P53 Target Research Articles

SPINK2 Protein Expression Is an Independent Adverse Prognostic Marker in AML and Is Potentially Implicated in the Regulation of Ferroptosis and Immune Response.

There is an urgent need for the identification as well as clinicopathological and functional characterization of potent prognostic biomarkers and therapeutic targets in acute myeloid leukemia (AML). Using immunohistochemistry and next-generation sequencing, we investigated the protein expression as well as clinicopathological and prognostic associations of serine protease inhibitor Kazal type 2 (SPINK2) in AML and examined its potential biological functions. High SPINK2 protein expression was an independent adverse biomarker for survival and an indicator of elevated therapy resistance and relapse risk. SPINK2 expression was associated with AML with an NPM1 mutation and an intermediate risk by cytogenetics and European LeukemiaNet (ELN) 2022 criteria. Furthermore, SPINK2 expression could refine the ELN2022prognostic stratification. Functionally, an RNA sequencing analysis uncovered a potential link of SPINK2 with ferroptosis and immune response. SPINK2 regulated the expression of certain P53 targets and ferroptosis-related genes, including SLC7A11 and STEAP3, and affected cystine uptake, intracellular iron levels and sensitivity to erastin, a specific ferroptosis inducer. Furthermore, SPINK2 inhibition consistently increased the expression of ALCAM, an immune response enhancer and promoter of T-cell activity. Additionally, we identified a potential small-molecule inhibitor of SPINK2, which requires further characterization. In summary, high SPINK2 protein expression was a potent adverse prognostic marker in AML and might represent a druggable target.

Influence of intestinal microbiota on autophagy in colorectal cancer.

e15505 Background: Gut microbiota favors butyrate-forming bacteria and maintains homeostatic balance which is revoked during the development of colorectal cancer (CRC)-associated dysbiosis. Butyrate is a short chain fatty acid that acts as a lysine deacetylase inhibitor and is known to activate the AMPK pathway as well as inhibiting inflammation and ROS production. The role of AMPK activation in diabetes-associated complications including colorectal cancer is poorly understood. This project examines the role of dysbiosis and butyrate on CRC onset and disease progression. Methods: C57BL/6- Apctm1Tyj/J ( APC) mice that spontaneously develop CRC were used and the study included four groups of mice (n = 3 for each): (1) Control C57BL/6, (2) APC, (3) APC treated with probiotics rich in butyrate forming bacteria and (4) APC treated with butyrate. Probiotics and butyrate were administered by oral gavage and intraperitoneally, respectively. Feces were collected at early (when CRC is developed at 8 weeks of age) and late (at 16 weeks of age, day of sacrifice) timepoints, and microbial composition was identified by 16S rRNA sequencing. Mice colons following sacrifice were also harvested for molecular, anatomical, and histological analyses. Results: The butyrate kinase gene was assessed in fecal samples. Butyrate forming bacteria were significantly reduced in APC mice but elevated in APC receiving probiotics when compared to control mice. Anatomically, polyp number was reduced in groups receiving probiotics and butyrate when compared to APC mice without treatment (p < 0.01 for each). Histologically, butyrate and probiotics ameliorated the histological changes observed in in APC mice as assessed by PAS staining. Furthermore, we observed a decrease in goblet cells, and increased number of crypts on distal colon histology. The expression of NADPH Oxidase 4 (NOX4) and TIGAR, a P53 target protein expression were significantly increased in APC mice and restored following the administration of probiotics or butyrate. ROS production assessed by HPLC showed similar pattern across our 4 groups. The expression of the autophagy markers Beclin-1, ATG-12 and LC3-β were restored to control levels after probiotics and butyrate administration. 16S rRNA sequencing of fecal samples revealed significant differences in microbial composition between groups, most significantly at 16 weeks with Lachnospiraceae (ASV 175), Muribaculaceae (ASV 53) and Muribaculaceae (ASV 584) were decreased in APC when compared to control mice ( < -7 Log2 fold change each). Conclusions: Collectively, our data suggests colorectal cancer is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria. This disruption leads to over-expression of NOX4, and ROS production associated with alteration in autophagy, thus worsening gastrointestinal complications and CRC. Restoring butyrate forming bacteria might serve as an effective adjuvant to therapy in CRC.

