{"paper_id":"2df02849-aac1-41fa-bdf2-6712ea3ecf8e","body_text":"Title: Lung cancer-enriched p53 mutants occupy canonical p53 target genes without activating \ntranscription, revealing a distinct loss-of-function behavior \n \nMason A. Tracewell1, Hailey N. Shankle1, †, Samantha M. Barnada1, †, Khushali S. Vyas1, Kevin \nM. Kim1, Theodhora Qyshkollari1, Jonathan E. Karlin1, Julie A. Barta2, and Steven B. McMahon1,*. \n \n1Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas \nJefferson University, Philadelphia, Pennsylvania. \n2 Division of Pulmonary and Critical Care Medicine , Sidney Kimmel Medical College, Thomas \nJefferson University, Philadelphia, Pennsylvania. \n \n† These authors have contributed equally to this work. \n \n*Corresponding author: Steven B. McMahon, Department of Biochemistry and Molecular Biology, \nSidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania. Email: \nsteven.mcmahon@jefferson.edu \n \nAbstract \nLung cancer is the most common cause of cancer-related death in the U.S. and globally. \nCigarette smoking remains the leading risk factor for lung cancer, in part by inducing loss -of-\nfunction mutations in tumor suppressor genes, including TP53. While most cancers share a set \nof common “hotspot” mutations in p53, lung cancer exhibits an additional, distinct cluster of \nhotspot mutations. This cluster is typified by the missense mutations TP53:p.V157F and \nTP53:p.R158L. While canonical hotspot mutations cause br oad misfolding of p53 or eliminate \nspecific DNA contact residues, mechanistic studies of the lung cancer mutants reported here \ndemonstrate that they retain the ability to bind the same genomic sites as wild-type p53. Despite \nactively binding to traditional p53 target genes, the lung cancer mutants are defective in activating \ntranscription. To our knowledge, this represents the first demonstration of functional inactivation \nof the p53 tumor suppressor at a point after DNA binding, but prior to  target gene ac tivation. \nRelevant to the sequential inactivation of each p53 allele during cancer progression, the lung \ncancer mutants block the activity of a wild -type p53 allele when co -expressed in a dominant \nnegative manner . Identification of this loss -of-function mechanism has key implications for \ntherapeutic strategies aimed at restoring p53 function in lung cancer.  \n \nIntroduction \nFunctional inactivation of the p53 tumor suppressor protein is an essential step in the \nprogression of most human cancers  [1-3]. Distinct from most tumor suppressors, inactivation of \np53 typically involves retention of the entire TP53 gene in cancer cells  [4, 5]. Instead of gene \ndeletion, single amino acid alterations in the p53 protein are introduced as a consequence of point \nmutations in the underlying locus [6]. This results in tumor cells producing a full-length p53 protein \nthat is non-functional due to the inactivating properties of a single missense mutation [7]. \nAs a sequence -specific transcription factor, wild-type p53 function relies on a highly \nstructured central domain in which several amino acids make direct hydrogen bonds with DNA  \n[8-10]. Many of the DNA contact residues in p53 are hotspots for mutation in cancer, presumably \nbecause alterations at these sites prevent the protein from binding to DNA  [10]. Additional \nmutational hotspots in p53 occur at amino acids that trigger protein misfolding, leading to a loss \nof DNA binding by p53 and a defect in the  transcription of target genes related to key cellular \nprocesses such as cell cycle control, DNA damage repair, and apoptosis [11-13]. These \nobservations have led to a decades -long search for strategies to “correct” the function of the \nmutant p53 protein in human cancer  [14]. At the biochemical level, these reactivation strategies \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nhave focused on refolding p53 into a conformation that restores sequence-specific DNA binding, \nwith numerous clinical trials based on this strategy [15, 16]. \nWe report here that human lung cancer contains a set of missense mutations in the p53 \nDNA-binding domain that debilitate p53 function by a distinct mechanism.  These lung cancer \nmutations do not impair the ability of p53 to bind its normal sites in the genome and would not \nrespond to current reactivation strategies. Instead, these lung cancer-enriched mutations impair \nthe ability of p53 to induce the transcription of downstream target genes in a non-productive DNA \nbinding manner. The identification of a uniq ue biochemical mode of p53 inactivation has broad \nimplications for the efficacy of reactivation strategies as a therapeutic approach in human cancer. \n \nMaterials and Methods: \n \nCell Culture \nHuman NCI-H460 (ATCC HTB -177), NCI-H1299 (ATCC CRL -5083), and NCI-H2087 (ATCC \nCRL-5922) cells were purchased from the American Type Culture Collection (ATCC, Manassas, \nVA). Following the receipt of the cell lines, aliquots of passages 2–5 were frozen in liquid nitrogen. \nNew aliquots were thawed every 4–6 months for use in experiments. NCI-H460 and NCI-H2087, \ncells were maintained in RPMI 1640 (Corning) supplemented with 10% Benchmark fetal bovine \nserum ( 100-106, Gemini) or 10% tetracycline  (TET) negative fetal bovine serum ( 100-800, \nGeminiBio) for the TET -inducible system, 1X Glutamine (100X, A2916801, Gibco) under  37°C \nand 5% CO2 conditions. NCI-H1299 cells were maintained in Dulbecco’s Modified Eagle Medium \n(Corning) supplemented with 10% tetracycline negative fetal bovine serum (100-800, GeminiBio), \n1X Glutamine (100X, A2916801, Gibco) under 37°C and 5% CO2 conditions. Cells were passaged \ntwice weekly, and Mycoplasma contamination was monitored by a PCR detection kit (30-1012K, \nATCC). \nTetracycline-inducible p53-expressing stable cell lines were generated in NCI-H1299 and \nNCI-H460 cells via lentiviral infection using ViraPower HiPerform Lentiviral Expression System \n(Invitrogen) [17]. p53 expression vectors were generated in pLenti6.3/V5 -DEST cloning vectors \n(Invitrogen). Expression vectors encoding for Val158 to F (V157F) and Arg158 to L (R158L) p53 \nmutants (V157F; R158L) were generated using the QuikChange II Site-Directed Mutagenesis kit \n(Agilent). Expression of wild-type ( WT) p53, V157F p53, and R158L  p53 were induced  with \ntetracycline (T7660, Millipore Sigma).  \n \nCellular Images \nCellular images were captured by the EVOS XL Core Imaging System (AMEX1000, Invitrogen by \nThermo Fisher Scientific).  \n \nCamptothecin (CPT) Treatment \nCamptothecin (J62523.03, Thermo Fisher Scientific) was used at 2 M to investigate p53 \nresponse to DNA damage. For TET+CPT conditions, cells were treated with TET for 16 hours to \nallow for induction of p53, and then CPT was added for 6, 24, or 48 hours. \n \nImmunoblotting \nCells were harvested and lysed in a Nonidet P -40-based whole-cell lysis buffer supplemented \nwith a protease inhibitor cocktail ( P8340, Sigma-Aldrich). Lysate concentration was determined \nusing bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against p53 \n(sc-126, Santa Cruz Biotechnology), GAPDH (#5174, Cell Signaling Technology), and Caspase-\n3 (#9662, Cell Signaling Technology). All the antibodies were used 1:1000 in TBS-T.  \n \nCellTiter-Glo 2.0 Cell Viability Assay \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nThe CellTiter-Glo 2.0 cell viability assay (G9242, Promega) assessed cell viability by quantifying \nthe amount of ATP, indicating metabolically active cells. 10-000-25,000 cells were plated per well \ninto a 96 -well plate (781971, BrandTech) and allowed to adhere overnight.  Per \nexperiment, cells were treated with the indicated treatments and time  respectively. The media \nwas removed, and 100L of fresh media was added per well. 100L per well of CellTiter-Glo 2.0 \nreagent was added, and the contents were mixed by a shaker. The plate was incubated at room \ntemperature for 10 minutes.  Luminescence wa s determined using a PolarStar Optima plate \nreader (BMG LabTech) at room temperature. \n \nCell Quantification \nCells were collected with Accutase (07922, Innovative Cell Technologies) and spun down at \n1300rpm for 5 minutes. Cells were resuspended with media and 25L per sample was diluted 1:1 \nwith trypan blue (2680430, Invitrogen). Cells were quantified using Countess 3 (Invitrogen). \n \nCell Cycle Analysis by Flow Cytometry \nCells were collected with Accutase and spun down at 1300rpm for 5 minutes. Cells were washed \ntwo times with phosphate buffer saline (PBS) (10010-023, Gibco), and then resuspended in PBS. \n500,000 cells were placed in a 15mL conical tube with a total volume of 300 L PBS. Cells were \nfixed by adding 200 proof ethanol dropwise, while vortexing the cells to prevent clumping, to a \nfinal concentration of 70% ethanol. Cells were incubated on ice for 30 minutes with vortexing \nevery 5 minutes. Cells were spun down at 1600rpm for 5 minutes and supernatant was aspirated. \nCells were resuspended in 50 L RNAse solution (5 g RNAse in 50 L PBS, R1253, Thermo \nFisher Scientific) and 100 L propidium iodide (PI) staining solution (556463, BD Biosciences). \nCells were stained for 15 minutes in the dark at room temperature. 