P53 R273H Research Articles

NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner.

The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain of function (GoF). It is known that the aggregation of mutant p53 can induce p53 GoF. The p73 activators RETRA and NSC59984 have an anti-cancer effect in p53 mutation cells, but we found that p73 activators were not effective in all head and neck squamous cell carcinoma (HNSCC) cell lines, with different p53 mutants. A comparison of the gene expression profiles of several regulator(s) in mutant HNSCC cells with or without aggregation of p53 revealed that nicotinamide phosphoribosyltransferase (NAMPT) is a key regulator of mutant p53 aggregation. An NAMPT inhibitor, to reduce abnormal aggregation of mutant p53, used in combination with a p73 activator, was able to effectively repress growth in HNSCC cells with p53 GoF mutants. This study, therefore, suggests a potential combination therapy approach for HNSCC with a p53 GoF mutation.

Oligomerization of Mutant p53 R273H is not Required for Gain-of-Function Chromatin Associated Activities.

The TP53 gene is often mutated in cancer, with missense mutations found in the central DNA binding domain, and less often in the C-terminal oligomerization domain (OD). These types of mutations are found in patients with the rare inherited cancer predisposition disorder called Li-Fraumeni syndrome. We previously found that mutant p53 (mtp53) R273H associates with replicating DNA and promotes the chromatin association of replication-associated proteins mini-chromosome maintenance 2 (MCM2), and poly ADP-ribose polymerase 1(PARP1). Herein, we created dual mutants in order to test if the oligomerization state of mtp53 R273H played a role in chromatin binding oncogenic gain-of-function (GOF) activities. We used site-directed mutagenesis to introduce point mutations in the OD in wild-type p53 (wtp53), and mtp53 R273H expressing plasmids. The glutaraldehyde crosslinking assay revealed that both wtp53 and mtp53 R273H formed predominantly tetramers, while the single OD mutant A347D, and the dual mtp53 R273H-A347D, formed predominantly dimers. The R337C, L344P, mtp53 R273H-R337C, and mtp53 R273H-L344P proteins formed predominantly monomers. Wtp53 was able to activate the cyclin-dependent kinase gene p21/waf and the p53 feedback regulator MDM2. As expected, the transactivation activity was lost for all the single mutants, as well as the mtp53 R273H-dual mutants. Importantly, mtp53 R273H and the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P were able to interact with chromatin. Additionally, the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P, maintained strong interactions with MCM2 and PARP1. Our findings suggest that while mtp53 R273H can form tetramers, tetramer formation is not required for the GOF associated chromatin interactions.

Abstract 2487: disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities

Abstract Background & Goals: The p53 tumor suppressor is one of the most important determinants of cell fate and is critical for tumor suppression. It functions by regulating the expression of a plethora of genes via sequence-specific DNA binding and also by transcription-independent, mitochondria-mediated mechanisms. Not surprisingly, the TP53 gene, which encodes p53, is frequently mutated in human cancer. For example, TP53 is mutated in about 75% of HER2-positive breast cancer and about 85% of triple negative breast cancer. The majority of TP53 mutations are missense mutations, changing one amino acid per mutant, most of which occur in its DNA-binding domain. p53 mutants show loss of tumor suppressor functions or gain of oncogenic functions. We recently found that peptidase D (PEPD), also known as prolidase, binds via its C-terminal sequence to the proline-rich domain (PRD) of wild type p53 and that disrupting the binding activates p53. The enzymatic activity of PEPD is not required for this function. Our objective in the present study is to determine whether PEPD also regulates p53 mutants. We focused on some of the most common oncogenic p53 mutants, including R175H, R248Q, R273H and R280K. Results: Our study shows that 1) each p53 mutant binds directly to PEPD and binds to PEPD in cells in a significant quantity, 2) disrupting their binding in cells by PEPD knockdown reactivates each p53 mutant, unleashing both transcription-dependent and -independent tumor suppressing activities, 3) after leaving PEPD, p53 mutants undergo acetylation, mono-ubiquitination and phosphorylation, which drive their refolding and reactivation, 4) Lysine 373 acetylation is essential for their reactivation, and 5) p53 mutants and p53WT show almost no difference in PEPD-binding and response to PEPD KD. Conclusions & Therapeutic Relevance: In conclusion, we have identified a cellular mechanism by which the tumor suppressor functions of p53 mutants are strongly reactivated, which is neither cell-specific nor mutant-specific. Mainly, p53 mutants bind to PEPD in cancer cells, and disrupting the complex causes posttranslational modifications of mutants freed from PEPD, which drive their refolding and unleash their tumor suppressor activities. This discovery advances our understanding of the function and regulation of p53 mutants and breaks new ground for developing novel cancer treatment strategies. Citation Format: Lu Yang. disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2487.

Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

P53 Gain-of-Function Mutants and Steroids in Ovarian Cancer Cell Metastasis

High-grade serous ovarian cancer (HGSOC) is a heterogeneous disease for which there currentlyis no cure. Because p53 is mutated in >90% of all ovarian cancer, we studied specific gain-of-function (GOF) p53 mutants and steroid hormones for tumor morphology and metastasis in vivo. For this, we analyzed ALST (WT p53), SKOV3 (p53 null), TYK-NU (p53-R175H), OVCAR3 (p53-R248Q) and OVCA420 (p53-R273H) cell line xenografts in Foxn1-/- mice. ALST cells failed tometastasize, likely due to the known apoptotic effects of WT p53. SKOV3 and the p53-GOF celllines metastasized to the omentum and exhibited distinct morphologies: SKOV3, epithelial-like;TYK-Nu, vascular-like; OVCAR3, epithelial/mesenchymal; and OVCA420 epithelial exclusive. Despite different morphologies and p53 status, each tumor type contained large, Polyploid GiantCancer Cells (PGCCs) that are stem-like cells undergoing endoreplication. A specificphosphorylated, active form of β−catenin (pCTNNB1-S31/S37/T41; pCTNNB1) co-localizedselectively with GOF p53 and the mitotic stress regulatory kinase pMSK1-T581 in mitotic cells andPGCCs, indicating that in addition to GOF p53 mutants, pCTNNB1 and pMSK1 play a role intumor progression. To determine if ALST cells could be rendered metastatic, the p53 GOFmutants R175H and R273H were stably expressed in these cells. Remarkably, the ALST(WT/R273H) cells, but not the ALST (WT/R175H) cells, formed solid tumors on the ovary, visceralfat and uterus; but not on the omentum where OVCA420 (p53-R273H) cells formed tumors. TheALST (WT/R273H) tumors harbored discreet populations of pCTNNB1+ mitotic cells and PGCCsand exhibited distinct growth-promoting responses to estradiol while showing growth-inhibitoryeffects of DHT and nuclear AR in the tumor-associated stromal cells. Stably expressing p53-R175H or p53-R273H GOF mutants in SKOV3 (p53 null) cells enhanced tumor progression withthe R273H mutant being most aggressive. The parental SKOV3 tumors also contained pCTNNB1that was associated with mitotic cells and PGCCs. In the SKOV3 p53-R175H cells - and moreimpressively in the p53-R273H cells - pCTNNB1 co-localized with p53 in PGCCs. Whereas ESR1staining was diffuse in the ALST-R273H cells, it was nuclear in the SKOV3 cell lines. However,tumor promotion by estrogen and inhibition by DHT were observed in each SKOV3 cell line,suggesting that stromal cells may also contribute to the steroid dependent effects. Collectively, 1)elevated levels of specific phosphorylated forms of CTNNB1 and MSK1 identify mitotic cells andPGCCs within the tumors and new targets for therapeutic approaches; 2) knowing the status ofp53, and the presence and localization of pCTNNB1 and pMSK1 and steroid hormone receptorsin ovarian tumors suggest combinatorial approaches should be considered to combat the mostlethal gynecological cancer. NIH-CA181808; NIH-HD097321 (JSR).

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI–H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation.TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI–H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA.Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).

Reducing Fatty Acid Oxidation Improves Cancer-free Survival in a Mouse Model of Li-Fraumeni Syndrome.

Germline mutations of TP53, which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid β-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the p53 R172H knock-in mutation (p53172H/H , homolog of the human TP53 R175H LFS mutation) with myoglobin-knockout (MB-/- ) mice known to have decreased FAO. MB-/- p53172H/H double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the p53 R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of MB. The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of MB. To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of p53172H/H mice using a Cre-loxP-mediated strategy. The heterozygous knockout of CPT2 resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with MB disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention. PREVENTION RELEVANCE: Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of TP53, dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.

Molecular dynamics study on the inhibition mechanisms of ReACp53 peptide for p53–R175H mutant aggregation

p53 mutant aggregation can lead to loss-of-function (LoF), dominant-negative (DN) and gain-of-function (GoF) effects, involved in tumor growth.

Berberine Inhibits Cell Proliferation by Interfering with Wild-Type and Mutant P53 in Human Glioma Cells.