ArhGAP11A mediates amyloid-β generation and neuropathology in an Alzheimer’s disease-like mouse model

Amyloid-β (Aβ) plays an important role in the neuropathology of Alzheimer's disease (AD), but some factors promoting Aβ generation and Aβ oligomer (Aβo) neurotoxicity remain unclear. We here find that the levels of ArhGAP11A, a Ras homology GTPase-activating protein, significantly increase in patients with AD and amyloid precursor protein (APP)/presenilin-1 (PS1) mice. Reducing the ArhGAP11A level in neurons not only inhibits Aβ generation by decreasing the expression of APP, PS1, and β-secretase (BACE1) through the RhoA/ROCK/Erk signaling pathway but also reduces Aβo neurotoxicity by decreasing the expressions of apoptosis-related p53 target genes. In APP/PS1 mice, specific reduction of the ArhGAP11A level in neurons significantly reduces Aβ production and plaque deposition and ameliorates neuronal damage, neuroinflammation, and cognitive deficits. Moreover, Aβos enhance ArhGAP11A expression in neurons by activating E2F1, which thus forms a deleterious cycle. Our results demonstrate that ArhGAP11A may be involved in AD pathogenesis and that decreasing ArhGAP11A expression may be a promising therapeutic strategy for AD treatment.

Genetic heterogeneity in p53-null leukemia increases transiently with spindle assembly checkpoint inhibition and is not rescued by p53.

Chromosome gains or losses often lead to copy number variations (CNV) and loss of heterozygosity (LOH). Both quantities are low in hematologic "liquid" cancers versus solid tumors in data of The Cancer Genome Atlas (TCGA) that also shows the fraction of a genome affected by LOH is ~ one-half of that with CNV. Suspension cultures of p53-null THP-1 leukemia-derived cells conform to these trends, despite novel evidence here of genetic heterogeneity and transiently elevated CNV after perturbation. Single-cell DNAseq indeed reveals at least 8 distinct THP-1 aneuploid clones with further intra-clonal variation, suggesting ongoing genetic evolution. Importantly, acute inhibition of the mitotic spindle assembly checkpoint (SAC) produces CNV levels that are typical of high-CNV solid tumors, with subsequent cell death and down-selection to novel CNV. Pan-cancer analyses show p53 inactivation associates with aneuploidy, but leukemias exhibit a weaker trend even though p53 inactivation correlates with poor survival. Overexpression of p53 in THP-1 does not rescue established aneuploidy or LOH but slightly increases cell death under oxidative or confinement stress, and triggers p21, a key p53 target, but without affecting net growth. Our results suggest that factors other than p53 exert stronger pressures against aneuploidy in liquid cancers, and identifying such CNV suppressors could be useful across liquid and solid tumor types.

Role of p53 in Cisplatin-Induced Myotube Atrophy.

Chemotherapy-induced sarcopenia is an unfavorable prognostic factor implicated in the development of postoperative complications and reduces the quality of life of patients with cancer. Skeletal muscle loss due to cisplatin use is caused by mitochondrial dysfunction and activation of muscle-specific ubiquitin ligases Atrogin-1 and muscle RING finger 1 (MuRF1). Although animal studies suggest the involvement of p53 in age-, immobility-, and denervation-related muscle atrophy, the association between cisplatin-induced atrophy and p53 remains unknown. Herein, we investigated the effect of a p53-specific inhibitor, pifithrin-alpha (PFT-α), on cisplatin-induced atrophy in C2C12 myotubes. Cisplatin increased the protein levels of p53, phosphorylated p53, and upregulated the mRNA expression of p53 target genes PUMA and p21 in C2C12 myotubes. PFT-α ameliorated the increase in intracellular reactive oxygen species production and mitochondrial dysfunction, and also reduced the cisplatin-induced increase in the Bax/Bcl-2 ratio. Although PFT-α also reduced the cisplatin-induced increase in MuRF1 and Atrogin-1 gene expression, it did not ameliorate the decrease in myosin heavy chain mRNA and protein levels and muscle-specific actin and myoglobin protein levels. We conclude that cisplatin increases muscle degradation in C2C12 myotubes in a p53-dependent manner, but p53 has minimal involvement in the reduction of muscle protein synthesis.

