteins and a control IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. f H1975-RT/tet-AFOS cells treated with vehicle or DOX for 7 days and then exposed to 1 μM osimertinib for 2 weeks were analyzed for osimertinib sensitivity by Alamar Blue staining (Supplementary Fig. ). The results (mean±SD of 4 determinations) are expressed as osimertinib IC50 μM values. g Six-week old nude mice were injected subcutaneously in the flank with 5 × 10 6 H1975-RT cells. Mice were pair-matched into three groups of 5 mice each when tumors reached 100–150 mm 3 that were treated with vehicle control (red), osimertinib (orange), and GO-203 plus osimertinib (blue) for the indicated days. Tumor volumes are expressed as the mean±SEM for five mice

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: Binding Assay, Control, Amplification, Staining, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: MUC1-C expression induced in the inflammatory memory response to osimertinib treatment is PBRM1/PBAF dependent. a ATAC-seq was performed in (i) triplicates on H1975 and H1975-RT cells, and (ii) duplicates on H1975-RT/CsgRNA and H1975-RT/MUC1sgRNA cells. Representative genome browser snapshots are shown for the MUC1 and STAT1 genes. Highlighted are the MUC1 pELS-1 (-541 to -551 bp upstream to the TSS) and pELS-2 (-1904 to -1909 bp upstream to the TSS) regions. Also highlighted is the STAT1 promoter region that is activated by an auto-inductive STAT1 binding motif. b H1975 and H1975-RT cells treated with 1 μM osimertinib for 1-3 days were analyzed for BRG1 (left) and PBRM1 (right) mRNA levels by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for untreated H1975 cells (assigned a value of 1). c Soluble chromatin from H1975 and H1975-RT cells treated with vehicle or 1 μM osimertinib for 2 days was precipitated with anti-PBRM1 or IgG. The DNA samples were amplified by qPCR for pELS-1 and pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. d Soluble chromatin from H1975 and H1975-RT cells was precipitated with antibodies against the indicated proteins and a control IgG. The DNA samples were amplified by qPCR for pELS-1 and pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. e Soluble chromatin from H1975-RT/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with antibodies against the indicated proteins and a control IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. f H1975-RT/tet-MUC1shRNA cells were treated with vehicle or DOX for 7 days. Soluble chromatin was precipitated with anti-PBRM1 or IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. g H1975-RT/tet-AFOS cells were treated with vehicle or DOX for 7 days. Chromatin was precipitated with anti-PBRM1 or IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. h Lysates from H1975-RT/CshRNA and H1975/PBRM1shRNA cells treated with 1 μM osimertinib for 2 days were immunoblotted with antibodies against the indicated proteins

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Amplification, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: MET-amplified MGH170 cells are dependent on MUC1-C for MET inhibitor resistance and survival. a MGH170 cells treated with 1 μM osimertinib for the indicated days were analyzed for MUC1-C, STAT1 and PBRM1 mRNA levels by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for untreated cells (assigned a value of 1). b Soluble chromatin from MGH170 cells treated with vehicle or 1 μM osimertinib for 3 days was precipitated with antibodies against the indicated proteins or a control IgG. The DNA samples were amplified by qPCR for pELS-1 and pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. c Soluble chromatin from MGH170/tet-MUC1shRNA cells treated with vehicle or DOX and 1 μM osimertinib for 3 days was precipitated with antibodies against the indicated proteins or a control IgG. The DNA samples were amplified by qPCR for pELS-2. The results (mean ± SD of 3 determinations) are expressed as percent input. d MGH170/CshRNA and MGH170/STAT1shRNA cells treated with 1 μM osimertinib were analyzed for clonogenic survival. Shown are representative photomicrographs of stained colonies. The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for untreated cells (assigned a value of 1). e MGH170/tet-AFOS cells treated with vehicle or DOX for 14 days were analyzed for clonogenic survival. Shown are representative photomicrographs of stained colonies. The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for untreated cells (assigned a value of 1). f MGH170 cells treated with 0.5 μM osimertinib and/or 0.5 μM savolitinib for 2 days were analyzed for the indicated mRNA levels by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for untreated cells (assigned a value of 1). g MGH170/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days and then with 0.5 μM osimertinib and/or 0.5 μM savolitinib for 2 days were analyzed for the indicated mRNA levels by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for untreated cells (assigned a value of 1). h Six-week old nude mice were injected subcutaneously in the flank with 5 × 10 6 MGH170 cells. Mice pair-matched into three groups of 5 mice each when tumors reached 100–150 mm 3 were treated with vehicle control (red), OSI + SAV (orange), and OSI + SAV plus GO-203 (blue) for the indicated days. Tumor volumes are expressed as the mean±SEM for five mice