350 L of PBS was added to \nthe cell suspension, and then the solution was pipetted into a FACS tube with a cell strainer \n(352235, Falcon). The data was collected by flow cytometry on a Cyto FLEX (Beckman Coulter) \nusing CytExpert (v1.2 Beckman Coulter). Data was analyzed with FlowJo (10.10.0) to perform the \ncell cycle analysis. Reported data are from a population of 30,000 cells. \n \nApoptosis Analysis by Flow Cytometry \nApoptosis was measured using the FITC annexin V apoptosis detection kit (556547, BD \nPharmingen) following manufacturer’s instructions. Cells were collected with Accutase and spun \ndown at 1300rpm for 5 minutes. Cells were washed two times with PBS, and then resuspended \nin 1X binding buffer. 5L of FITC annexin V and 5L of PI staining solution was added to 100L \nof cell suspension containing 100,000 cells. Samples were incubated at room temperature for 15 \nminutes in the dark. 400 L of 1X binding buffer was added to each sample. Samples were \npipetted into a FACS tube with a cell strainer. The data was collected by flow cytometry on a \nCytoflex using Cytexpert. The data was analyzed with FlowJo, and reported  data are from a \npopulation of 30,000 cells. \n \nClick-it Reaction \nH2087 cells were collected with trypsin (25053CI, Corning) and plated on chamber slides (80427, \nIbidi) to adhere for 24 hours at a confluency of ~70% . Cells were labelled with media containing \n5M 5 -ethynyl-2′-deoxyuridine (EdU, 7207, Tocris) for 1.5 hours, followed by 30 minutes of \nregular media. Cells were washed with PBS and then fixed for 15 minutes with 3.7% formaldehyde \n(252549, Sigma-Aldrich). Cells were washed with PBS and then permeabilized with 0.25% Triton \nX-100 (21568-0010, Acros Organics ) for 10 minutes.  Cells were washed and blocked with 1% \nBSA (BP1605-100, Thermo Fisher Scientific) for 10 minutes. Click-it reaction was performed for \n30 minutes in the dark using 0.1M copper sulfate (C1297, Sigma -Aldrich), 1mM Biotin Azide \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n(900891, Sigma-Aldrich), 0.1M Ascorbic Acid (255564, Sigma -Aldrich) and 0.1x PBS,  and then \nthe cells were washed with PBS. DNA biotinylation enabled proximity ligation assay. \n \nProximity Ligation Assay \nPerformed proximity ligation assay (PLA) following manufacturer’s protocol for Duolink In Situ \nDetection Reagents (DUO92008, Sigma -Aldrich). Cells were blocked for 10 minutes with 1x \nwestern blocking solution (11921673001, Roche) and 1.5% normal donkey serum (017-000-121, \nJackson ImmunoResearch). The following primary antibodies (one rabbit and one mouse per \ncondition) were added at a 1:1000 dilution: rabbit IGG (NI01, EMD Millipore), Anti -Biotin (rabbit, \nAB53494, Abcam), or p53 (mouse, sc-126, Santa Cruz Biotechnology). The samples were placed \nin a humidity chamber at 4C overnight and remained in the humidity chamber until mounting was \ncompleted. \nThe samples were washed with PBS for 15 minutes and blocked briefly with 1x western \nblocking solution. The samples were incubated with PLA probes diluted in 1x western blocking \nsolution for 1 hour at 37 C. The samples were washed with Buffer A, and the ligation step was \nperformed for 30 minutes at 37C. The samples were washed with Buffer A, and the amplification \nstep was performed for 1 hour and 40 minutes at 37 C. The slides were washed with PBS and \nstored in mounting medium with DAPI (50011, Ibidi). The slides were stored at 4C until imaging \nusing the DeltaVision Ultra widefield microscope system. \n \nLuciferase Reporter Assay \nThe pGL3 firefly luciferase reporter plasmids made for p21 (GAACATGTCCcAACATGTTg) and \nBAX (GGGCAgGCCCCGGGCTTGCTg) response elements have been described previously [18, \n19]. Cells were transfected with 1g of firefly luciferase reporter plasmid (p21 or BAX), 250ng of \np53 expression vectors (WT, V157F, R158L, or EV), and 200ng of pRL -TK Renilla luciferase \ncontrol reporter vector (E2241, Promega) using Lipofectamine 3000 ( L3000015, Invitrogen) \naccording to manufacturer’s instructions. Dual-luciferase reporter assay system kit (E1910, \nPromega) was used for data collection. Cells were harvested 24 hours post -transfection with \npassive lysis buffer. Luminescence of samples was detected by a GloMax® Explorer (3.2.3, \nPromega) and recorded in relative light units (RLU).  \n \nRNA-sequencing \nH1299 cells containing a TET-inducible plasmid for WT, V157F, or R158L p53 were treated with \nTET or TET+CPT as described above and collected. Total RNA was extracted using the RNeasy \nPlus Mini Kit (74134, Qiagen) following the manufacturer’s protocol. RNA was quantified, and the \nRNA integrity  number (RIN)  was verified using an  RNA ScreenTape® for Tapestation 4150 \n(v4.1.1, Agilent). All samples used for library preparation and subsequent sequencing had a RIN \nvalue above 8.0. Directional RNA libraries were pr epared using 1μg of total RNA as input and \nusing NEBNext® Poly(A) mRNA  Magnetic Isolation  Module (E7490L, New England Biolabs) , \nNEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina ® (E7760L, New England \nBiolabs), and NEBNext® Multiplex Oligos Dual Index Primers for Illumina (E7600S and E7780S, \nNew England Biolabs) according to the manufacturer’s instructions. Libraries were sequenced on \na NextSeq 2000 (Illumina) generating single-end 150 bp reads. \n \nRNA-sequencing Analysis \nFastQC (https://github.com/s-andrews/FastQC) was used for quality control of all raw fastq files \nand adapters were removed using TrimGalore! (https://github.com/FelixKrueger/TrimGalore). \nReads mapping to each gene in each sample were quantified to abundanc e using Kallisto [20]. \nAbundance was then converted to raw gene count via DESeq2 [21]. The raw gene count for each \nsample was used to determine differential gene expression (p -value < 0.05; FDR <5%) . \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nAbundance was used to determine transcript per million for data visualization. All statistical \nanalyses were performed using Kallisto v0.50.0, DESeq2 v1.40.2  [21], R v4.2.3 and Prism \n1v0.1.1. \n \nReverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis \nCells were collected and total RNA was extracted using the RNeasy Plus Mini Kit following \nmanufacture’s protocol. cDNA was created from 2 μg of RNA using the High -Capacity cDNA \nReverse Transcription Kit ( 4368814, Thermo Fisher  Scientific) following manufacturer’s \ninstructions. RT-qPCR was performed on the cDNA using the Fast SYBR TM Green Master Mix \n(4385612, Thermo Fisher Scientific) following manufacturer’s instructions on the Step One Plus \nReal Time -PCR system (v2.3 , Applied Biosystems ). mRNA levels between samples were \nnormalized to GAPDH transcript levels. Primer sequences are listed in Supplemental Table 1. \n \nChromatin Immunoprecipitation (ChIP) \nChIP was performed as previously described [22] with minor alterations. Approximately 15 million \ncells were cross-linked with 1% formaldehyde for 10 minutes rocking at room temperature, and \nthen quenched with 125mM glycine (BP381, Fisher Scientific) for 5 minutes. The cells were \nwashed twice with 1X PBS. Chromatin was extracted from the fixed cells as described in [23]. \nThe fixed cell pellet was lysed with ChIP lysis buffer 1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; \n1mM EDTA; 10% Glycerol; 0.5% Igepal CA-630; 0.25% Triton X-100), rocked for 10 minutes, and \nthen spun down at 2000xRCF for 5 minutes. The supernatant was aspirated, and the pellet was \nlysed with ChIP lysis buffer 2 (10 mM Tris -HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM \nEGTA), rocked for 5 minutes, and then spun down at 2000xRCF for 5 minutes. The supernatant \nwas aspirated, and the nuclei pellet was lysed with ChIP lysis buffer 3 (10 mM Tris-HCl, pH 8; \n100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na -Deoxycholate; 0.5% N-lauroylsarcosine). \nChromatin was sheared to an average base-pair length of 100-300 using the Q800R2 Sonicator \n(QSonica) kept at 4 °C with circulating chiller ( 4905-110, QSonica), with 50% amplitude, and a  \npulse of 20 seconds on and 20 seconds off, for 20 minutes of sonication on time. 80 μL TE with \n1% SDS was added to 20μL for each sonicated sample. 