Journal: Cell Discovery

Article Title: ASO-based PKM splice-switching therapy increases anti-CTLA-4 antibody efficacy in pancreatic ductal adenocarcinoma

doi: 10.1038/s41421-026-00882-9

Figure Lengend Snippet: a IC 50 of ASO1-TMO. Three classical PDAC cell lines (SUIT-2, PANC-1, and AsPC-1), two basal-like PDAC cell lines (Hs 766 T and BxPC-3), and the MIA PaCa-2 cell line were used for IC 50 determination. Cells were transfected with Lipofectamine and varying concentrations (0, 25, 50, 100, and 200 nM) of ASO1-TMO on Day 0, 10 3 cells were plated on a 96-well plate on Day 1, and cell viability was monitored by a colorimetric assay on Day 3. b Radioactive RT-PCR showing the extent of PKM splice switching after MIA PaCa-2 cells were transfected with the indicated concentrations of ASO1-TMO for 2 days (left). ImageJ was used to quantify the expression of the PKM2, PKM1, and PKMds isoforms (right). c Radioactive RT-PCR results showing the extent of PKM splice switching after BxPC-3 cells were transfected with various concentrations of ASO1-TMO as described in b for 2 days (left). Isoform quantification in b (right). d Western blot analysis showing the extent of isoform switching after MIA PaCa-2 or BxPC-3 cells were transfected with 50 nM ASO for 3 days. NTC, no-treatment control. e Viability of MIA PaCa-2 cells at each time point. Cells were transfected with 50 nM ASO on Day 0, 0.5 × 10 3 cells were plated on a 96-well plate on Day 1, and the OD450 was monitored daily by a colorimetric assay. f PK activity was measured on Day 3 after transfection with 50 nM ASO. g Lactic acid levels were measured on Day 3 after transfection with 50 nM ASO. Statistical analysis: unpaired two-sided t test ( a , b , c , f , g ); two-way ANOVA ( e ).

Article Snippet: MIA PaCa-2 (ATCC, CRL-1420), BxPC-3 (ATCC, CRL-1687), PK1 (RCB1972), Hs 766 T (ATCC, HTB-134), SUIT-2 (JCRB1094), PANC-1 (ATCC, CRL-1469), and AsPC-1 (ATCC, CRL-1682) cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Transfection, Colorimetric Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Activity Assay

Journal: Cell Discovery

Article Title: ASO-based PKM splice-switching therapy increases anti-CTLA-4 antibody efficacy in pancreatic ductal adenocarcinoma

doi: 10.1038/s41421-026-00882-9

Figure Lengend Snippet: a Schematic diagram of the animal experiment. ASO1-TMO and ASO2-TMO target the human PKM exon 10 regions (red and blue, respectively) that we previously targeted with MOE ASOs . ASO2-TMO targets an SRSF3 binding site that is conserved between humans and mice (yellow). b Whole-animal live imaging of luciferase-expressing MIA PaCa-2 cells transplanted into the pancreas. Luminescence images of the transplanted mice on the indicated days are shown with a color scale in photons/sec/cm 2 /steradian. A total of 14 mice were randomized to each treatment group. c Quantification of the luciferase signal in ( b ). Statistical analysis: two-way ANOVA. d Images of representative tumors from animals treated as indicated on Day 23. The duodenum, pancreas, tumor, and spleen are shown. Dashed lines delineate the tumor. Scale bar, 1 cm. e Quantification of tumor weight on Day 23 (after the duodenum, normal pancreas, and spleen were removed). f ASO1-TMO and ASO2-TMO induce PKM splice switching in pancreatic tumors, as determined by radioactive RT-PCR of RNA from pancreatic tumor samples from tumor-bearing mice on Day 23. g Western blot analysis of PKM1, PKM2, and Vinculin in orthotopic tumor samples from mice treated with SCR-TMO, ASO1-TMO, or ASO2-TMO. h Representative H&E and IHC images of a tumor, showing the expression of PKM1 and PKM2. i Representative IF staining images of tumor sections from mice treated with saline, SCR-TMO, ASO1-TMO, or ASO2-TMO showing the expression of PKM2 (green), the proliferation marker Ki67 (red), and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 50 µm. Statistical analysis: unpaired two-sided t -test ( e , f ); two-way ANOVA ( c ).