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: Amplification, Quantitative RT-PCR, Control, Staining, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: MUCIN 1 confers inflammatory memory of tyrosine kinase inhibitor resistance in non-small cell lung cancer

doi: 10.1038/s41392-025-02482-7

Figure Lengend Snippet: MUC1-C expression in EGFR mutant NSCLCs associates with response to osimertinib. The designated NSCLC cell lines ( a ) and patient-derived cells ( b ) were treated with the indicated concentrations of M1C ADC for 7 days and analyzed for cell viability by Alamar Blue staining. The results (mean±SD of six determinations) are expressed as relative cell viability (% control). Indicated are the anti-MUC1-C ADC IC50 values. c Mice bearing established MGH170 PDX tumors were pair-matched into two groups of six mice each when tumors reached 100 mm 3 . Control vehicle or M1C ADC (7.5 mg/kg) was administered intravenously in the tail vein on the indicated days. Tumor volumes are expressed as the mean ± SD. Kaplan–Meier curves for PFS ( d ) and OS ( e ) of patients with EGFR mutant NSCLCs treated with osimertinib according to high (red) or low (blue) levels of MUC1-C expression. P values were obtained using the log rank test. f Association of MUC1-C low and high levels with STAT1 expression. Kaplan–Meier curves for PFS ( g ) and OS ( h ) of patients with EGFR mutant NSCLCs treated with osimertinib according to (i) MUC1-C/STAT1 low/low (blue), (ii) MUC1-C/STAT1 high/low or low/high (green), and (iii) MUC1-C/STAT1 high/high (red) levels of expression. i Schemas depicting the MUC1-C-driven inflammatory memory response that confers resistance of NSCLC cells to osimertinib. MUC1-C homodimers form complexes with EGFR and other RTKs, such as MET, at the NSCLC cell membrane. In establishing TKI resistance (left), targeting EGFR with osimertinib induces MUC1-C-mediated activation of the MUC1 gene in an auto-inductive pathway. This response to osimertinib includes occupancy of MUC1 memory domains (i) pELS-1 by MUC1-C and STAT1, and (ii) pELS-2 by MUC1-C, AP-1 (JUN) and PBAF (BRG1, PBRM1). In turn, upregulation of MUC1-C induces STAT1 and thereby the formation of MUC1-C/STAT1 complexes that regulate the expression of downstream ISGs in association with DNA damage resistance and immune evasion. – , – In conferring the memory response after a drug holiday (right), the MUC1 pELS-1 and pELS-2 domains exhibit increases in (i) chromatin accessibility and (ii) H3K27ac and H3K4me1 deposition. In this way, cells are poised to respond to subsequent treatment with osimertinib, as well as MET-TKIs and the EGFR-TKI TQB3804, with rapid induction of (i) MUC1-C, STAT1 and the IFN type I/II pathways, and (ii) recurrence of refractory disease. The findings that targeting MUC1-C genetically or pharmacologically suppresses this memory response of TKI resistance have uncovered MUC1-C as a target for M1C ADC, as well as small molecule inhibitor, treatment of NSCLC patients with refractory tumors. Images generated using BioRender software

Article Snippet: Established MGH170 PDX tumors were treated with PBS or GO-203 at a dose of 12 μg/gm body weight in the absence and presence of oral osimertinib (OSI) (MedChemExpress, #HY-15772A) and savolitinib (SAV) administration each at doses of 10 mg/kg/day.

Techniques: Expressing, Mutagenesis, Derivative Assay, Staining, Control, Membrane, Activation Assay, Generated, Software