IntroductionGlioma is the most common malignant brain tumor. TP53 is the most common mutant gene in human cancer. Wild-type p53 (wtp53) is a tumor suppressor protein whereas mutant p53 (mutp53) is an oncoprotein that promotes tumor cell proliferation. Our aim was to examine the inhibitory effects of berberine on the proliferation of human glioma cells via regulation of wtp53, mutp53, and their downstream molecules.MethodsWe selected wtp53 cells (U87 cells) and mutp53 cells (U251 cells termed p53 R273H) to examine the inhibitory effects of berberine on human glioma cells. We used the CCK-8 kit to detect the toxic effect of berberine. Flow cytometry was used to detect the effect of berberine. Clone formation test was used to test the inhibitory effect of berberine on the proliferation of glioma cells. Western blot was used to detect the changes of related proteins such as p53, p-p53, p21 and cyclin D1. Lentivirus transduction was used to transduce wild-type p53 into U251 cells to further examine the effect of berberine. The nude mouse subcutaneous tumor model was used to detect the effect of berberine on inhibiting the proliferation of glioma cells in vivo.ResultsBerberine promoted the phosphorylation of wtp53, increased the expression of p21 protein, reduced cyclin D1 content, and caused G1 phase arrest in U87 cells. Berberine also reduced mutp53 content and caused G2 phase arrest in U251 cells with a concurrent decrease in p21, cyclin D1, and cyclin B1 content. Transduction with wtp53 enhanced the effects on cell cycle arrest. Further, berberine significantly inhibited glioma growth in vivo mouse tumor model.DiscussionGlioma is a group of heterogeneous brain tumors with unique biological and clinical characteristics. Berberine can inhibit glioma cells through a variety of ways. Our research indicated that berberine inhibited the proliferation of glioma cells by interfering with wtp53 and mutp53. This indicates that berberine could be used as a potential drug to treat wild-type and mutant p53 glioma.

BRAF G469A, BRAF L597V, FGFR1 N546K, FGFR1 K656E and P53 R273H mutations: How are they distributed in Moroccan patients with pediatric Low-Grade Gliomas?

BackgroundLow-Grade Gliomas are common brain tumors in children. They are known for their heterogeneity, especially their molecular heterogeneity. This makes their definition and classification based only on histological criteria difficult and lacking in precision. If this presented a limit to progress in the understanding of these tumors, advances in molecular biology and molecular genetics have allowed us to deepen our knowledge of the nature of these tumors and the way they develop, which helps us considerably in improving their classification, diagnosis and treatment. AimWe identified the distribution of BRAF G469A, BRAF L597V, FGFR1 N546K, FGFR1 K656E and P53 R273H mutations in Moroccan patients with pLGGs, according to their sex, age, brain tumor location and subtype and we compared the results with their distribution in Moroccan patients with pHGGs. MethodologyWe studied BRAF G469A, BRAF L597V, FGFR1 N546K, FGFR1 K656E and P53 R273H mutations in 89 pLGG tissues by comparing them with 123 hLGG tissues, using PCR-RFLP and TaqMan. Data was analyzed by using IBM SPSS software v24. ResultsBy studying the involvement of the BRAF G469A mutation, the BRAF L597V mutation, the FGFR1 N546K mutation, the FGFR1 K656E mutation and the P53 R273H mutation in the development of Pediatric Low-Grade Gliomas, we obtained significant results for the BRAF L597V mutation. 6.7% of the pLGG patients studied were carriers of this mutation (P = 0.0450). (2/2) of these patients with the tumor localized in the hemispheres, (1/8) in the SAI nervous system and (3/29) in the spinal cord were positive. As for the P53 R273H mutation, (19/78) of the pLGG positive cases were Pilocytic Astrocytomas, (1/4) Pilomyxoid Astrocytomas and (4/6) Diffuse Gliomas. By studying the different interactions between the mutations, we obtained significant interactions between the presence of the BRAF L597V mutation and the absence of the BRAF G469A mutation (P = 0.0415), the presence of the BRAF L597V mutation and the absence of the FGFR1 N546K mutation (P = 0.0448) and the presence of the FGFR1 K656E and P53 R273H mutations (P = 0.0056). ConclusionIn our future studies, we plan to continue in the same vein and explore other mutations suspected to be involved in the development of pediatric Low-Grade Gliomas. Studying them would be an opportunity to understand the origin of these tumors but also a field to improve their classification and thus their diagnosis and treatment.