FIT links c-Myc and P53 acetylation by recruiting RBBP7 during colorectal carcinogenesis.

Colorectal cancer (CRC) poses one of the most serious threats to human health worldwide, and abnormally expressed c-Myc and p53 are deemed the pivotal driving forces of CRC progression. In this study, we discovered that the lncRNA FIT, which was downregulated in CRC clinical samples, was transcriptionally suppressed by c-Myc in vitro and promoted CRC cell apoptosis by inducing FAS expression. FAS is a p53 target gene, and we found that FIT formed a trimer with RBBP7 and p53 that facilitated p53 acetylation and p53-mediated FAS gene transcription. Moreover, FIT was capable of retarding CRC growth in a mouse xenograft model, and FIT expression was positively correlated with FAS expression in clinical samples. Thus, our study elucidates the role of the lncRNA FIT in human colorectal cancer growth and provides a potential target for anti-CRC drugs.

16 Biological Effects of Benzo-[A]-Pyrene and Cerium Nanoparticles on the Human Placental Barrier

Abstract The human placenta is a transitory organ essential for fetal development, whose functions can be disrupted by xenobiotics in maternal blood. Benzo-[a]-pyrene (B[a]P) is a carcinogenic, mutagenic and reprotoxic pollutant as well as an endocrine disruptor that can be internalized in the human body by respiratory exposure. Cerium dioxide nanoparticles (CeO2 NP) have been introduced into our environment, mainly for their catalytic properties and share the same emission sources as B[a]P (cigarette smoke and diesel engine exhaust). The aim of our study is to determine the impact of these two atmospheric pollutants on the human placenta in concomitant exposure, in order to get closer to the environmental reality. Chorionic villi and villous cytotrophoblasts (VCT) from human placentas at term of pregnancy were exposed in vitro to the pollutants. The internalization of the pollutants was observed by confocal and Raman microscopy. Cytotoxicity was assessed using WST-1 test and extracellular LDH assay. Placental endocrine activity was assessed by ELISA. The signaling pathways impacted have been studied by Western Blot, RT-qPCR and IHC. CeO2 NP and B[a]P can be internalized within the chorionic villi. B[a]P alone or in co-exposure with CeO2 NP activate the metabolic pathway of the aryl hydrocarbon receptor (AhR), causing DNA damage. B[a]P also stabilizes the stress transcription factor p53 and its transcriptional target p21. Although these two pollutants do not cause major toxicity on term human trophoblasts after in vitro exposures, cellular stress markers are induced.

Multitargeted molecular docking and dynamics simulation studies of flavonoids and volatile components from the peel of Citrus sinensis L. (Osbeck) against specific tumor protein markers

