Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: (A) Volcano plot showing gene expression changes in PC-9 cells after treatment with osimertinib (Osi). The x-axis denotes fold expression change, and the y-axis shows p-value of the change. Genes with a log₂ fold expression change ≥ 1 and an adjusted p-value < 0.05 are labeled. (B) KEGG pathway enrichment analysis of genes downregulated by Osi in PC-9 cells. Pathways are ranked based on –log₁₀ (p-value). (C) PC-9 and HCC827 cells were treated with the indicated concentrations of osimertinib for 24 hrs. Protein expression of RRM1 and RRM2 was assessed by western blot, with GAPDH as a loading control. Results shown are representative of three independent experiments. (D) Intracellular dNTP levels in PC-9 and HCC827 cells were quantified with or without 24-hour Osi treatment. Data are presented as mean ± S.D. from three independent biological replicates.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Gene Expression, Expressing, Labeling, Western Blot, Control

Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: (A) Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) was performed using an anti-RNAPII (8WG16) antibody to evaluate recruitment of RNA polymerase II to the RRM2 promoter, with or without osimertinib (Osi) treatment. Relative enrichment was normalized to the ACTB promoter. (B) ChIP-qPCR using HA-tagged MYBL2-overexpressing cell lines was conducted to assess MYBL2 binding to the RRM2 promoter. Promoter occupancy is shown relative to input. Data are presented as mean ± s.e.m. (n = 3). ***p < 0.001. (C) RRM2 mRNA expression was quantified by RT-qPCR in PC-9 and HCC827 cells following EGFR knockdown using siRNA. Transcript levels were normalized to GAPDH. (D) The impact of EGFR knockdown on MYBL2 recruitment to the RRM2 promoter was evaluated by ChIP-qPCR in HA-MYBL2-expressing cells transfected with control or EGFR siRNA. Promoter enrichment was normalized to input. Data are shown as mean ± s.e.m. (n = 3). ***p < 0.001. All data are representative of at least three independent experiments.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Expressing, Quantitative RT-PCR, Knockdown, Transfection, Control

Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: (A) PC-9 and HCC827 cells were treated with 5 nM osimertinib (Osi) for the indicated durations. Protein levels of RRM1, RRM2, and RRM2B were assessed by western blotting, with GAPDH used as a loading control. Results are representative of three independent experiments. (B) Time-course analysis of RRM2 and RRM2B mRNA expression in PC-9 and HCC827 cells treated with 5 nM Osi. Transcript levels were measured by RT-qPCR, normalized to GAPDH, and expressed relative to untreated controls. Data represent three independent experiments. (C) Intracellular dNTP levels in PC-9 cells with or without RRM2B knockdown following 0, 24, or 48 hours of Osi treatment. dNTP levels were normalized to untreated controls. Data shown are representative of three independent experiments. (D) Quantification of DNA damage, expressed as comet tail moment, from comet assays in HCC827 and PC-9 cells treated with DMSO (control), osimertinib (10 nM), siRRM2B, or the combination of osimertinib (5 µM) and siRRM2B for 24 hours.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Knockdown

Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: (A) A549, PC-9, and HCC827 cells were treated with 5 nM osimertinib (Osi). Protein levels of phosphorylated CHK1 (p-CHK1), phosphorylated CHK2 (p-CHK2), total CHK1, and CHK2 were analyzed by western blotting. GAPDH served as a loading control. Data are representative of three independent experiments. (B) Cells were treated as in panel A, with MG132 added 2 hours before harvest. Protein levels of p-CHK1, p-CHK2, CHK1, and CHK2 were assessed by western blotting. ACTB was used as a loading control. Data represent three independent experiments. (C) Proliferation assays in HCC827 and PC-9 cells treated with increasing concentrations of osimertinib alone or in combination with PV1019 (CHK2 inhibitor, concentration specified). (D) PC-9 cells were treated with DMSO, 5 nM Osi, 10 µM PV1019, or the combination for the indicated times. Nuclear fractions were analyzed by western blotting for RRM1, RRM2, RRM2B, POLD1, POLH, and TNNT3. ACTB and Histone H3 were used as loading controls. (E) PC-9 cells were treated with DMSO, 5 nM Osi, 10 nM LY2606368 (CHK1/CHK2 inhibitor), or the combination for the indicated times. Nuclear extracts were analyzed as in panel D. Data are representative of three independent experiments.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Control, Concentration Assay