Gb3-cSrc complex in glycosphingolipid-enriched microdomains contributes to the expression of p53 mutant protein and cancer drug resistance via β-catenin-activated RNA methylation.

Glucosylceramide synthase (GCS) is a key enzyme catalyzing ceramide glycosylation to generate glucosylceramide (GlcCer), which in turn serves as the precursor for cells to produce glycosphingolipids (GSLs). In cell membranes, GSLs serve as essential components of GSL‐enriched microdomains (GEMs) and mediate membrane functions and cell behaviors. Previous studies showed that ceramide glycosylation correlates with upregulated expression of p53 hotspot mutant R273H and cancer drug resistance. Yet, the underlying mechanisms remain elusive. We report herewith that globotriaosylceramide (Gb3) is associated with cSrc kinase in GEMs and plays a crucial role in modulating expression of p53 R273H mutant and drug resistance. Colon cancer cell lines, either WiDr homozygous for missense‐mutated TP53 (R273H+/+) or SW48/TP53‐Dox bearing heterozygous TP53 mutant (R273H/+), display drug resistance with increased ceramide glycosylation. Inhibition of GCS with Genz‐161 (GENZ 667161) resensitized cells to apoptosis in these p53 mutant‐carrying cancer cells. Genz‐161 effectively inhibited GCS activity, and substantially suppressed the elevated Gb3 levels seen in GEMs of p53‐mutant cells exposed to doxorubicin. Complex formation between Gb3 and cSrc in GEMs to activate β‐catenin was detected in both cultured cells and xenograft tumors. Suppression of ceramide glycosylation significantly decreased Gb3‐cSrc in GEMs, β‐catenin, and methyltransferase‐like 3 for m6A RNA methylation, thus altering pre‐mRNA splicing, resulting in upregulated expression of wild‐type p53 protein, but not mutants, in cells carrying p53 R273H. Altogether, increased Gb3‐cSrc complex in GEMs of membranes in response to anticancer drug induced cell stress promotes expression of p53 mutant proteins and accordant cancer drug resistance.

Abstract 3429: Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer

Abstract We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1 (Xiao et. al., Cancer Research 2019). The missense R273H GOF mtp53, without a functional central DNA binding domain, has the capacity to interact tightly with chromatin but it is unclear what regions of mtp53 are responsible for the chromatin-based DNA replication activities. GOF mtp53 proteins possess an intact C-terminal domain (CTD) and oligomerization domain (OD). In wild-type p53 the CTD and OD domain regulate non-specific DNA binding, post-translational modification, and protein tetramerization. We are targeting the CTD and OD regions of mtp53 to test if their disruption limits oncogenic GOF activity. We generated CRISPR/Cas9 edited R273H CTD and OD in the TNBC cell line MDA-MB-468, and also exogenously expressed such dual mutant proteins in p53 null cancers, to evaluate the roles played by oligomerization and non-specific DNA binding in mtp53 interactions with replicating DNA and PARP1. We determined the oligomerization state of R273H mtp53 using the glutaraldehyde crosslinking assay and observed that GOF mtp53 R273H mtp53 in breast cancer cells exists as a tetramer. Experiments are in progress to evaluate cell cycle progression, replication initiation, and mtp53 chromatin interactions in cells expressing missense mtp53 proteins with co-associated mutated CTDs or ODs. Our preliminary data suggests that disrupting the mtp53 CTD inhibits its interactions with chromatin and progression through the S-phase. Unpublished data on the roles played by the mtp53 CTD and OD in DNA replication will be presented at the 2020 AACR meeting. Acknowledgements: This work was funded by grants from the Breast Cancer Research Foundation BCRF-18-011 and BCRF-19-011 to Jill Bargonetti. Devon Lundine and Gu Xiao also received project support from TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership, Award Number U54 CA221704(5) from the National Cancer Institute. Special thanks to Ella Freulich for help with CRISPR/Cas9 work and James J. Manfredi for mutant p53 plasmids to exogenously express protein. Citation Format: Devon Lundine, George Annor, Viola Ellison, Gu Xiao, Carol Prives, Jill Bargonetti. Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3429.