Article Snippet: MIA PaCa-2 (ATCC, CRL-1420), BxPC-3 (ATCC, CRL-1687), PK1 (RCB1972), Hs 766 T (ATCC, HTB-134), SUIT-2 (JCRB1094), PANC-1 (ATCC, CRL-1469), and AsPC-1 (ATCC, CRL-1682) cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Binding Assay, Imaging, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Saline, Marker

Journal: The Journal of Cell Biology

Article Title: Membrane-Type 1 Matrix Metalloproteinase Cleaves Cd44 and Promotes Cell Migration

doi:

Figure Lengend Snippet: Shedding of endogenous CD44H in human pancreatic tumor cell line, MIA PaCa-2. (A) MIA PaCa-2 cells (3 × 10 5 ) were cultured in a six-well plate in serum-free medium in the presence or absence of the proteinase inhibitors as indicated. After 48 h, the cell lysate (bottom) and the conditioned medium (top) were subjected to Western blot analyses. CD44 was detected by the mouse anti–human CD44 mAb. Concentrations of the inhibitors were adjusted as follows: 50 nM for TIMP-1, 50 nM for TIMP-2, 10 μM for BB94, 1.0 μM for E-64, and 1.0 mM for AEBSF. (B) Expression of genes for CD44 and MT-MMPs were examined by RT-PCR using specific primers as described in Materials and Methods. Sizes of the amplified fragments were 461 bp for CD44, 589 bp for MT1-MMP, 578 bp for MT2-MMP, 461 bp for MT3-MMP, 334 bp for MT4-MMP, 564 bp for MT5-MMP, and 500 bp for GAPDH. PCR product of CD44 indicates that CD44 expressed in MIA PaCa-2 is CD44H.

Article Snippet: Cell lines from human pancreatic carcinoma (MIA PaCa-2), breast carcinoma (ZR-75-1), and osteosarcoma (MG-63) were obtained from American Type Culture Collection and cultured in RPMI-1640 medium (Life Technologies) supplemented with 10% FBS and kanamycin.

Techniques: Cell Culture, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: (A) Dose-dependent effect of MRTX849 on the proliferation of MiaPaCa-2 and H358 cells based on live cell imaging. AsPC-1 cells were treated with different concentrations of MRTX1133, and the cell growth was monitored. Error bars represent mean and SD from triplicates. Experiments were done at three independent times. (B) Effect of the mutant-specific KRAS inhibitors, MRTX849 and MRTX1133 on the proliferation of MiaPaCa-2-MR, H358-MR and AsPC-1-MR cells that have developed acquired resistance following long-term selection. Mean and SD were determined from triplicates, and the experiments were carried out 3 independent times. (C) Live cell imaging to examine the effect of MRTX849 on the proliferation of UM53 and RS4774 cell lines. Error bars represent mean and SD from triplicates. Experiments were done at three independent times. (D) Western blot analysis on MiaPaCa-2-MR, UM53 and RS4774 cells to evaluate the effect of MRTX849 on ERK phosphorylation following 48-hour exposure at the indicated concentration. (E) Differential effects of KRAS knockdown on the proliferation of MiaPaCa-2-WT, AsPC-1-WT, MiaPaCa-2-MR, AsPC-1-MR and UM53 cells. Error bars indicate mean and SD from triplicates. *** represents p<0.0001 as determined by 2-way ANOVA. Experiments were done at three independent times. (F) In vivo efficacy of MRTX849 on xenografts derived from MiaPaCa-2-WT and MiaPaCa-2-MR cells and on the tumor growth of RS4774 PDX. Error bars represent mean and SEM. *** represents p<0.0001 as determined by 2-way ANOVA. (G) Representative images of immunohistochemical staining on the vehicle- and MRTX849-treated tissues to determine the phosphorylation status of ERK. Scale bar represents 50 microns.

Article Snippet: MiaPaCa-2 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic–antimycotic.