2μg/μL RNase and 2μg/μL Proteinase K \n(25530-015, Thermo Fisher Scientific) were added to each sample and incubated for 1 hour at \n65°C. Samples were purified with the Monarch® PCR & DNA Cleanup Kit (T1030L, New England \nBiolabs) following manufacture’s protocol. The chromatin shearing efficiency of the samples was \nchecked by D1000 ScreenTape® Tapestation 4150 (v4.1.1, Agilent). \nThe remainder of the sonicated samples kept on ice, had Triton X -100 ( 21568-0010, \nThermo Fisher Scientific) added to a final concentration of 1%. The samples were centrifuged at \n20,000xRCF for 10 minutes at 4°C. The supernatant was collected, and 2% input samples were \ncollected and stored at -20°C. The remaining sheared chromatin was diluted 1:3 in ChIP lysis \nbuffer 3 with 1% Triton X-100. The sheared chromatin was incubated with 15μg antibodies (p53, \nAHO0152, Thermo Fisher Scientific; mIGG, sc -2025, Santa Cruz Biotechnology) and \nDynabeadsTM Protein G beads (10004D, Thermo Fisher Scientific) rotating overnight at 4°C. The \nbeads were washed three times with ChIP wash buffer (0.1% SDS; 1% Triton X -100; 10mM \nEDTA; 150mM NaCl; 20mM Tris-HCl pH 8.0). The beads were washed a final time with final ChIP \nwash buffer (0.1% SDS; 1% Triton X -100; 10mM EDTA; 500mM NaCl; 20mM Tris -HCl pH 8.0). \nThe beads were resuspended in 1X TE containing 1% SDS and incubated at 65°C for  10 min, \nand this was repeated twice to elute all th e immunocomplexes. The inputs were diluted to the \nsame volume as the ChIP samples, and the inputs and ChIP samples were incubated overnight \nat 65°C to reverse cross -linking. The samples were digested with Proteinase K (0.5 μg/μL) for 1 \nhour at 65°C, and then the DNA was purified using the ChIP DNA Clean & ConcentratorTM (D5205, \nZymo Research). \n \nChIP-qPCR \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nAfter the samples were purified by the ChIP DNA Clean & Concentrator TM kit, the samples were \nresuspended with water to a total volume of 100 μL. Samples were analyzed by qPCR with 2 μL \nof each ChIP sample per well in triplicate for each ChIP -qPCR primer, ChIP -qPCR primer \nsequences are found in Supplemental Table 2. \n \nChIP-sequencing \nAfter the samples were purified by the ChIP DNA Clean & Concentrator TM kit, the samples were \nquantified with 1X dsDNA HS Assay Kit (Q33231, Thermo Fisher Scientific) on the Qubit 4 \n(Thermo Fisher Scientific). Barcoded libraries were prepared with NEBNext ® UltraTM II DNA \nLibrary Prep Kit for Illumina® using NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers \nSet 1) (E7600S, New England BioLabs). Libraries were sequenced on a NextSeq 2000 (Illumina) \ngenerating single-end ~138 bp reads.  \n \nChIP-sequencing Analysis \nFastQC (https://github.com/s-andrews/FastQC) was used for quality control of all raw fastq files \nand adapters were removed using TrimGalore! (https://github.com/FelixKrueger/TrimGalore). The \nsequences were then aligned to  human reference genome  hg19 using the Burrows -Wheeler \nAlignment tool with the MEM algorithm [24]. Aligned reads were filtered with a mapping quality \ngreater than 10 (MAPQ >10) and PCR duplicates were removed. Peaks were called using MACS2 \n[25] with default parameters and FDR < 5%. All statistical analyses were performed using \nBEDTools [26], deepTools [27], R v4.2.3, and Prism 1v0.1.1. ChIP peaks were visualized in \nIntegrative Genomics Viewer [28] on genome build hg19. Only one representative ChIP replicate \nwas used for data visualization purposes. \n \nPublicly Accessible ChIP-sequencing \nFor the publicly accessible data used, FASTQ files for each sample were downloaded from GEO: \nGSE238181 [35] and GSE59176 [36]. FastQC (https://github.com/s-andrews/FastQC) was used \nfor quality control of all raw fastq files and adapters were removed using TrimGalore! \n(https://github.com/FelixKrueger/TrimGalore). The sequences were then aligned to  human \nreference genome hg19 using the Burrows-Wheeler Alignment tool with the MEM algorithm [24]. \nAligned reads were filtered with a mapping quality greater than 10 (MAPQ >10) and PCR \nduplicates were removed. Bigwigs were created for visualization in Integrative Genomics Viewer \n[28]. \n \nMotif Analysis \nFasta files for the regions of interest were produced using BEDTools [26]. Motif analysis of all p53 \nbound regions was performed using MEME-ChIP [29], The MEME Suite [30]. The motif discovery \nand enrichment mode were performed in classic mode using Human motifs. The number of motifs \ndiscovered was set to 10 with a maximum motif width set to 20. All other parameters were set to \ndefault.  \n \np53 Protein Purification \nUntagged, thermostable, N-terminally truncated human p53 (residues 94 -393) and its Val158 to \nF (V157F) and Arg158 to L (R158L) mutants were expressed in BL21 -CodonPlus (DE3)-RIL E. \ncoli (Stratagene). 6L of bacteria cell cultures were grown at 37°C until th ey reached an \nOD600 equal to 0.5–0.8. Cells were then shifted to 18°C. Protein expression was induced by the \naddition of 0.2mM isopropyl -β-D-thiogalactoside (IPTG). Cells were harvested by centrifugation \n(20 minutes at 7500 × g and 4°C), resuspended in cell lysis buffer [20 mM bis-tris propane (BTP, \npH 6.8), 200mM NaCl, 2mM DTT, and 0.5mM Tris (2 -carboxyethyl) phosphine hydrochloride \n(TCEP)] and homogenized by an Emulsiflex C5 cell disruptor (Avestin). Cell lysates were \ncentrifuged at 35,000 ×  g for 30 min utes at 4°C  and cleared cell extracts were loaded onto a \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nHiTrap SP cation exchange column (GE Healthcare) pre -equilibrated with a lysis buffer. The \nproteins were eluted using a linear gradient of NaCl from 200mM to 1M concentration. Eluted \nproteins were then loaded onto a HiTrap Q anion exchange column (GE Healt hcare) to remove \nresidual nucleic acids, and the protein-containing flow-through was collected. Finally, the pooled \nprotein was concentrated and loaded onto a HiLoad Superdex 200 16/60 size exclusion column \n(GE Healthcare) and eluted in a buffer containing 150mM NaCl, 20mM BTP (pH 6.8), and 0.5mM \nTCEP. Final protein concentrations were estimated by measuring absorbance at 280nm. \n \nColloidal Blue Staining \nColloidal blue staining was performed with a colloidal blue staining kit (LC6025, Invitrogen) \nfollowing manufacturer protocol for Novex® Tris-glycine gels. Samples were loaded into the gel \nin addition to either Benchmark Protein Ladder (110747012, Thermo Fisher Scientific) or \nPrecision Plus Protein Dual Color Standards  (#1610374, Bio -Rad) for a ladder. The \nelectrophoresis process was conducted at 100V. The gel was stained for 3 hours with colloidal \nblue solution and then destained for 8 hours with deionized water before being imaged.  \n \nSurface Plasmon Resonance (SPR) \nSPR experiments were performed using a Biacore X100 instrument (GE Healthcare) at 25 C \nusing streptavidin coated sensor chips (Sensor chip SA, Biacore  X100; GE Healthcare). Sensor \nchips were primed with running buffer (20mM BTP (pH 6.8); 200mM NaCl; 50µg/mL BSA; 0.005% \nTween-20; 0.5mM TCEP) until resonance units (RUs) on all flow cells were stable. Biotinylated \ndsDNAs were resuspended at a final concentration of 10nM in running buffer and immobilized on \nthe SA sensor chip by injecting at a flow rate of 10µL/min until RUs reached 250. Each experiment \nutilized two flow cells; DNA was immobilized on one flow cell, and the other flow cell served as a \nreference. DNA sequences listed in Supplemental Table 3. \nTo determine p53 DNA binding affinity constants, p53 protein solutions (1, 10, 25, 50, 100, \nand 200nM protein concentrations, diluted in running buffer) were delivered to the flow cell with \nimmobilized dsDNA and reference cell at a flow rate of 30µL/min for 300 seconds to measure \nassociation, followed by dissociation where only running buffer was flowed at 30µL/min for 240 \nseconds. Between experiments, the sensor chip surface was regenerated by two 120 -second \ninjections of running buffer containing 500mM Na Cl at 30µL/min to remove any remaining p53 \nprotein from the dsDNA. \nKinetic parameters (association (ka), dissociation (kd), and affinity (KD)) were obtained \nusing BIAevaluation software 2.1 (GE Healthcare). First, RUs collected for the flow cell containing \nimmobilized dsDNA were subtracted by RUs obtained from the reference cell. Then, sensorgrams \nwere globally fitted for all p53 protein concentrations to the Langmuir binding model of simple 1:1 \nbimolecular interaction. Goodness of fit was evaluated based on the χ2 value and visual \ninspection.  \n \nStatistical Analysis \nData are expressed as mean standard deviation. All statistical analysis was performed using \nGraphPad Prism 10 Version 10.3.1 (464) unless stated otherwise. The data are a representation \nof three independent biological replicates unless stated otherwise.  \n \nResults \n \nThe V157F p53 mutant binds DNA in endogenously expressing cells \nThe V157F p53 mutant regulates a novel transcriptome [31, 32]. However, the mechanism \nbehind this regulation is unknown . To determine whether the V157F p53 mutant binds DNA to \nperform gain-of-function activities, proximity ligation assay (PLA) was performed. The human lung \nadenocarcinoma line H2087 is homozygous for the V157F p53 mutation. In these cells, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nsignificantly increased proximity between p53 and DNA was observed (Figure 1A, Supplemental \nFigure 1). In fact,  quantification of these images demonstrated that the  H2087 experimental \ncondition (p53 + Biotin)  had significantly higher PLA signal compared to the three control \nconditions (Figure 1B). These results suggest that the V157F p53 mutant is in close proximity to \nDNA and may be bound to DNA. \nLung cancer cells accumulate p53 mutations in a small cluster of amino acids (V157, \nR158, A159) that are not commonly mutated in other forms of cancer. Given their physical \nproximity, it was of interest to assess whether other missense mutations at this cluster have similar \nbiological effects. V157F and R158L missense mutations of p53 are the most common alterations \nin this lung cancer cluster, and we previously showed that these two missense mutations have a \nsimilar impact on oxidative stress response [32, 33]. ChIP-seq was performed to determine where \nV157F and R158L  mutant p53 may be bound  in the genome . H2087 (V157F  p53), H66 1 \n(homozygous for the R158L p53  mutant allele ), and H460 (WT  p53) cells were treated with  \ncamptothecin (CPT). CPT binds and inhibits topoisomerase I , causing DNA damage during S-\nphase of the cell cycle and leading to activation of WT p53 [34]. V157F p53 (H2087) and R158L \np53 (H661) were bound at many of the same  sites as WT p53 (H460), including canonical WT \np53 target genes for both cell cycle arrest genes , such as p21 and RRM2B , and pro-apoptotic \ngenes, such as BAX and PUMA (Figure 1C). In H460 (WT p53) cells, 282 peaks were identified \nat promoters. Of these 282 promoters bound by WT p53, 241 were bound by V157F (H2087) and \n223 were bound by R158L (H661). While V157F and R158L p53 mutants are bound at the same \nlocation as WT p53, they have less signal intensity at these sites compared to WT p53.  \nTwo p ublicly available ChIP -seq datasets (GSE238181 [35] and GSE59176 [36 ]) that \ninclude data from  WT p53 cell lines  (MOLM-13 and  MCF7) and mutant p53 cell lines (NB -\n4:R248Q, Mono -Mac-6:R273H, HCC70 :R248Q, BT549 :R249S, MDA -MB-468:R273H) were \nutilized to demonstrate that mutant p53 variants are not commonly bound at WT p53 loci. Of these \npublicly available ChIP-seq data, WT p53 was bound at the same loci as  the WT p53 (H460), \nV157F p53 (H2087), and R158L p53 (H661) (Figure 1C). The five different p53 mutants from the \npublicly available ChIP-seq data had no binding at WT p53 target genes (Figure 1C). The results \nfrom a motif analysis for WT p53 (H460), V157F p53 (H2087), and R158L p53 (H661) are shown, \nand all three have a strikingly similar positional weight matrix for the p53 canonical binding \nsequence (Figure 1D-F). These results suggest that the V157F and R158L p53 mutants bind at \nWT p53 target genes, which is unique for p53 mutants. \nTo verify the finding that V157F p53 mutant binds at WT p53 target genes from the ChIP-\nseq analysis, ChIP -qPCR was also performed on H2087  (V157F) and H460 (WT) cells. The \nbinding sites of WT p53 at target genes p21, RRM2B, BAX, and PUMA were used to verify that \nmutant p53 binds to the same genomic region as WT p53 (Figure 1G-J). V157F p53 (H2087) was \nbound at canonical WT p53 target genes at levels similar to WT p53 with CPT treatment (Figure \n1G-J). CPT treatment increased WT p53  (H460) binding at canonical WT p53 target genes  as \nexpected, and V157F p53 (H2087) was bound at similar levels with or without CPT stimulus  \n(Figure 1G-J). These results suggest that the V157F p53 mutant is able to bind at canonical WT \np53 target genes, which is an unprecedented finding for a p53 mutant. \n \nV157F and R158L p53 mutants bind DNA similar to WT p53 in vitro \nTo further support the PLA and ChIP data, t he interaction kinetics between p53 and \ndifferent DNA sequences w ere studied using a surface plasmon resonance (SPR) approach. A \nthermostable, N-truncated version of p53 was used for these experiments, previously shown to \nretain the same DNA binding ability as WT p53 protein (Figure 2A) [18, 37, 38]. WT, V157F, and \nR158L versions of p53 were purified for use with SPR (Figure 2B). Salt concentrations of 200mM \nand 275mM were utilized as they have been previously shown in published works to greatly \ndecrease non-specific binding [18]. WT p53 target genes p21 and BAX, along with a scrambled \nDNA sequence as a control, were used to test the binding affinity for the p53 variants at salt \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nconcentrations of 200mM and 275mM, and protein concentrations ranging from 1nM to 200nM \n(Figure 2C, Supplemental Figure 2). The interaction between the p21 response element sequence \nand either of the p53 mutants resulted in a slightly lower maximum resonance unit (RU) value \nthan that of the interaction between the p21 response element and WT p53 (Figure 2D). There \nwas no difference in binding interaction between the three p53 variants and the BAX response \nelement (Figure 2E). All three p53 variants did not have a strong binding affinity for the scrambled \nsequence as expected (Figure 2F). The DNA binding affinity for each of the p53 variants was \ndetermined and expressed through the dissociation constant KD (Table 1). The KD for p53 \nmutants at WT p53 target gene response elements are strikingly similar to the KD of WT p53.  \nThe binding interactions between the three p53 variants and WT p53 target genes AEN, \nPLK3, and PUMA were also tested, and the data suggests that the p53 mutants bind similarly to \nWT p53 at these genes (Supplemental Figure 3A-B, Supplemental Table 4). This is evidence that \nthe mutant protein can bind WT p53 target gene sequences but raises the question as to whether \nthey can form tetramers. This was tested by using a crosslinking agent, glutaraldehyde, and \nperforming gel electrophoresis to determine whether tetramers are forming for each purified p53 \nprotein. The WT, V157F, and R158L p53 proteins can form dimers and tetramers, indicated by \nthe bands at 2x and 4x the purified protein molecular weight upon addition of  glutaraldehyde \n(Supplemental Figure 4). These results suggest that V157F and R158L p53 mutants  can \ntetramerize and interact with WT p53 response elements similarly to WT p53.  \n \nV157F and R158L p53 mutants bind DNA when expressed in p53-null lung cancer cells \nThe results suggest that endogenous V157F p53 (H2087) and R158L (H661) are able to \nbind to DNA. However, to determine whether this observation was cell type-specific property, we \nutilized a H1299 tetracycline (TET)-inducible expression system to compare the DNA-binding \naffinity of mutant and WT p53 in a p53-null background. The benefits of this system allow for the \nprotein to be expressed at similar levels and in the same genetic background, thus removing most \ngenomic variability for comparing binding affinities . A representative immunoblot of whole -cell \nlysate was used to confirm TET-induction of p53 in the H1299 TET-system (Figure 3A). Utilizing \nChIP-qPCR, the binding sites of WT p53 at target genes p21 and BAX were used to verify that \nmutant p53 binds to the same genomic region as WT p53 (Figure 3B). All conditions were found \nto bind DNA significantly over the IgG control for p21 (Figure 3B). WT, V157F, and R158L were \nall found to bind significantly over IgG control only in the CPT-treated conditions for BAX (Figure \n3B). These results suggest that the V157F and R158L p53 mutants are able to bind a t some \ndegree to WT p53 target genes in the H1299 TET-system. \nTo further investigate whether mutant p53 can bind DNA at the same areas as WT p53,  \nChIP-seq was performed on samples containing either TET-inducible WT p53, V157F p53, or \nR158L p53 induced with TET, and some conditions were additionally treated with CPT to induce \nDNA damage for robust p53 activity . ChIP-seq peaks for WT p53, V157F p53, and R158L p53 \nare shown with and without CPT treatment at WT p53 target genes p21, PLK3, BAX, and PUMA \n(Figure 3C). V157F p53 is bound at representative WT p53 target genes similarly to WT p53, and \nR158L p53 is clearly bound at WT p53 target genes but to a lesser extent than WT p53, indicated \nby a lower signal intensity (Figure 3C). The motif analysis for the CPT-treated conditions indicated \nthat WT p53, V157F p53, and R158L p53 all have a similar binding preference for the p53 \ncanonical binding sequence (Figure 3D-F). The KEGG p53 signaling pathway (hsa04115) was \nutilized to determine the overlap of WT p53 signaling genes identified as bound between WT p53, \nV157F p53, and R158L p53 from the ChIP-seq analysis [39-41]. A majority of the genes in the \nKEGG p53 signaling pathway were identified as bound in the V157F p53 and WT p53 groups in \nthe presence or absence of CPT (Figure 3G-H). R158L p53 was found to be bound at a smaller \nsubset of the p53 signaling genes compared to WT p53 or V157 F p53 (Figure 3G -H). These \nresults suggest that V157F  p53 and R158L p53  expressed in a TET -inducible system bind to \ncanonical WT p53 target genes at levels comparable to  WT p53 under endogenous conditions. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nThese data support that this binding is a biological feature of these lung-enriched p53 mutants \nrather than a cell type-specific property. \n \nV157F and R158L p53 mutants fail to induce WT p53 target genes \nTo assess whether the p53 mutants (V157F or R158L) are able to induce the expression \nof WT p53 target genes that the mutant protein binds to, RNA -seq was conducted utilizing the \nH1299 TET-expression system. H1299 cells conditionally expressing either WT, V157F, or R158L \np53 were treated with CPT to induce DNA damage for robust p53 activation or left untreated. \nRNA-seq analysis identified 2286 differentially expressed genes (p < 0.05; FDR < 5%; log2FC +/- \n1.5) in the V157F p53 CPT -treated condition compared to the WT p53 CPT -treated condition \n(Figure 4A). Additionally, 2797 genes were differentially expressed (p < 0.05; FDR < 5%; log2FC \n+/- 1.5) in the R158L p53 CPT-treated condition compared to the WT p53 CPT-treated condition \n(Figure 4B). Canonical WT p53 target genes were a mong both sets of differentially expressed \ngenes. In the mutant p53 conditions,  p21, PLK3, BAX, and PUMA were significantly \ndownregulated in both the TET and TET+CPT-treated groups compared to the corresponding WT \np53 condition, with the exception of PUMA for the R158L CPT -treated group, which was not \nsignificantly different from the WT p53 CPT-treated group (Figure 4C). These data suggest that \nthe V157F or R158L p53 mutants are unable to induce expression of WT p53 target genes to a \nsimilar level of WT p53 even though they bind to the same target sequence. \nTo confirm the results from the RNA-seq that V157F and R158L p53 mutants are unable \nto express WT p53 target genes, RT-qPCR was performed utilizing the H1299 TET-expression \nsystem. TET expression of WT p53 alone or in combination with CPT treatment induces \nexpression of WT p53 target genes p21, PLK3, BAX, and PUMA (Figure 4D). TET expression of \nV157F p53 or R158L p53 alone or in combination with CPT treatment does not induce expression \nof WT p53 target genes p21, PLK3, BAX, and PUMA ( Figure 4D). CPT treatment alone without \nTET expression of p53 has little impact on the expression of WT p53 target genes as expected \n(Figure 4D). The expression of WT p53 target genes p21, PLK3, BAX, and PUMA was verified in \nendogenously expressing cell lines H460 (WT p53), H2087 (V157F p53), and H661 (R158L p53) \nutilizing RT-qPCR. Following CPT treatment, both mutant p53 cell lines had significantly lower \nexpression of WT p53 target genes than WT p53 expressing cells (H460)  (Supplemental Figure \n5). These data support the RNA-seq findings and suggest that the V157F and R158L p53 mutants \nare unable to induce functional expression of WT p53 target genes. \nA luciferase reporter assay was utilized to  further validate that these p53 mutants are \nunable to induce the expression of representative WT p53 cell cycle control and apoptotic target \ngenes, p21 and BAX , respectively. The same quantity of luciferase reporter plasmid and p53 \nexpression vector encoding either WT p53, V157F p53, R158L p53, or vector alone (EV) was \ntransfected into a H1299 p53 -null cell. The cells were lysed, and the lysate was immunoblotted \nfor p53 to show similar p53 levels across the samples (Figure 4E). WT p53 significantly induced \nthe expression of both p21 and BAX ( Figure 4F-G). The expression of either V157F or R158L \nmutant p53 does not significantly induce the expression of either p21 or BAX compared to empty \nvector control (Figure 4F-G). These results suggest that the V157F and R158L p53 mutants do \nnot induce the expression of canonical WT p53 cell cycle control and apoptotic target genes. \n \nV157F and R158L p53 mutants are defective for apoptosis induction \nWe sought t o determine whether the observed non -productive binding of these lung -\nenriched p53 mutants results in a biological consequence. WT p53 has been shown to cause cell \ncycle arrest and cell death by apoptosis when cells are treated with a DNA-damaging agent [42-\n44]. CPT was used as the DNA-damaging agent to trigger apoptosis and determine if mutant p53 \ncould induce apoptosis in the same capacity as WT p53. This was first assessed by a cell viability \nassay in the H1299 TET -expression system ( Figure 5A). In CPT -treated conditions, WT p53 \nexpression significantly reduced cell viability compared to either V157F or R158L p53 (Figure 5A). \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nLive cell numbers were also quantified , and under CPT-treated conditions, WT p53 exhibited a \nsignificantly lower live cell population compared to either V157F or R158L p53 , consistent with \nproductive apoptosis (Figure 5B). Flow cytometry with a nnexin V and propidium iodide (PI) \nstaining was also used to functionally measure apoptosis. The TET-only cell populations had \ncomparable levels of annexin V and PI staining via flow cytometry among the p53 samples (Figure \n5C). CPT-treated c ells expressing WT p53 had a rightward shift of the cell population to the \nsecond and third quadrants indicating an increase in PI-positive cells, annexin V-positive cells, or \ndouble positive cells (Figure 5C). The increase in annexin V-positive cells was drastically higher \nin CPT -treated cells expressing WT p53 compared to cells expressing V157F or R158L p53 \n(Figure 5C). These data suggest that V157F and R158L p53 induce apoptosis at a reduced \ncapacity compared to WT p53 following CPT treatment. \nTo determine whether mutant p53 ha s an impact on cell cycle arrest, DNA content was \nanalyzed in fixed cells by flow cytometry. The TET-only cell populations had similar percentages \nof cells in each cycle cell stage: Sub G1, G1, S, and G2/M (Figure 6A-B, Supplemental Table 5). \nAfter treatment of CPT for 24 hours, cells expressing WT p53 had a drastic increase in cells with \nSub G1 and G1 DNA content compared to cells expressing V157F or R158L p53 (Figure 6A-B, \nSupplemental Table 5). The cells expressing V157F or R158L p53 with treatment of CPT for 24 \nhours had a substantial increase in cells with S -phase DNA content and appear ed to be in S -\nphase cell cycle arrest (Figure 6A-B, Supplemental Table 5 ). After treatment with CPT for 48 \nhours, cells with V157F or R158L p53 had an increase in cells with Sub G1 and G2/M DNA content \ncompared to the 24-hour CPT-treated conditions (Figure 6A-B, Supplemental Table 5). However, \nthe 48-hour CPT-treated cells expressing WT p53 had the most cells with Sub G1 DNA content, \nindicating a higher percentage of cells were apoptotic compared to cells with  V157F or R158L \np53 (Figure 6A-B, Supplemental Table 5). Cell imaging studies further support the flow cytometry \ndata, where cells expressing V157F or R158L p53 undergo cell cycle arrest, and cells expressing \nWT p53 exhibit increased apoptosis  (Supplemental Figure 6A). We next investigated whether \nmutant p53 exhibited a gain-of-function contributing to increased cell cycle arrest. However, cell \ncycle arrest was CPT-dependent and not mutant p53-dependent, indicating that CPT induces cell \ncycle arrest independently of p53 status  (Supplemental Figure 6B). In contrast, CPT induced \napoptosis specifically in WT p53-expressing cells, whereas mutant p53-expressing cells remained \narrested and did not undergo apoptosis to the same extent as WT p53 cells. These data indicate \nthat V157F and R158L p53 exhibit a reduced ability to promote CPT-induced apoptosis compared \nto WT p53, consistent with a loss-of-function rather than a gain-of-function phenotype. \n \nV157F and R158L p53 mutants exhibit a dominant-negative phenotype \nIt was determined that the V157F and R158L p53 mutants participate in non-productive \nDNA binding at canonical WT p53 target genes . Considering a  dominant-negative (DN) \nphenotype is common amongst p53 mutants, it raises the question of whether these p53 mutants \nmay impact WT p53 function through a DN effect when co -expressed. To assess whether these \np53 mutants exert a DN phenotype on WT p53 , the H1299 (WT p53) system was utilized. WT \np53 was expressed using TET , and either mutant p53 or empty vector w ere expressed via lipid \ntransfection ( Figure 7A). A dual-luciferase reporter assay  was performed as a readout for \ntranscription of the p21 and BAX genes. The presence of V157F, R158L, or EV alone showed no \nsignificant increase in transcription signal for both p21 and BAX (Figure 7B, 7C). WT p53 induction \nvia TET resulted in a significant increase in transcription of both p21 and BAX in the EV condition \nas expected  (Figure 7B-C). Co-expression of either V157F or R158L p53 mutant with TET -\ninduced WT p53 resulted in a significant reduction in transcription for both p21 and BAX genes \n(Figure 7B-C). To further support this observation, expression of WT p53 target genes p21, PLK3, \nBAX, and PUMA were measured by RT-qPCR (Figure 7D-G). Co-expression of either V157F or \nR158L p53 mutant with TET -induced WT p53 also resulted