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

Journal: Oncogene

Article Title: X-linked cancer-associated polypeptide (XCP) from lncRNA1456 modulates PHF8 histone demethylase activity to regulate the epigenome, gene expression, and cellular pathways in breast cancer

doi: 10.1038/s41388-026-03740-w

Figure Lengend Snippet: A , B Xenograft assays showing subcutaneous tumor growth of MCF-7 ( A ) or MDA-MB-231 ( B ) breast cancer cells ectopically expressing dox-inducible FLAG-GFP or XCP-FLAG ( left ). Tumor weight at end of experiment is shown ( right ). Animals injected with MCF-7 cells had E2-pellet implantations at the back of the neck to promote growth of MCF-7 cells in vivo. All animals were fed doxycycline in their chow. Each point represents the mean ± SEM. For MCF-7, GFP n = 7 and XCP n = 7; For MDA-MB-231, GFP n = 8 and XCP n = 8. Significance was calculated using unpaired t-test. C , D Expression of FLAG-GFP or XCP-FLAG in tumor tissue was validated from MCF-7 ( C ) or MDA-MB-231 ( D ) xenograft tumors by Western blot ( left ) and RT-qPCR ( right ). For Western blot, 3 representative tumors from each group are shown. β-tubulin was used as a loading control. For RT-qPCR, the average of all tumors from each group is shown. RNA levels were quantified by normalizing to housekeeping gene RPL19 mRNA. Each bar represents the mean + SEM. For MCF-7, GFP n = 7 and XCP n = 7; For MDA-MB-231, GFP n = 8 and XCP n = 8. [See also Fig. ].

Article Snippet: MCF-7 (ATCC; RRD:CVCL_0031) cells were kindly provided by Benita S. Katzenellenbogen (University of Illinois, Urbana-Champaign, Champaign, IL) and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC; RRID:CVCL_0062).

Techniques: Expressing, Injection, In Vivo, Western Blot, Quantitative RT-PCR, Control

Journal: Oncogene

Article Title: X-linked cancer-associated polypeptide (XCP) from lncRNA1456 modulates PHF8 histone demethylase activity to regulate the epigenome, gene expression, and cellular pathways in breast cancer

doi: 10.1038/s41388-026-03740-w

Figure Lengend Snippet: A , B Heatmaps ( left ) and Gene Ontology analysis ( right ) showing XCP-mediated gene expression changes in MCF-7 ( A ) and MDA-MB-231 ( B ) xenograft tumors. C , D Box plots showing gene set analysis of XCP-induced genes and their expression levels stratified into breast cancer subtypes using PAM50 gene set analysis ( left ) and stratified by ER status ( right ) in MCF-7 ( C ) and MDA-MB-231 ( D ) xenograft tumors. Observed differences are significant as determined by an ANOVA comparison of the means ( P value < 0.00001). [See also Fig. ].

Article Snippet: MCF-7 (ATCC; RRD:CVCL_0031) cells were kindly provided by Benita S. Katzenellenbogen (University of Illinois, Urbana-Champaign, Champaign, IL) and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC; RRID:CVCL_0062).

Techniques: Gene Expression, Expressing, Comparison

Journal: Journal of Biological Chemistry

Article Title: Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5

doi: 10.1074/jbc.m602556200

Figure Lengend Snippet: FIGURE 3. Identification of a CRM1-responsive NES in OREBP/TonEBP. A, effectofLMB(10ng/ml)onthesubcellularlocalizationoftheindicatedOREBP mutants expressed in HeLa cells. Representative fluorescence images of fixed cells expressing FLAG-OREBP1–581 and live cells expressing OREBP1–581-GFP. For isotonic treatment, cells transfected with the indicated expression plas- mids were cultured in isotonic medium with or without LMB for 5 h. For hypo- tonic treatment, cells were pretreated with hypertonic medium for 90 min to induce nuclear translocation of the fusion proteins with or without LMB. Sub- sequently, cells were cultured in hypotonic medium for another 90 min with or without LMB. FLAG-OREBP1–581 recombinant protein was visualized after fixation using a FLAG antibody and a FITC-labeled secondary antibody and wasanalyzedbyfluorescencemicroscopy.OREBP1–581-GFPrecombinantpro- tein was visualized by green fluorescence microscopy in live cells. B, align- ment of OREBP/TonEBP nuclear export signal with several characterized NES motifs. The conserved residues are highlighted. C, amino acid sequences of the OREBP/TonEBP NES and illustration of the point mutant. The mutated residues are in boldface. D, functional analysis of the NES. Representative flu- orescence images of fixed HeLa cells. Cells expressing the indicated mutants were treated with hypotonic or isotonic medium for 90 min. Recombinant protein was visualized after fixation with a FLAG antibody and a FITC-labeled secondary antibody and was analyzed by fluorescence microscopy. Images were examined with 40 objective. Scale bar, 100 m.

Article Snippet: CRM1 cDNA (OriGene Technologies, Inc.) was cloned in-frame into Myc tag expression vector pCMV-Tag-3C (Stratagene).

Techniques: Fluorescence, Expressing, Transfection, Cell Culture, Translocation Assay, Recombinant, Labeling, Microscopy, Mutagenesis, Functional Assay

Journal: Journal of Biological Chemistry

Article Title: Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5

doi: 10.1074/jbc.m602556200

Figure Lengend Snippet: FIGURE 5. In vivo interaction of NES with CRM1. A, BiFC analysis of interaction between CRM1 and OREBP/TonEBP. Representative fluorescence images of HeLa cells co-transfected with and expressing the indicated plasmids. At 16 h after transfection, cells were treated with hypertonic medium for 2 h and fixed. Fluorescence emission of the cells was imaged. B, quantitation of BiFC signal. The median of yellow fluorescent protein (YFP)/CFP ratios in each group was derivedfrommorethan100transfectedcells.C,expressionofVN173andVC155fusionproteinsasdeterminedbyWesternblotting.HeLacellsweretransfected with plasmids encoding pHA-OREBP1–581VC and pHA-OREBP1–581L4AVC and pHA-CRM1-VN, respectively. Cell lysates were loaded for the determination of protein expression using immunoblotting analysis with anti-HA (for pHA-CRM1-VN), anti-OREBP (for OREBP1–581VC and OREBP1–581L4AVC), and anti-tubulin antibodies. D, effect of overexpressed CRM1 on subcellular localization of OREBP/TonEBP mutants. Myc-CRM1 expression plasmid was transfected into HeLa cells along with expression plasmids for FLAG-tagged OREBP/TonEBP mutants. Cells were treated with hypertonic medium for 2 h, fixed, and stained with anti-FLAG antibody to determine subcellular localization of OREBP/TonEBP mutant proteins (green) and anti-Myc antibody to detect recombinant CRM1 by indirect immunofluorescence microscopy (red). The cells were counterstained with DAPI to visualize nuclei. Images were examined with 40 objective. Scale bar, 100 m.

Article Snippet: CRM1 cDNA (OriGene Technologies, Inc.) was cloned in-frame into Myc tag expression vector pCMV-Tag-3C (Stratagene).

Techniques: In Vivo, Fluorescence, Transfection, Expressing, Quantitation Assay, Western Blot, Plasmid Preparation, Staining, Mutagenesis, Recombinant, Immunofluorescence, Microscopy