Journal: bioRxiv

Article Title: Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

doi: 10.64898/2025.12.24.696437

Figure Lengend Snippet: Stepwise drug escalation assay to generate resistance to combined treatment with cell cycle checkpoint inhibitors (PV1019 or LY2606368) and osimertinib in (A) PC-9 and (B) HCC827 and cells. Cells were exposed to increasing concentrations of osimertinib, starting at 10 nM and escalating to 1000 nM over several weeks. At each step, proliferating cells were expanded until resistance was confirmed by sustained growth in 1000 nM osimertinib, assessed via trypan blue exclusion assay. (C) Tumor growth rates of HCC827 xenografts treated with DMSO (vehicle control, n=8), osimertinib (n=8), LY2606368 at 1 mg/kg or 3 mg/kg (n=5 each), or a combination of osimertinib with LY2606368 at each dose (n=7 and n=9, respectively). Tumor volumes were measured twice weekly and are presented as mean ± SEM. Statistical significance was determined using Student’s t -test. (D) Relative mRNA expression levels of RRM2B, RRM2, MYBL2, TNNT3, and CHK1 in tumor tissues following treatment with DMSO or osimertinib, as measured by quantitative RT-PCR.

Article Snippet: RRM1 (#10526-1-AP), RRM2 (11661-1-AP), RRM2B (#18005-1-AP), CHK1 (#25887-1-AP), CHK2 (#13954-1-AP), EGFR (#66455-1-Ig), beta Actin (#20536-1-AP), HA tag (#51064-2-AP), Histone H3 (#17168-1-AP), GAPDH (#60004-1-Ig), POLD1(#15646-1-AP), POLH (#28133-1-AP), MYBL2 (#18896-1-AP) and TNNT3 (#19729-1-AP) were purchased from Proteintech.

Techniques: Trypan Blue Exclusion Assay, Control, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: WEE1 inhibitor exerts synergistic effect with KRAS G12C inhibitor via MYBL2-RRM2 axis in KRAS G12C -mutant lung cancer

doi: 10.1038/s41419-025-07992-4

Figure Lengend Snippet: A GSEA analysis comparing sotorasib, adavosertib, and combination groups to the control group, as well as comparing the combination group to sotorasib and adavosertib groups. B NES values of E2F targets and G2M checkpoint pathways in GSEA analysis, with pairwise comparisons among the four treatment groups. C Venn diagram of the top 20 genes in E2F targets pathway comparing the combination group to sotorasib group, adavosertib group and control group. D Venn diagram of the top 20 genes in G2M checkpoint pathway comparing the combination group to sotorasib group, adavosertib group and control group. E Flowchart illustrating the identification of RRM2 transcription factors using TCGA LUAD and GSE31210 and GSE30219 datasets. F The correlation between MYBL2, CENPA, FOXM1, E2F1, ZNF367, and HMGA1 with RRM2 in RNA-seq data. G, H The correlation between MYBL2 and RRM2 in TCGA LUAD ( G ) and our RNA-seq ( H ). I The schematic diagram for two MYBL2 binding motifs on RRM2 promoter (FL) and related truncated (ST) and site-mutated promoter (S-mut) constructs. J Relative luciferase reporter activity of RRM2 promoter (−2000 ~ + 60) while co-transfected with MYBL2 expression plasmid for 48 h in H2122 and H23 cells. K Relative luciferase reporter activity of the truncated and site-mutated RRM2 promoters while co-transfected with MYBL2 expression plasmid for 48 h in H2122 cells. L The expression of MYBL2 and RRM2 proteins in H2122, H23 and H358 cells under control, sotorasib, adavosertib or combination treatment for 24 h. M The expression of phosphorylation and total levels of ERK and AKT proteins in H2122, H23 and H358 cells under control, sotorasib, adavosertib or combination treatment for 24 h.

Article Snippet: For IHC staining in human LUAD tissue, MYBL2 and RRM2 protein levels in the tumor and normal lung samples were assessed by IHC using anti-MYBL2 (1:400 dilution, Proteintech) and anti-RRM2 (1:400 dilution, ABclonal).