Techniques: Live Cell Imaging, Mutagenesis, Selection, Western Blot, Phospho-proteomics, Concentration Assay, Knockdown, In Vivo, Derivative Assay, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: (A) GSEA analysis illustrating the differential effect of MRTX849 on the genes associated with E2F target pathway and the proteins associated with G2/M checkpoint in cell cycle machinery. (B) Heat map represents the differential effect of MRTX849 in downregulating the indicated genes and proteins that are involved in cell cycle from MiaPaCa-2-WT and MiaPaca-2-MR cells following 48-hour treatment. (C) Biochemical analysis on the indicated sensitive (WT) and resistance cell lines to compare the effect of MRTX849 on RB phosphorylation and cyclin A expression at different doses after treating the cells for 48 hours. (D) GSEA analysis highlighting pathways associated with genes and proteins that are differentially expressed between MiaPaCa-2-MR and MiaPaCa-2-WT cells. (E) Volcano plots of differentially expressed genes and proteins in MiaPaCa-2-MR versus MiaPaCa-2-WT cells. (F) Heat map illustrating the effects of indicated targeted therapeutic agents on normalized fold change in cell growth in combination with DMSO or mutant-selective KRAS inhibitors. (G) Synergistic interactions between KRAS inhibitors and erlotinib or AZD4547 in AsPC-1-MR, UM53, and MiaPaCa-2-MR cells. Heat maps depict normalized fold change in growth rate following treatment with increasing concentrations of KRAS inhibitors in combination with erlotinib or AZD4547. Bliss synergy scores were calculated using the SynergyFinder online platform.

Article Snippet: MiaPaCa-2 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic–antimycotic.

Techniques: Phospho-proteomics, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: (A) Comparison of the antiproliferative effects of mutant-selective KRAS inhibitors and RMC-6236 in the indicated cell lines. Error bars represent mean and SD from triplicates. Experiments were done 3 independent times. *** represents p<0.0001 as determined by 2-way ANOVA. (B) EC 50 values of mutant specific KRAS inhibitors and RMC-6236 in multiple cell lines. Error bar represents mean and SD from 2 independent experiments. *** represents p<0.0001 as determined by unpaired student t-test. (C) GSEA analysis highlighting the top pathways that were differentially impacted in AsPC-1-MR cells following the treatment with MRTX1133 (100 nM) and RMC-6236 (50 nM) for 48 hours. (D) Heatmap depicts the differential expression of the indicated genes associated with MEK, MTOR and cell cycle pathways following the treatment with MRTX1133 and RMC-6236 in AsPC-1-MR cells. (E) Immunoblot analysis comparing the effect of mutant-specific KRAS inhibitors and RMC-62366 on the indicated proteins after exposing the cells to different drug concentrations up to 48 hours. (F) Long term colony formation assay in AsPC-MR and MiaPaCa-2-MR cells following the treatment with RMC-6236. AsPC-1-MR cells were treated with 50 nM RMC-6236 for 19 days and MiaPaCa-2-MR cells were treated 100 nM of RMC-6236 for 9 days. (G) Differential effect of RMC-6236 on the proliferation of MiaPaCa-2-WT and MiaPaCa-2-RR cells following the treatment with different drug concentrations. Error bars represent mean and SD from triplicates. Experiment was done at 3 independent times. (H) Biochemical analysis to compare the effect of RMC-6236 on the indicated proteins between the MiaPaCa-2-WT and MiaPaCa-2-RR cells following 48-hour exposure.

Article Snippet: MiaPaCa-2 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic–antimycotic.

Techniques: Comparison, Mutagenesis, Quantitative Proteomics, Western Blot, Colony Assay

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: Venn diagram showing drugs that enhanced the efficacy of mutant-selective KRAS inhibitors in MiaPaCa-2-MR, AsPC-1-MR, and UM53 cells, identified through combinatorial drug screening. Common hits included the CDK4/6 inhibitors palbociclib, abemaciclib, and G1T38. Correlation plot comparing the fold change in growth rate for individual drugs in combination with DMSO or mutant-selective KRAS inhibitors. (B) Synergistic interactions between palbociclib and KRAS or RAS inhibitors in AsPC-1-MR, UM53, and MiaPaCa-2-MR cells. Isobolograms were generated based on normalized fold change in growth rate following treatment with increasing concentrations of palbociclib in combination with KRAS or RAS inhibitors. Bliss synergy scores were calculated using the SynergyFinder online platform. (C) Western blot analysis in MiaPaCa-2-MR, UM53 and RS4774 following the treatment with palbociclib (100 nM) in combination with MRTX849 (250 nM) up to 48 hours. AsPC-1-MR cells were treated with palbociclib (100 nM) in combination with MRTX1133 (250 nM). (D) Western blotting in UM53 cells to examine the effect of palbociclib (100 nM) in combination with the RAS inhibitor, RMC-6236 (25 nM) on the cell cycle proteins following 48-hour treatment. (E) Effect of Palbociclib in combination with MRTX849 on the growth of spheroids derived from MiaPaCa-2-MR and UM53 cells. Error bars represent mean and SEM from triplicates. *** represents p<0.0001 and ** represents p<0.001 as determined by 2-way ANOVA. Experiments were done at 3 independent times.