Citrus sinensis (L.) Osbeck (Rutaceae), commonly known as the sweet orange, is a popular and widely consumed fruit with several medicinal properties. The present study aimed to perform the in silico screening of 18 flavonoids and eight volatile components from the peel of C. sinensis against apoptotic and inflammatory proteins, metalloprotease, and tumor suppressor markers. Flavonoids obtained higher probabilities than volatile components against selected anti-cancer drug targets. Hence, the data from the binding energies against the essential apoptotic and cell proliferation proteins substantiate that they may be promising compounds in developing effective candidates to block cell growth, proliferation, and induced cell death by activating the apoptotic pathway. Further, the binding stability of the selected targets and the corresponding molecules were analyzed by 100 ns molecular dynamics (MD) simulations. Chlorogenic acid has the most binding affinity against the important anti-cancer targets iNOS, MMP-9, and p53. The congruent binding mode to different drug targets focused on cancer shown by chlorogenic acid suggests that it may be a compound with significant therapeutic potential. Moreover, the binding energy predictions indicated that the compound had stable electrostatic and van der Waal energies. Thus, our data reinforce the medicinal importance of flavonoids from C. sinensis and expand the need for more studies, seeking to optimize results and amplify the impacts of further in vitro and in vivo studies. Communicated by Ramaswamy H. Sarma

Butein inhibits cancer cell growth by rescuing the wild-type thermal stability of mutant p53

p53 is a transcription factor that activates the expression of various genes involved in the maintenance of genomic stability, and more than 50% of cancers harbor inactivating p53 mutations, which are indicative of highly aggressive cancer and poor prognosis. Pharmacological targeting of mutant p53 to restore the wild-type p53 tumor-suppressing function is a promising strategy for cancer therapy. In this study, we identified a small molecule, Butein, that reactivates mutant p53 activity in tumor cells harboring the R175H or R273H mutation. Butein restored wild-type-like conformation and DNA-binding ability in HT29 and SK-BR-3 cells harboring mutant p53-R175H and mutant p53-R273H, respectively. Moreover, Butein enabled the transactivation of p53 target genes and decreased the interactions of Hsp90 with mutant p53-R175H and mutant p53-R273H proteins, while Hsp90 overexpression reversed targeted p53 gene activation. In addition, Butein induced thermal stabilization of wild-type p53, mutant p53-R273H and mutant p53-R175H, as determined via CETSA. From docking study, we further proved that Butein binding to p53 stabilized the DNA-binding loop-sheet-helix motif of mutant p53-R175H and regulated its DNA-binding activity via an allosteric mechanism, conferring wild-type-like the DNA-binding activity of mutant p53. Collectively, the data suggest that Butein is a potential antitumor agent that restores p53 function in cancers harboring mutant p53-R273H or mutant p53-R175H. SignificanceButein restores the ability of mutant p53 to bind DNA by reversing its transition to the Loop3 (L3) state, endows p53 mutants with thermal stability and re-establishes their transcriptional activity to induce cancer cell death.

PHLDA1 is a P53 target gene involved in P53-mediated cell apoptosis.

Pleckstrin homeolike domain, family A, member 1 (PHLDA1) is a multifunctional protein that plays diverse roles in A variety of biological processes, including cell death, and hence its altered expression has been found in different types of cancer. Although studies have shown a regulatory relationship between p53 and PHLDA1, the molecular mechanism is still unclear. Especially, the role of PHLDA1 in the process of apoptosis is still controversial. In this study, we found that the expression of PHLDA1 in human cervical cancer cell lines was correlated with the up-expression of p53 after treatment with apoptosis-inducing factors. Subsequently, the binding site and the binding effect of p53 on the promoter region of PHLDA1 were verified by our bioinformatics data analysis and luciferase reporter assay. Indeed, we used CRISPR-Cas9 to knockout the p53 gene in HeLa cells and further confirmed that p53 can bind to the promoter region of PHLDA1 gene, and then directly regulate the expression of PHLDA1 by recruiting P300 and CBP to change the acetylation and methylation levels in the promoter region. Finally, a series of gain-of-function experiments further confirmed that p53 re-expression in HeLap53-/- cell can up-regulate the reduction of PHLDA1 caused by p53 knockout, and affect cell apoptosis and proliferation. Our study is the first to explore the regulatory mechanism of p53 on PHLDA1 by using the p53 gene knockout cell model, which further proves that PHLDA1 is a target-gene in p53-mediated apoptosis, and reveals the important role of PHLDA1 in cell fate determination.