in a significant reduc tion in  \ntranscription for each WT p53 target gene  (Figure 7D-G). These data suggest that V157F and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nR158L mutant p53 both exert a DN phenotype on WT p53 when co -expressed in a  H1299 \nbackground. \nTo assess whether TET-induced mutant p53 would exert a DN phenotype on \nendogenously expressed WT p53, we  utilized the H460 ( pl:V157F p53) TET-system that \nexpresses endogenous WT p53 and plasmid-encoded V157F p53 (pl:V157F p53) following TET \ntreatment. WT p53 and subsequent transcription of WT p53 target genes were induced with CPT \ntreatment, and expression of V157F p53 was induced by TET. Expression of WT p53 target genes \np21, PLK3, BAX, and PUMA were measured by RT -qPCR (Figure 7H-K). Each WT p53 target \ngene had significantly increased expression with CPT treatment in the 0 M TET condition with \nno presence of the V157F p53 mutant (Figure 7H-K). Co-expression of V157F mutant p53 in the \n0.25M and 1M TET conditions caused a significant reduction in transcription signal of each of \nthe WT p53 target genes (Figure 7H-K). The biological impact of the DN phenotype was further \nassessed by immunoblotting for the apoptosis marker cleaved caspase-3 (CC3) [45] (Figure 7L). \nCo-expression of V157F mutant p53 in the 0.25 M and 1 M TET conditions reduced protein \nlevels of CC3 compared to CPT treatment alone  (Figure 7L). The biological impact was also \nassessed using a cell viability assay. Co -expression of V157F mutant p53 with CPT treatment \nsignificantly increased cell viability compared to CPT treatment alone in the H460 (pl:V157F p53) \nTET-system (Figure 7M). In the H460 (pl:WT p53) TET-system, expression of TET-induced WT \np53 had no impact on cell viability compared to CPT treatment alone (Figure 7N). Taken together, \nthese data show that the V157F p53 mutant and R158L p53 mutant have DN phenotypes when \nco-expressed with WT p53. \n \nDiscussion \n \nLoss of p53 function is a near obligatory step in the development and progression of \nhuman cancer [46]. Most frequently, p53 inactivation results from individual missense mutations \nthat cause either the generalized misfolding of the protein or the disruption of p53 binding to its \ncanonical genomic sites. Among the mutations that occur most commonly across all cancer types, \nmissense mutations at R175 and Y220 can  trigger protein misfolding, and mutations at DNA \ncontact residues, R248 and R273, eliminate sequence-specific DNA interactions [47]. In addition \nto these ubiquitous hotspot mutations, lung cancer contains an additional hotspot mutation cluster \nwhose activities whose impact is not well -understood. We report here that mutations within this \ncluster do not cause either p53 misfolding or disruption of its sequence -specific DNA binding \ncapacity. Instead, the function of these p53 mutants is inhibited at a discrete biochemical step \nsubsequent to target gene binding, but prior to transcriptional induction. The identification of non-\nproductive DNA binding by mutant p53 represents a significant advance in our understanding of \nthe biochemical mechanisms responsible for loss of tumor suppressor function in human cancer. \nNon-productive DNA binding by the lung -enriched V157F and R158L p53 mutants may \nrelate to these  mutants existing in two conformational states, mutant and “WT -like” [48]. The \nexistence of a “WT-like” state for each of these mutants may allow them to bind DNA at WT p53 \ntarget genes but not induce gene expression. It is unclear whether the mutant form prevents \ntransactivation of the target gene or if the “WT -like” state is unable to induce gene expression. \nTherapeutic strategies that force these p53 mutants into a “WT -like” conformation could be \nsufficient to restore WT function. Restoring WT p53 function is a central goal of many drug \ndevelopment programs targeting mutant p 53 [15]. The findings presented here may identify a \nnovel point of intervention for strategies aimed at reconfiguring specific p53 mutants into a “WT-\nlike” state. \nEarly in the development of most human cancers, incipient tumor cells often harbor one \nmutant p53 allele while retaining the other WT allele prior to loss of heterozygosity . As p53 \nfunctions as a tetramer, the co -expression of mutants and WT alleles result in the formation of \ninactive heterotetramers [49]. In some cases, even a single monomer carrying a conformational \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nor DNA-binding site mutation can inactivate the tetramer [50, 51]. It remains plausible that a DN \nphenotype could provide advantages in the earliest stages of tumorigenesis when mutant p53 \nand wild-type p53 are co -expressed, and help  drive the loss of heterozygosity [52, 53] . \nFurthermore, a DN effect has been shown to be the driving selection force for TP53 missense \nmutations in myeloid malignancies [54]. We postulate that the DN phenotype of V157F and R158L \nmay play a minor role in their enrichment in lung cancer. \nThe discovery that the lung cancer -enriched p53 mutations do not disrupt sequence -\nspecific DNA binding but instead block target gene transactivation raises the possibility that these \nmutations eliminate the ability of p53 to recruit transcriptional cofactors. Current efforts are \nfocused on understanding whether cofactor recruitment or other events in the transcription cycle \nare defective in lung cancer cells carrying these p53 mutants. \n \nAcknowledgements \nThe authors would like to thank Dr. Jason Hill and BioImaging Core for their help and use of their \nfacilities. The authors thank the Genomic Facility at the Wistar Institute for the Next Generation \nIllumina Sequencing.  The authors thank Dr. Charles Scott and Elizabeth McDuffie for their \nexpertise and use of their equipment. The authors thank Dr. Erik Debler and Dr. Hideharu \nHashimoto for assistance with p53 protein purification. The authors thank Dr. Alexander Mazo \nand Dr. Tyler Fenstermaker for their helpful advice. \n \nAuthor Contributions  \nMAT and SBM designed the project. MAT, HNS, SMB, KMK, TQ, and JEK contributed to \nexperiments. MAT, HNS, and SMB analyzed data. MAT and JAB provided critical advice and \ntools for the study. MAT and SM wrote the manuscript. All the authors read and approved the \nmanuscript. \n \nFunding \nThis study was supported by funding from Pennsylvania Commonwealth Universal Research \nEnhancement (CURE) and the Bioimaging Shared Resource of the Sidney Kimmel Cancer Center \n(NCI 5 P30 CA-56036). \n \nEthics Statement \nN/A \n \nConflict of Interest \nNone declared. \n \nData Availability \nThe RNA-sequencing and ChIP -sequencing data reported in this study are available in GEO: \nGSE318292 and GSE318294. \n \nReferences \n1. Kandoth, C., et al., Mutational landscape and significance across 12 major cancer types. \nNature, 2013. 502(7471): p. 333-339. \n2. Rivlin, N., et al., Mutations in the p53 Tumor Suppressor Gene: Important Milestones at \nthe Various Steps of Tumorigenesis. Genes Cancer, 2011. 2(4): p. 466-74. \n3. Kastenhuber, E.R. and S.W. Lowe, Putting p53 in Context. Cell, 2017. 170(6): p. 1062-\n1078. \n4. Oren, M. and C. Prives, p53: A tale of complexity and context. Cell, 2024. 187(7): p. \n1569-1573. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n5. Perri, F., S. Pisconti, and G. Della Vittoria Scarpati, P53 mutations and cancer: a tight \nlinkage. Ann Transl Med, 2016. 4(24): p. 522. \n6. Baugh, E.H., et al., Why are there hotspot mutations in the TP53 gene in human cancers? \nCell Death Differ, 2018. 25(1): p. 154-160. \n7. Petitjean, A., et al., TP53 mutations in human cancers: functional selection and impact \non cancer prognosis and outcomes. Oncogene, 2007. 26(15): p. 2157-65. \n8. Joerger, A.C. and A.R. Fersht, The tumor suppressor p53: from structures to drug \ndiscovery. Cold Spring Harb Perspect Biol, 2010. 2(6): p. a000919. \n9. Kitayner, M., et al., Structural Basis of DNA Recognition by p53 Tetramers. Molecular \nCell, 2006. 22(6): p. 741-753. \n10. Cho, Y., et al., Crystal structure of a p53 tumor suppressor-DNA complex: understanding \ntumorigenic mutations. Science, 1994. 265(5170): p. 346-55. \n11. Pilley, S., T.A. Rodriguez, and K.H. Vousden, Mutant p53 in cell-cell interactions. Genes \nDev, 2021. 35(7-8): p. 433-448. \n12. Hafner, A., et al., The multiple mechanisms that regulate p53 activity and cell fate. \nNature Reviews Molecular Cell Biology, 2019. 20(4): p. 199-210. \n13. Solares, M.J. and D.F. Kelly, Complete Models of p53 Better Inform the Impact of \nHotspot Mutations. Int J Mol Sci, 2022. 23(23). \n14. Stein, Y., R. Aloni-Grinstein, and V. Rotter, Mutant p53 oncogenicity: dominant-\nnegative or gain-of-function? Carcinogenesis, 2020. 41(12): p. 1635-1647. \n15. Nishikawa, S. and T. Iwakuma, Drugs Targeting p53 Mutations with FDA Approval and \nin Clinical Trials. Cancers (Basel), 2023. 15(2). \n16. Hu, J., et al., Targeting mutant p53 for cancer therapy: direct and indirect strategies. \nJournal of Hematology & Oncology, 2021. 