Article Snippet: MiaPaCa-2 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic–antimycotic.

Techniques: Mutagenesis, Drug discovery, Generated, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: (A) Live cell imaging to monitor the proliferation of MiaPaCa-2-MR, AsPC-1-MR and UM53 cells following the treatment with INX-315 in combination with mutant-specific KRAS inhibitors. The effect of INX-315 in combination with RMC-6236 on the proliferation of MiaPaCa-2-MR and UM53 cells. Error bars represent mean and SD from triplicates. *** represents p<0.0001 as determined by 2-way ANOVA. (B) The impact of INX-315 in combination with MRTX849 on the growth of spheroids derived from MiaPaCa-2-MR cells. Error bars were determined from mean and SEM from triplicates. *** represents p<0.0001 as determined by 2-way ANOVA. Experiment was done at 3 independent times. Representative images of the spheroids from MiaPaCa-2-MR cells. (C) Western blotting on the indicated proteins from MiaPaCa-2-MR, UM53 and RS4774 cells following the treatment with INX-315 in combination with MRTX849 up to 48 hours. (D) Stack plots illustrating the % of cell population at each phase of cell cycle based on PI profile following the treatment with MRTX849 in combination palbociclib or INX-315. (E) Bivariate flow cytometry analysis of UM53 cells showing the effects of palbociclib or INX-315 in combination with MRTX849 or RMC-6236 on BrdU incorporation. (F) Immunofluorescence analysis of phospho–histone H3 (S10) in UM53 cells pretreated with DMSO or INX-315 in combination with MRTX849 for 48 hours and subsequently treated with nocodazole (250 nM) for 24 hours. Scale bar represents 75 microns. (G) Comparison of cellular outgrowth in the indicated cell lines following removal of KRAS or RAS inhibitors in combination with palbociclib or INX-315 after 4 days of treatment. Error bars represent mean and SD from triplicates. *** represent p<0.0001 as determined by 2-way ANOVA.

Article Snippet: MiaPaCa-2 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic–antimycotic.

Techniques: Live Cell Imaging, Mutagenesis, Derivative Assay, Western Blot, Flow Cytometry, BrdU Incorporation Assay, Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: (A) In vivo effect of palbociclib in combination with MRTX849 on tumor growth in MiaPaCa-2-MR xenografts and RS4774 PDX. Error bars were determined based on mean and SEM. *** represents p value <0.0001 as determined by 2-way ANOVA. (B) Column graphs illustrate the tumor weights from MiaPaCa-2-MR xenografts and RS4774 PDX following the treatment with palbociclib in combination with MRTX849. *** p<0.0001, ** p<0.01 as determined by unpaired student t-test. (C) Effect of palbociclib in combination with MRTX849 on the mice body weights. Error bar represents mean and SEM. (D) Immunohistochemical analysis of RB phosphorylation in tumor tissues from MiaPaCa-2-MR xenografts and RS4774 PDX models following treatment with palbociclib in combination with MRTX849. Corresponding H&E-stained sections are shown. Scale bar represents 100 microns. (E) Effect of INX-315 in combination MRTX849 on tumor progression in mice bearing MiaPaCa-2-MR xenografts. The change in mice body weights following the combination treatment was monitored. (F) Immunohistochemical analysis of phospho-RB and phospho–histone H3 in tumor tissues following 5 days of treatment with palbociclib or INX-315 in combination with MRTX849. Scale bar represents 100 microns.

Article Snippet: MiaPaCa-2 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic–antimycotic.

Techniques: In Vivo, Immunohistochemical staining, Phospho-proteomics, Staining