Synthesis and Antineoplastic Activity of a Dimer, Spiroindolinone Pyrrolidinecarboxamide.

The mutation or function loss of tumour suppressor p53 plays an important role in abnormal cell proliferation and cancer generation. Murine Double Minute 2 (MDM2) is one of the key negative regulators of p53. p53 reactivation by inhibiting MDM2-p53 interaction represents a promising therapeutic option in cancer treatment. Here, to develop more effective MDM2 inhibitors with lower off-target toxicities, we synthesized a dimer, spiroindolinone pyrrolidinecarboxamide XR-4, with potent MDM2-p53 inhibition activity. Western blotting and qRT-PCR were performed to detect the impact of XR-4 on MDM2 and p53 protein levels and p53 downstream target gene levels in different cancers. Cancer cell proliferation inhibition and clonogenic activity were also investigated via the CCK8 assay and colony formation assay. A subcutaneous 22Rv1-derived xenografts mice model was used to investigate the in vivo anti-tumour activity of XR-4. The results reveal that XR-4 can induce wild-type p53 accumulation in cancer cells, upregulate the levels of the p53 target genes p21 and PUMA levels, and then inhibit cancer cell proliferation and induce cell apoptosis. XR-4 can also act as a homo-PROTAC that induces MDM2 protein degradation. Meanwhile, the in vivo study results show that XR-4 possesses potent antitumour efficacy and a favourable safety property. In summary, XR-4 is an interesting spiroindolinone pyrrolidinecarboxamide-derivative dimer with effective p53 activation activity and a cancer inhibition ability.

An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression.

We analyze the African-centric Y107H hypomorphic variant and show that it confers increased cancer risk; we use Y107H in order to identify PADI4 as a key tumor-suppressive p53 target gene that contributes to an immune modulation signature and that is predictive of cancer survival and the success of immunotherapy. See related commentary by Bhatta and Cooks, p. 1518. This article is highlighted in the In This Issue feature, p. 1501.

P53-dependent DNA repair during the DNA damage response requires actin nucleation by JMY

The tumour suppressor p53 is a nuclear transcription factor with key roles during DNA damage to enable a variety of cellular responses including cell cycle arrest, apoptosis and DNA repair. JMY is an actin nucleator and DNA damage-responsive protein whose sub-cellular localisation is responsive to stress and during DNA damage JMY undergoes nuclear accumulation. To gain an understanding of the wider role for nuclear JMY in transcriptional regulation, we performed transcriptomics to identify JMY-mediated changes in gene expression during the DNA damage response. We show that JMY is required for effective regulation of key p53 target genes involved in DNA repair, including XPC, XRCC5 (Ku80) and TP53I3 (PIG3). Moreover, JMY depletion or knockout leads to increased DNA damage and nuclear JMY requires its Arp2/3-dependent actin nucleation function to promote the clearance of DNA lesions. In human patient samples a lack of JMY is associated with increased tumour mutation count and in cells results in reduced cell survival and increased sensitivity to DNA damage response kinase inhibition. Collectively, we demonstrate that JMY enables p53-dependent DNA repair under genotoxic stress and suggest a role for actin in JMY nuclear activity during the DNA damage response.

The role of truncated p53 isoforms in the DNA damage response.

The tumour suppressor p53 is activated following genotoxic stress and regulates the expression of target genes involved in the DNA damage response (DDR). The discovery that p53 isoforms alter the transcription of p53 target genes or p53 protein interactions unveiled an alternative DDR. This review will focus on the role p53 isoforms play in response to DNA damage. The expression of the C-terminally truncated p53 isoforms may be modulated via DNA damage-induced alternative splicing, whereas alternative translation plays an important role in modulating the expression of N-terminally truncated isoforms. The DDR induced by p53 isoforms may enhance the canonical p53 DDR or block cell death mechanisms in a DNA damage- and cell-specific manner, which could contribute to chemoresistance in a cancer context. Thus, a better understanding of the involvement of p53 isoforms in the cell fate decisions could uncover potential therapeutic targets in cancer and other diseases.