Techniques: Control, RNA Sequencing, Binding Assay, Construct, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: WEE1 inhibitor exerts synergistic effect with KRAS G12C inhibitor via MYBL2-RRM2 axis in KRAS G12C -mutant lung cancer

doi: 10.1038/s41419-025-07992-4

Figure Lengend Snippet: A, B Comparison of MYBL2 ( A ) and RRM2 ( B ) expression in normal lung tissue and early-stage lung adenocarcinoma tumors or in tumor tissues from non-relapsed and relapsed lung cancer patients in the GSE31210 dataset. C, D Comparison of MYBL2 ( C ) and RRM2 ( D ) expression at different stages in the GSE31210 dataset. E, F Relapse free survival probability of two groups classified by MYBL2 ( E ) or RRM2 ( F ) median expression levels in the GSE31210 dataset. G The expression of MYBL2 and RRM2 proteins in normal lung epithelial cells (NOR) and NSCLC cells (NSCLC). H Representative IHC images of MYBL2 and RRM2 expression in LUAD tissue microarray. I RRM2 expression levels in high and low expression groups based on the median value of MYBL2. J The correlation between MYBL2 and RRM2 IHC H score in LUAD tissue microarray. K Comparison of MYBL2 and RRM2 expression at different stages in the LUAD tissue microarray. L Percent survival probability of two groups classified by MYBL2 or RRM2 median expression levels in the LUAD tissue microarray. M Percent survival probability of four patient groups classified by the median expression levels of MYBL2 or RRM2 in the LUAD tissue microarray.

Article Snippet: For IHC staining in human LUAD tissue, MYBL2 and RRM2 protein levels in the tumor and normal lung samples were assessed by IHC using anti-MYBL2 (1:400 dilution, Proteintech) and anti-RRM2 (1:400 dilution, ABclonal).

Techniques: Comparison, Expressing, Microarray

Journal: Cell Death & Disease

Article Title: WEE1 inhibitor exerts synergistic effect with KRAS G12C inhibitor via MYBL2-RRM2 axis in KRAS G12C -mutant lung cancer

doi: 10.1038/s41419-025-07992-4

Figure Lengend Snippet: A The expression of MYBL2 and RRM2 proteins in H2122 and H23 cells under mock, siMYBL2 or siRRM2 treatment for 72 h. B Representative images of colony formation in H2122 and H23 cells under mock, siMYBL2 or siRRM2 treatment for 10–14 days. C Representative images and proportion of EdU incorporation assay in H2122 cells under mock, siMYBL2 or siRRM2 treatment for 48 h. n = 3 per group. D Representative images and proportion of γH2AX positive cells in H2122 cells under mock, siMYBL2 or siRRM2 treatment for 48 h. n = 3 per group. E Representative images of cell apoptosis assay in H2122 and H23 cells under mock, siMYBL2 or siRRM2 treatment for 48 h. F Cell apoptosis proportion in H2122 and H23 cells under mock, siMYBL2 or siRRM2 treatment for 48 h. n = 3 per group. G The expression of MYBL2 and RRM2 proteins in H2122 and H23 cells under empty vector (EV), MYBL2 or RRM2 plasmids treatment for 48 h. H Representative images of colony formation H2122 and H23 cells under EV, MYBL2 or RRM2 plasmids treatment for 10–14 days. I The cell viability of H2122 cells overexpressing EV, MYBL2, and RRM2 under control, sotorasib, adavosertib or combination treatment for 72 h. n = 3 per group. J The cell growth of H2122 and H23 cells overexpressing EV, MYBL2, and RRM2 under combination of sotorasib and adavosertib treatment for 72 h. K Representative images of colony formation in H2122 and H23 cells overexpressing EV, MYBL2, and RRM2 under combination of sotorasib and adavosertib treatment for 10-14 days. L, M Representative images ( L ) and proportion ( M ) of EdU positive cells in H2122 cells overexpressing EV, MYBL2, and RRM2 under control, sotorasib, adavosertib or combination treatment for 48 h. n = 3 per group. Data are mean ± SD of three independent replicates.

Article Snippet: For IHC staining in human LUAD tissue, MYBL2 and RRM2 protein levels in the tumor and normal lung samples were assessed by IHC using anti-MYBL2 (1:400 dilution, Proteintech) and anti-RRM2 (1:400 dilution, ABclonal).

