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: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

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: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

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) Representative DNA fiber images from the indicated cell lines under different treatment conditions. Cells were labeled sequentially with IdU (red) for 1 hour without treatment, followed by CldU (green) in the presence of osimertinib, dNTP supplementation, or their combination. DNA fibers were then isolated and spread to analyze replication dynamics. (B) Quantification of replication fork progression, shown as the ratio of CldU to IdU track lengths for individual DNA fibers. (C) Representative comet assay images showing DNA damage in HCC827 and PC-9 cells after 24-hour treatment with DMSO (control), osimertinib (5 µM), dNTPs (25 µM), or the combination of osimertinib and dNTPs. (D) Quantification of DNA damage, expressed as comet tail moment, under the indicated conditions. Data reflect the extent of double-strand DNA breaks in response to treatment.

Article Snippet: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

Techniques: Labeling, Isolation, Single Cell Gel Electrophoresis, 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: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

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) PC-9 and HCC827 cells were treated with 5 nM osimertinib (Osi) for the indicated time points, and nuclear extracts were prepared. TNNT3 protein levels were analyzed by western blotting; ACTB and Histone H3 were used as loading controls. Data shown are representative of three independent experiments. (B) PC-9 and HCC827 cells were transfected with control or TNNT3 siRNA 24 hours prior to Osi treatment. Protein levels of TNNT3 and RRM2B were analyzed by western blotting, with ACTB as a loading control. Results are representative of three independent experiments. (C) Relative RRM2B mRNA expression was quantified by RT-qPCR in TNNT3-depleted cells following transfection with TNNT3 siRNA and treatment with Osimertinib. Transcript levels were normalized to GAPDH. (D) Representative images from colony formation assays in PC-9 and HCC827 cells transfected with control siRNA, RRM2B siRNA, or FLAG-tagged RRM2B, and treated with varying concentrations of Osi. (E) Quantification of colony density in treated cells from panel C. Data reflect the impact of RRM2B modulation on cellular resistance to Osi.

Article Snippet: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

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

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: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

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: The PC-9 and HCC827 cell lines were obtained from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ mL), and streptomycin (100 μg/mL), at 37 °C and 5% CO2.

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