Extracellular vesicles derived from cervical cancer cells carrying MCM3AP-AS1 promote angiogenesis and tumor growth in cervical cancer via the miR-93/p21 axis

Tumor cells can promote angiogenesis by secreting extracellular vesicles (EVs). Meanwhile, tumor-derived EVs can carry long non-coding RNAs to activate pro-angiogenic signaling in endothelial cells. Here, we investigated the role of long non-coding RNA MCM3AP-AS1 carried by cervical cancer (CC) cell-derived EVs in the angiogenesis and the resultant tumor growth in CC, as well as the potential molecular mechanisms. LncRNAs significantly expressed in CC cell-derived EVs and CC were screened, followed by prediction of downstream target genes. EVs were isolated from HcerEpic and CaSki cell supernatants, followed by identification. The expression of MCM3AP-AS1 in CC was analyzed and its interaction with miR-93-p21 was confirmed. Following co-culture system, the role of MCM3AP-AS1 carried by EVs in HUVEC angiogenic ability, CC cell invasion and migration in vitro along with angiogenesis and tumorigenicity in vivo was assayed. MCM3AP-AS1 was overexpressed in CC cell-derived EVs as well as in CC tissues and cell lines. Cervical cancer cell-derived EVs could transfer MCM3AP-AS1 into HUVECs where MCM3AP-AS1 competitively bound to miR-93 and upregulate the expression of the miR-93 target p21 gene. Thus, MCM3AP-AS1 promoted angiogenesis of HUVECs. In the similar manner, MCM3AP-AS1 enhanced CC cell malignant properties. In nude mice, EVs-MCM3AP-AS1 induced angiogenesis and tumor growth. Overall, this study reveals that CC cell-derived EVs may transport MCM3AP-AS1 to promote angiogenesis and tumor growth in CC.

Synergistic anticancer activity of combined ATR and ribonucleotide reductase inhibition in Ewing's sarcoma cells.

Ewing's sarcoma is a highly malignant childhood tumour whose outcome has hardly changed over the past two decades despite numerous attempts at chemotherapy intensification. It is therefore essential to identify new treatment options. The present study was conducted to explore the effectiveness of combined inhibition of two promising targets, ATR and ribonucleotide reductase (RNR), in Ewing's sarcoma cells. Effects of the ATR inhibitor VE821 in combination with the RNR inhibitors triapine and didox were assessed in three Ewing's sarcoma cell lines with different TP53 status (WE-68, SK-ES-1, A673) by flow cytometric analysis of cell death, mitochondrial depolarisation and cell cycle distribution as well as by caspase 3/7 activity determination, by immunoblotting and by real-time RT-PCR. Interactions between inhibitors were evaluated by combination index analysis. Single ATR or RNR inhibitor treatment produced small to moderate effects, while their combined treatment produced strong synergistic ones. ATR and RNR inhibitors elicited synergistic cell death and cooperated in inducing mitochondrial depolarisation, caspase 3/7 activity and DNA fragmentation, evidencing an apoptotic form of cell death. All effects were independent of functional p53. In addition, VE821 in combination with triapine increased p53 level and induced p53 target gene expression (CDKN1A, BBC3) in p53 wild-type Ewing's sarcoma cells. Our study reveals that combined targeting of ATR and RNR was effective against Ewing's sarcoma in vitro and thus rationalises an in vivo exploration into the potential of combining ATR and RNR inhibitors as a new strategy for the treatment of this challenging disease.

MicroRNA-494 augments fibrotic transformation of human retinal pigment epithelial cells and targets p27 with cell-type specificity.