14(1): p. 157. \n17. Monteith, J.A., et al., A rare DNA contact mutation in cancer confers p53 gain-of-\nfunction and tumor cell survival via TNFAIP8 induction. Mol Oncol, 2016. 10(8): p. \n1207-20. \n18. Farkas, M., et al., Distinct mechanisms control genome recognition by p53 at its target \ngenes linked to different cell fates. Nature Communications, 2021. 12(1): p. 484. \n19. Resnick-Silverman, L., et al., Identification of a novel class of genomic DNA-binding \nsites suggests a mechanism for selectivity in target gene activation by the tumor \nsuppressor protein p53. Genes Dev, 1998. 12(14): p. 2102-7. \n20. Bray, N.L., et al., Near-optimal probabilistic RNA-seq quantification. Nature \nBiotechnology, 2016. 34(5): p. 525-527. \n21. Love, M.I., W. Huber, and S. Anders, Moderated estimation of fold change and \ndispersion for RNA-seq data with DESeq2. Genome Biology, 2014. 15(12): p. 550. \n22. Barnada, S.M., et al., ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial-\nto-mesenchymal transition in cranial neural crest specification. Am J Hum Genet, 2024. \n111(10): p. 2232-2252. \n23. Schmidt, D., et al., ChIP-seq: using high-throughput sequencing to discover protein-DNA \ninteractions. Methods, 2009. 48(3): p. 240-8. \n24. Li, H. and R. Durbin, Fast and accurate short read alignment with Burrows–Wheeler \ntransform. Bioinformatics, 2009. 25(14): p. 1754-1760. \n25. Zhang, Y., et al., Model-based Analysis of ChIP-Seq (MACS). Genome Biology, 2008. \n9(9): p. R137. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n26. Quinlan, A.R. and I.M. Hall, BEDTools: a flexible suite of utilities for comparing \ngenomic features. Bioinformatics, 2010. 26(6): p. 841-842. \n27. Ramírez, F., et al., deepTools: a flexible platform for exploring deep-sequencing data. \nNucleic Acids Research, 2014. 42(W1): p. W187-W191. \n28. Robinson, J.T., et al., Integrative genomics viewer. Nature Biotechnology, 2011. 29(1): p. \n24-26. \n29. Machanick, P. and T.L. Bailey, MEME-ChIP: motif analysis of large DNA datasets. \nBioinformatics, 2011. 27(12): p. 1696-1697. \n30. Bailey, T.L., et al., The MEME Suite. Nucleic Acids Research, 2015. 43(W1): p. W39-\nW49. \n31. Barta, J.A. and S.B. McMahon, Lung-Enriched Mutations in the p53 Tumor Suppressor: \nA Paradigm for Tissue-Specific Gain of Oncogenic Function. Molecular Cancer \nResearch, 2019. 17(1): p. 3-9. \n32. Barta, J.A., et al., The lung-enriched p53 mutants V157F and R158L/P regulate a gain of \nfunction transcriptome in lung cancer. Carcinogenesis, 2020. 41(1): p. 67-77. \n33. Tracewell, M.A., et al., Somatic p53 mutations that are markedly overrepresented in lung \ncancer confer resistance to ROS-induced cell death. Carcinogenesis, 2025. \n34. Liu, L.F., et al., Mechanism of action of camptothecin. Ann N Y Acad Sci, 2000. 922: p. \n1-10. \n35. Gerritsen, M., et al., Presence of mutant p53 increases stem cell frequency and is \nassociated with reduced binding to classic TP53 binding sites in cell lines and primary \nAMLs. Exp Hematol, 2022. 110: p. 39-46. \n36. Zhu, J., et al., Gain-of-function p53 mutants co-opt chromatin pathways to drive cancer \ngrowth. Nature, 2015. 525(7568): p. 206-211. \n37. Emamzadah, S., et al., Reversal of the DNA-binding-induced loop L1 conformational \nswitch in an engineered human p53 protein. J Mol Biol, 2014. 426(4): p. 936-44. \n38. Petty, T.J., et al., An induced fit mechanism regulates p53 DNA binding kinetics to confer \nsequence specificity. Embo j, 2011. 30(11): p. 2167-76. \n39. Kanehisa, M., Toward understanding the origin and evolution of cellular organisms. \nProtein Sci, 2019. 28(11): p. 1947-1951. \n40. Kanehisa, M., et al., KEGG: biological systems database as a model of the real world. \nNucleic Acids Res, 2025. 53(D1): p. D672-d677. \n41. Kanehisa, M. and S. Goto, KEGG: kyoto encyclopedia of genes and genomes. Nucleic \nAcids Res, 2000. 28(1): p. 27-30. \n42. Aubrey, B.J., et al., How does p53 induce apoptosis and how does this relate to p53-\nmediated tumour suppression? Cell Death & Differentiation, 2018. 25(1): p. 104-113. \n43. Chen, J., The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and \nProgression. Cold Spring Harb Perspect Med, 2016. 6(3): p. a026104. \n44. Chipuk, J.E. and D.R. Green, Dissecting p53-dependent apoptosis. Cell Death & \nDifferentiation, 2006. 13(6): p. 994-1002. \n45. Porter, A.G. and R.U. Jänicke, Emerging roles of caspase-3 in apoptosis. Cell Death & \nDifferentiation, 1999. 6(2): p. 99-104. \n46. Vogelstein, B., S. Sur, and C. Prives, P53: The most frequently altered gene in human \ncancers. Nat Educ, 2010. 3. \n47. Chen, X., et al., Mutant p53 in cancer: from molecular mechanism to therapeutic \nmodulation. Cell Death & Disease, 2022. 13(11): p. 974. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n48. Lei, J., et al., Insights into Allosteric Mechanisms of the Lung-Enriched p53 Mutants \nV157F and R158L. Int J Mol Sci, 2022. 23(17). \n49. Willis, A., et al., Mutant p53 exerts a dominant negative effect by preventing wild-type \np53 from binding to the promoter of its target genes. Oncogene, 2004. 23(13): p. 2330-\n2338. \n50. Chan, W.M., et al., How many mutant p53 molecules are needed to inactivate a \ntetramer? Mol Cell Biol, 2004. 24(8): p. 3536-51. \n51. Chène, P., In vitro analysis of the dominant negative effect of p53 mutants. J Mol Biol, \n1998. 281(2): p. 205-9. \n52. Butera, A. and I. Amelio, Deciphering the significance of p53 mutant proteins. Trends in \nCell Biology, 2024. \n53. Giacomelli, A.O., et al., Mutational processes shape the landscape of TP53 mutations in \nhuman cancer. Nature Genetics, 2018. 50(10): p. 1381-1387. \n54. Boettcher, S., et al., A dominant-negative effect drives selection of TP53 missense \nmutations in myeloid malignancies. Science, 2019. 365(6453): p. 599-604. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nFigure 1: V157F and R158L p53 mutants bind at WT p53 target genes in endogenous cells. \nA, Proximity ligation assay (PLA) was performed on H2087 (V157F p53) cells. Samples included the \nexperimental condition (p53 + Biotin), rabbit I gG in place of the biotin antibody (Rb I gG), a sample with no \nbiotin azide added to the click-it reaction to mark the DNA (no Biotin), and a sample where p53 was depleted \nfrom the cells with shRNA (p53 KD). Images shown are of merged DAPI (blue) and PLA signal (red) \nchannels, and PLA signal alone (white). B, PLA images containing 100-300 cells per image were quantified \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nby the area of the PLA signal over the nuclei count in the image. p53 + Biotin was significantly different \nfrom all three controls by one-way ANOVA, * p-value <0.05. C, ChIP-seq was performed on H460 (WT p53) \nand H2087 (V157F p53) cells treated with CPT ( 2M) for 6 hours (Tracewell, et al.). Data from two other \npublicly available datasets were used (GSE238181  [35] and GSE59176 [36]) that feature cell lines with \nboth WT p53 and different p53 mutants. Signal intensity of p53 binding at representative p53 targ et genes \np21, PLK3, BAX, and PUMA visualized in Integrative Genomics Viewer [28]. Peak scaling was grouped \nseparately for each experiment. WT p53 tracks are blue, and p53 mutant tracks are red/orange.  D-F, \nEnriched query motifs at p53 (WT, V157F, or R158L) bound regions identified by MEME-ChIP [29, 30]. The \nblack line highlights the conserved p53 consensus core sequence “C(A/T)(A/T)G”. G-J, ChIP-qPCR was \nperformed on H2087 and H460 (WT p53) cells. The ChIP samples were analyzed by qPCR with primers \ndesigned at WT p53 binding sites of p53 target genes: p21, RRM2B, BAX, and PUMA. A two-way ANOVA \nwas performed to determine significant differences in binding between the p53 ChIP sample and the \ncorresponding IgG control, *** p-value <0.001, ** p-value <0.01, ns: non-significant. \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nFigure 2: V157F and R158L p53 mutants bind to p53 target genes similar to WT p53. \nA, Schematic of full -length WT p53 and protein purified N-p53 construct used for subsequent SPR \nexperiments. B, Coomassie Brilliant Blue staining for protein purified N-p53 constructs of WT, V157F, and \nR158L p53. C, SPR binding data in resonance units (RU) for WT, V157F, and R158L p53. 1nM -200nM \nprotein concentrations were used at a salt concentration of 275mM. Binding was checked using p21 and \nBAX response elements, as well as a scrambled control DNA sequence. D-F, Comparisons between WT \nand mutant p53 displaying the 200nM protein concentration at 275mM salt concentration for p21 (D), BAX \n(E), and scrambled (F). \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nTable 1: KD values derived from SPR data for WT p53 and mutant p53 binding interactions with different \nDNA sequences at different salt concentrations. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nFigure 3: V157F and R158L p53 mutants bind at WT p53 target genes in TET-inducible system. \nA, Representative i mmunoblot of whole cell lysates collected from samples used for subsequent \nexperiments. The samples consisted of H1299 cells with TET-inducible plasmids for WT, V157F, or R158L \np53. Cells were treated with TET alone (1g/mL) or TET (1g/mL) followed by CPT (2M) for an additional \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n6 hours. The samples had successful induction of p53, and GAPDH was used as a loading control. B, ChIP-\nqPCR was performed on H1299 cells with TET -inducible plasmids for WT, V157F, or R158L p53 treated \nwith TET alone (1 M) and treated with CPT ( 2M) for 6 hours or left untreated. The ChIP samples were \nanalyzed by qPCR with primers designed at WT p53 binding sites of p53 target genes: p21 and BAX. Data \nvalues given in percentage input. Significance is shown comparing  the p53 ChIP sample to the \ncorresponding IgG control, Two-way ANOVA, *** p <0.001, ** p <0.01, * p <0.05, ns: non-significant. C, \nChIP-seq was performed on H1299 cells containing a TET-inducible plasmid for WT, V157F, or R158L p53 \nthat were treated with TET (1 M) alone for 22 hours or TET (1M) for 16 hours and then CPT (2M) for 6 \nhours. Signal intensity of p53 binding at representative p53 target genes p21, PLK3, BAX, and PUMA \nvisualized in Integrative Genomics Viewer [28]. Peak scaling was grouped separately for each condition; \nWT p53 (blue), V157F p53 (red), and R158L p53 (purple). D-F, Enriched motifs at p53 (WT, V157F, or \nR158L) bound regions in the TET+CPT-treated condition identified by MEME-ChIP [29, 30]. The black line \nhighlights the conserved p53 consensus core sequence “C(A/T)(A/T)G”. G-H, Venn diagrams displaying \nthe number of genes in the KEGG p53 signaling pathway (hsa04115) [39-41] that are bound in WT, V157F, \nand R158L p53 in both TET-treated and TET+CPT-treated conditions via ChIP-seq.  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \n \nFigure 4: V157F and R158L p53 mutants do not express WT p53 target genes \nH1299 cells containing a TET-inducible plasmid for WT, V157F, or R158L p53 were treated with TET (1M) \nalone or TET (1M) and then CPT (2M) for 24 hours. A, Volcano plot of differentially expressed genes in \nthe V157F p53 mutant expressed CPT -treated condition relative to the WT p53 expressed CPT -treated \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\ncondition determined by DESeq2 [21]. Pink points represent differentially expressed genes (p < 0.05; FDR \n< 5%; Log 2Fold Change +/ -1.5, n=2286). B, Volcano plot of differentially expressed genes in the R158L \np53 mutant expressed CPT -treated condition relative to the WT p53 expressed CPT -treated condition \ndetermined by DESeq2 [21] . Green points represent differentially expressed genes (p < 0.05; FDR < 5%; \nLog2Fold Change +/-1.5, n=2797). C, Violin plots displaying Log2TPM of WT p53 target genes p21, PLK3, \nBAX, and PUMA for TET and TET+CPT treated conditions. Significance is shown for comparison to WT \np53 in the respective treatment group,  Two-way ANOVA, *** p <0.001, ** p <0.01, ns: non-significant. D, \nRNA was isolated from H1299 cells containing a TET-inducible plasmid for WT, V157F, or R158L p53. The \ncells were treated with TET only (1 M), CPT only (2 M), TET and CPT, or left untreated. RTq -PCR was \nperformed on the cDNA samples for the WT p53 target genes: p21, PLK3, BAX, and PUMA. Significance \nis shown for comparison to the UT condition, Two-way ANOVA, *** p <0.001, ns: non-significant. E, Whole-\ncell lysates were collected from H1299 cells 24 hours post -transfection with a luciferase reporter plasmid \nfor p21 or BAX and 500ng of expression plasmid for WT p53, V157F p53, R158L p53, or empty vector (EV). \nImmunoblot of the whole -cell lysates for p53 and loa ding control of GAPDH. F-G, Lysates from ( E) were \nmeasured for luminescence in relative light units (RLU) for either p21 (F) or BAX (G). Significance is shown \nfor WT p53 compared to other conditions, One-way ANOVA, *** p <0.001, ns: non-significant. \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nFigure 5: V157F and R158L p53 mutant cells do not induce apoptosis at the same rate as WT p53 cells in \nresponse to CPT treatment. \nFor (A-C), H1299 cells containing a TET-inducible plasmid for WT, V157F, or R158L p53 were treated with \nTET (1M) alone, or treated with TET (1 M) and CPT (2 M) was additionally added for either 24 or 48 \nhours. A, Cell viability was measured at 48 hours post-addition of CPT. A two-way ANOVA was performed \nto determine significant differences in cell viability between conditions, significance is shown for comparing \nTET+CPT conditions, ** p -value <0.01. B, Cell quantification was performed on the cells 48 hours post -\naddition of CPT. A two-way ANOVA was performed to determine significant differences between conditions, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nsignificance is shown for comparing TET+CPT conditions, *** p -value <0.001. C, Cells were collected and \nincubated with a staining solution of FITC Annexin V and propidium iodide (PI). The stained cells were \nanalyzed by flow cytometry. \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nFigure 6: V157F and R158L p53 mutant cells undergo cell cycle arrest, whereas WT p53 cells undergo \napoptosis after CPT treatment. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\nA, H1299 cells containing a TET -inducible plasmid for WT, V157F, or R158L p53 were treated with TET \n(1M) alone, or treated with TET (1 M) and CPT (2M), which was additionally added for either 24 or 48 \nhours. Cells were collected, and cell cycle analysis by flow cytometry was performed.  Data analysis and \nhistogram creation were done in FlowJo (10.10.0) . One-way ANOVA, Two-way ANOVA, p < 0.05.  B, Pie \ncharts representing the percentage of cells in each stage of the cell cycle (Sub G1, G1, S, and G2/M) are \nshown from the cell cycle analysis performed in (A).  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\n \nFigure 7: V157F and R158L p53 mutants have a dominant-negative phenotype. \nA, H1299 cells with a TET-inducible plasmid for WT p53 were transfected with a luciferase reporter plasmid \nfor p21 or BAX, and 250ng of expression plasmid for V157F p53, R158L p53, or empty vector (EV). Samples \nwere treated with TET (1 M) to induce expression of WT p53 or left untreated. Immunoblot of the whole -\ncell lysates for p53 and GAPDH as a loading control. B-C, Lysates from (A) were measured for \nluminescence in relative light units (RLU) for either p21 (B) or BAX (C). Two -way ANOVA was used for \nstatistical analysis, *** p-value <0.001, ns: non-significant. D-G, H1299 cells with a TET -inducible plasmid \nfor WT p53 were treated with TET (1 M) to induce WT p53 expression or left untreated. Cells were also \ntransfected with 250ng of expression plasmid for V157F p53, R158L p53, or empty vector (EV). RNA was \nisolated from the samples, and RTq -PCR was performed on the cDNA samples for the WT p53 t arget \ngenes: p21 (D), PLK3 (E), BAX (F), and PUMA (G). H-K, H460 (WT p53) cells with a TET-inducible plasmid \nfor V157F p53 (pl:V157F p53) were treated with TET (0.25 M or 1 M) or left untreated (0 M). The cells \nwere also treated with CPT (2 M) or equivalent amount of DMSO (0.01%) for 6 hours. RNA was isolated \nfrom the cells, and RTq-PCR was performed on the cDNA samples for the WT p53 target genes: p21 (H), \nPLK3 (I), BAX (J), and PUMA (K). Two-way ANOVA was used for statistical analysis, significance is shown \nfor comparison to 0M TET + CPT condition, * p-value <0.05. L, H460 (WT p53) cells with a TET-inducible \nplasmid for V157F p53 (pl:V157F p53) were treated with TET (0.25M or 1M) or left untreated (0M). The \ncells were also treated with CPT (2 M) or equivalent amount of DMSO (0.01%) for 48 hours. Whole cell \nlysates of the cells were collected and probed for p53, cleaved caspase-3 (CC3), and GAPDH as a loading \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint \n\ncontrol. M, H460 (WT p53) cells with a TET-inducible plasmid for V157F p53 (pl:V157F p53) were treated \nwith TET (0.25M or 1M) or left untreated. The cells were also treated with CPT (2M) for 48 hours or left \nuntreated. Cell viability was measured at 48 hours post CPT treatment. A two-way ANOVA was performed \nto determine significant differences in cell viability between conditions, significance is shown for comparison \nto 0M TET + CPT condition, * p-value <0.05. N, H460 (WT p53) cells with a TET-inducible plasmid for WT \np53 (pl:WT p53) were treated with TET (0.25M or 1M) or left untreated. The cells were also treated with \nCPT (2M) or equivalent amount of DMSO (0.01%) for 48 hours. Cell viability was measured at 48 hours \npost-addition of CPT. A two-way ANOVA was performed to determine significant differences in cell viability \nbetween conditions, significance is shown for comparison to 0M TET + CPT condition, ns: non-significant. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.14.705936doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}