Techniques: Expressing, Apoptosis Assay, Plasmid Preparation, Control

Journal: Cell Death & Disease

Article Title: WEE1 inhibitor exerts synergistic effect with KRAS G12C inhibitor via MYBL2-RRM2 axis in KRAS G12C -mutant lung cancer

doi: 10.1038/s41419-025-07992-4

Figure Lengend Snippet: A Diagram illustrating the function of RRM2. B The cell viability of H2122 and H23 cells under control, sotorasib, adavosertib or combination treatment for 72 h with and without 10 μM dNTPs or dNs. n = 3 per group. C The cell growth of H2122 and H23 cells under combination of sotorasib and adavosertib treatment for 72 h with and without 10 μM dNTPs or dNs. D Representative images of colony formation in H2122 and H23 cells under combination of sotorasib and adavosertib treatment for 10–14 days with and without dNTPs or dNs. E, F Representative images ( E ) and proportion ( F ) of EdU positive in H2122 cells under control, sotorasib, adavosertib or combination treatment for 48 h with and without 10 μM dNTPs. n = 3 per group. G, H Representative images ( G ) and apoptosis proportion ( H ) of cell apoptosis assay in H2122 cells under control, sotorasib, adavosertib or combination treatment for 48 h with and without 10 μM dNTPs or dNs. n = 3 per group. I, J Representative images of cell cycle distribution and γH2AX positive cells in H2122 ( I ) and H23 ( J ) cells under combination treatment for 48 h with and without 10 μM dNTPs performed using flow cytometry. The cell cycle was divided into the 2 N, 4 N, S1, S2, S3, no replication S, and subG1 phases by PI and EdU. K Proportion of no replication S phase in H2122 and H23 cells under combination treatment for 48 h with and without dNTPs. n = 3 per group. L Proportion of γH2AX positive cells in H2122 and H23 cells under combination treatment for 48 h with and without dNTPs. n = 3 per group. Data are mean ± SD of three independent replicates.

Article Snippet: For IHC staining in human LUAD tissue, MYBL2 and RRM2 protein levels in the tumor and normal lung samples were assessed by IHC using anti-MYBL2 (1:400 dilution, Proteintech) and anti-RRM2 (1:400 dilution, ABclonal).

Techniques: Control, Apoptosis Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: WEE1 inhibitor exerts synergistic effect with KRAS G12C inhibitor via MYBL2-RRM2 axis in KRAS G12C -mutant lung cancer

doi: 10.1038/s41419-025-07992-4

Figure Lengend Snippet: A H2122 xenografts tumors treated with sotorasib, adavosertib, or a combination of both for 18 days shown in tumor growth. n = 5 mice, with 10 tumors per group. B Percent survival in H2122 xenografts tumors treated with sotorasib, adavosertib, or their combination for 18 days. Tumor volume exceeding 600 mm 3 is considered lethal. n = 10 tumors per group. C Waterfall plot analyses of changes in tumor volume at the end of treatment. n = 10 tumors per group. D Photograph of xenografts tumors treated with sotorasib, adavosertib, or a combination of both for 18 days. n = 10 tumors per group. E Tumor mass of xenografts tumors treated with sotorasib, adavosertib, or a combination of both for 18 days. n = 10 tumors per group. F Body weight of mice bearing H2122 xenografts tumors treated with sotorasib, adavosertib, or a combination of both for 18 days. n = 5 mice per group. G H&E staining, IHC staining, and TUNEL staining of xenografts tumors. Scale bar, 100 μm in the full image and 25 μm in the magnified image of the top right corner. For TUNEL staining, The blue fluorescence (DAPI) in tumor represented for tumor cells and red fluorescence (TUNEL, TMR red) represented for apoptosis tumor cells. Scale bar, 50 μm. H The staining expression of Ki67, MYBL2, RRM2, pH2AX and apoptosis index in xenografts tumors treated with sotorasib, adavosertib, or their combination for 18 days. n = 9 per group. Expression is calculated using three slices per group, with three randomly selected regions per slice. I The correlation between MYBL2 and RRM2 IHC H score in xenografts tumors. n = 12. J Abstract. In KRAS-G12C mutant NSCLC, the combination of G12Ci and WEE1i synergistically suppresses the MYBL2-RRM2 axis. This suppression leads to a reduction in dNTPs, triggers DNA replication stress, and results in the accumulation of DNA damage, ultimately culminating in apoptosis. Data are mean ± SD of independent replicates.

Article Snippet: For IHC staining in human LUAD tissue, MYBL2 and RRM2 protein levels in the tumor and normal lung samples were assessed by IHC using anti-MYBL2 (1:400 dilution, Proteintech) and anti-RRM2 (1:400 dilution, ABclonal).

Techniques: Staining, Immunohistochemistry, TUNEL Assay, Fluorescence, Expressing, Mutagenesis