Epiretinal membranes (ERMs) are the result of fibro-cellular proliferation that cause distortion and impairment of central vision. We hypothesized that select microRNAs (miRs) regulate retinal fibro-proliferation and ERM formation. Following IRB approval, a pilot study was performed in patients presenting for retina surgery with and without clinical ERMs. Total RNA was isolated from ERM tissue and controls from non-ERM vitreous and subjected to miR profiling via microarray analysis. MiR-494 was identified as the only miR selectively expressed at significantly greater levels, and in silico analysis identified p27 as a putative fibroproliferative gene target of miR-494. In vitro testing of miR-494 and p27 in fibrotic transformation was assessed in spontaneously immortalized human retinal pigment epithelial (RPE) and human Müller cell lines, stimulated to transform into a fibroproliferative state via transforming growth factor beta (TGFβ). Fibroproliferative transformation was characterized by de novo cellular expression of alpha smooth muscle actin (αSMA). In both RPE and Müller cells, both TGFβ and miR-494 mimic decreased p27 expression. In parallel experiments, transfection with p27 siRNA augmented TGFβ-induced αSMA expression, while only in RPE cells did co-transfection with miR-494 inhibitor decrease αSMA levels. These results demonstrate that miR-494 augments fibrotic transformation in both Müller cells and RPEs, however only in RPEs does miR-494 mediate fibrotic transformation via p27. As p27 is known to regulate cellular proliferation and differentiation, future studies should extend clinical testing of miR-494 and/or p27 as a potential novel non-surgical therapy for ERMs, as well as identify relevant miR-494 targets in Müller cells.

Abstract LB361: Li-Fraumeni syndrome-associated dimer-forming mutant p53 promotes transactivation-independent mitochondrial cell death

Abstract Mutations that occur within the oligomerization domain (OD) of the tumor suppressor p53 generally abolish p53 tetramerization and are associated with increased cancer susceptibility in Li-Fraumeni syndrome (LFS). Despite their prevalence, the impact of OD-mutant p53 proteins in cancer, especially beyond a loss of canonical p53 activity, has not been well elucidated. We sought to delineate the gain-of-function (GOF) vs. loss-of-function (LOF) activities of OD-mutant p53, specifically focusing on the LFS tumor-derived p53(A347D) variant. We obtained LFS patient-derived dermal fibroblasts heterozygous for the mutation and generated an allelic series of U2OS cancer cell lines expressing wild-type p53, heterozygous (p53+/AD) or homozygous (p53AD/AD) mutant p53 or no p53 (p53 KO). In contrast to wild-type p53, mutant p53(A347D) exclusively forms dimers in both fibroblasts and cancer cells and has lost the ability to bind and transactivate the majority of canonical p53 target genes, which yields comparable tumorigenic properties between mutant p53 and p53 KO cells. Mutant p53(A347D), however, displays neomorphic activities. Glycolysis and oxidative phosphorylation pathways are enriched in cells bearing p53(A347D) relative to both wild-type p53 and p53 KO cells. Furthermore, dimeric mutant p53 induces striking mitochondrial network aberration and preferentially associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Ultimately, we describe dimeric mutant p53 as wielding a double-edged sword: driving tumorigenesis through LOF while gaining enhanced apoptogenic activity, thereby providing a potential basis for select therapeutic approaches. Citation Format: Joshua H. Choe, Tatsuya Kawase, An Xu, Asja Guzman, David R. Tong, Aleksandar Z. Obradovic, Ana M. Low-Calle, Bita Alaghebandan, Ananya Raghavan, Kaitlin Long, Paul M. Hwang, Joshua D. Schiffman, Yan Zhu, Ruiying Zhao, Dung-Fang Lee, Chen Katz, Carol L. Prives. Li-Fraumeni syndrome-associated dimer-forming mutant p53 promotes transactivation-independent mitochondrial cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB361.

Abstract 2604: Functional analysis of p53 codon 72 polymorphism with cysteine substitution in cancer cells

Abstract The p53 codon p72 polymorphism with proline (p53P72)/arginine (p53R72) substitution is associated with cancer incidence. Cysteine(C) substitution at p53 codon 72 polymorphism (p53C72) is rarely studied in cancer patients. A patient who developed AML and who carries p53C72 was described (El-Deiry, 2022 WIN Symposium). The patient’s family has high rates of cancer susceptibility in the women some of whom carry FANCC alterations and other alleles. To examine the functional characteristics of different p53 alterations at codon 72, we generated plasmids expressing p53C72, p53 R72 or p53P72. The variants of p53 were overexpressed in p53-null cancer cells by transient transfection of the plasmids. We tested functions of the p53 codon 72 polymorphisms (p53C72, p53R72 and p53P72) in two cancer cell lines (Soas-2 and HCT116 p53 null) with different expression levels of the p53 variants at different time points. To examine the effect of codon 73 variants on p53 transactivation, we performed a PG13-Luciferase reporter assay and examined the endogenous p53 targets at the protein level. The luciferase-reporter assay showed that all the three variants of the p53 codon p72 variants increased PG-13 luciferase expression at early time points. The Western blot assay showed that the p53C72, the p53R73 and the p53P72 increased endogenous p21, DR5, puma and MDM2 at the protein levels at different time points. The p53C72 increased the p53 targets and PG13-driven luciferase expression much higher than the p53R72 and the p53P72. These results suggest that the p53 codon 72 variants (C72, R72 and P72) retain wild-type activity for p53-mediated transcription, and p53C72 has the highest potency among them. We examined the effect of the p53 variants on cell growth and death. PARP cleavage (a marker of cell apoptosis) was induced in cells overexpressing all the three p53 codon 72 variants. We observed a lesser potency of p53C72 to induce PARP cleavage compared to p53R72 and p53P72. Colony formation assay showed a suppressive effect of p53C72, p53R72 and p53P72 on cancer cell growth. Our studies demonstrate some differences of in wild-type functions of p53 p53C72, p53R72 and p53P72 alleles. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Functional analysis of p53 codon 72 polymorphism with cysteine substitution in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2604.

Abstract 4734: BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma

Abstract Glioblastoma (GBM) is the most notorious primary brain tumor. The median survival of GBM patients is only about one year and about 95% patients succumb after five years. This gloomy picture has not been improved for decades, even with extensive treatments including surgery, radiotherapy, and chemotherapy. Hence, a better understanding of the mechanisms underlying GBM development may provide new therapeutic opportunities. Primary GBM generally harbors a low mutational load when compared to other human cancers. Even P53—the guardian of the genome that is essentially disabled in nearly all human cancers, remains unmutated in about 71% of GBM cases. Moreover, these P53 wildtype GBM cases are as aggressive as those with P53 mutations. To explore the mechanism by which P53 wildtype GBM thrive in the presence of intact P53, we performed CRISPR screens in a panel of human cancer cell lines using an sgRNA library specifically targeting the functional domains of ~200 chromatin regulators. We discovered that BRD8’s bromodomain—a druggable domain as shown by the tremendous success in preclinical and clinical trial in diverse cancers—is a vulnerability specifically in P53 wildtype GBM cases. Our mechanistic studies demonstrate that BRD8 functions through the EP400 chromatin remodeling complex and hijacks the histone variant H2AZ at P53 target loci, enforcing a compact chromatin state that blocks P53’s accessibility to its targets. We show that targeting the BRD8 bromodomain releases H2AZ, opens up chromatin, engages P53-mediated transactivation, and triggers growth arrest. Consistent with these findings, BRD8 is highly expressed with H2AZ in patient-derived proliferating GBM cells, and is inversely correlated with the expression of P53 targets. Our work solves a long-standing mystery in P53 wildtype GBM, and presents a promising therapeutic target for the majority of GBM patients. Citation Format: Sherine Xueqin Sun, Olaf Klingbeil, Christopher Vakoc, Alea Mills. BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4734.