Abstract 2962: Molecular mechanisms underlying the sensitizing effect of mutant p53 protein expression for Auranofin treatment of NSCLC and PDAC cells

Abstract Introduction: Auranofin (AF) is an orally available, organogold compound that is FDA-approved for its use in rheumatoid arthritis. AF gained interest as an anti-cancer agent since it acts as a thioredoxin reductase (TrxR) inhibitor. Data extracted from The Cancer Genome Atlas show that TXNRD1 is abundantly expressed in lung adenocarcinoma patients and negatively affects their outcome, making TrxR1 an attractable target in non-small cell lung cancer (NSCLC) patients. The goal of this study was to determine if mutant p53 expression can be used as a predictive biomarker for the treatment of AF in NSCLC. In addition, we aimed to obtain a profound understanding of the molecular mechanisms that govern the sensitizing effect of mutant p53 towards AF-treated NSCLC cells by investigating the type of cell death. Material and methods: We determined IC50-values using the sulforhodamine-B assay for AF in a panel of NSCLC and pancreatic ductal adenocarcinoma (PDAC) cell lines with different p53 mutations and expression levels. In addition, we correlated these IC50-values to baseline expression levels of different antioxidant related proteins. Results were confirmed in the NSCLC cell line NCI-H1299 (p53 deletion, null) and its two isogenic derivates (mutant p53 R175H or R273H knock-in). mRNA-Seq of the isogenic cell line panel was performed after exposure to a medium dose of AF. Finally, the IncuCyte ZOOM system was used to determine the percentage of AF-induced cell death by the means of the Cytotox Green reagent after pretreatment of the tumor cells with a panel of apoptosis, necroptosis and ferroptosis inhibitors. Results: A strong inverse correlation was observed between mutant p53 protein expression and AF IC50-values and baseline Trx levels in the NSCLC and PDAC cells, indicating that mutant p53 can sensitize cancer cells for AF. This sensitizing effect was confirmed in the p53Mutant isogenic NSCLC cells. Transcriptome analysis revealed the up- and downregulation of specific genes involved in ferroptosis. More specifically, AF induced cell death through various mechanisms, including apoptosis and ferroptosis, which were dependent on the expression levels of p53 rather than the type of p53 mutation. In cells with high expression levels of mutant p53, AF induced ferroptosis and lipid peroxidation. At lower expression levels of p53, the apoptosis pathway was triggered by AF with an increase in caspase-3/7 positive cells and inhibition of cell death by the caspase inhibitor Z-VAD-FMK. Conclusion: Mutant p53 protein expression renders NSCLC and PDAC cells more susceptible to AF. In an isogenic setting, medium levels of mutant p53 sensitize NSCLC cells for apoptosis while cells with higher levels are effectively killed through ferroptosis. Altogether, AF presents a potential novel therapeutic strategy to efficiently kill p53 mutated NSCLC tumor cells by various types of cell death. Citation Format: Laurie Freire Boullosa, Filip Lardon, Evelien Smits, Christophe Deben. Molecular mechanisms underlying the sensitizing effect of mutant p53 protein expression for Auranofin treatment of NSCLC and PDAC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2962.

MDM2-C Functions as an E3 Ubiquitin Ligase.

BackgroundMouse double minute 2 (MDM2) is an E3 ubiquitin ligase that is over-expressed in many cancers and regulates target proteins through ubiquitination. Full-length MDM2 (MDM2-FL) is best known for targeting wild-type p53 for degradation by the proteasome, but the functions of the many splice variants of MDM2 are under-explored. The three well-studied alternative MDM2 isoforms are MDM2-A/ALT2, MDM2-B/ALT1, and MDM2-C/ALT3. MDM2-A and MDM2-B are capable of down-regulating MDM2-FL activity and have transforming activity in cancers with mutant p53. The MDM2 isoform MDM2-C is over-expressed in breast cancer and correlates with decreased survival in the context of mutant p53 expression. Therefore, MDM2-C requires further study to determine if it has biochemical activities similar to MDM2-FL. Hypothesis: We hypothesized that like MDM2-FL, the MDM2-C isoform (lacking exons 5–9 and containing a full C-terminal RING finger sequence) would maintain E3 ubiquitin ligase activity.Materials and MethodsIn order to explore the biochemical function of MDM2-C, we used an in vitro ubiquitination assay and a glutaraldehyde cross-linking assay.ResultsHere we report, for the first time, that MDM2-C has E3 auto-ubiquitin ligase activity, which can promote ubiquitination of wild-type p53 and mutant p53 R273H, and also can form a protein–protein interaction with p53 proteins.ConclusionThis information strongly positions MDM2-C as a protein with biochemical activities that may explain the varied outcomes observed in patients with high-level expression of MDM2-C in the presence of wild-type p53 versus mutant p53.

The Mutant p53-Driven Secretome Has Oncogenic Functions in Pancreatic Ductal Adenocarcinoma Cells

The cancer secretome is a rich repository of useful information for both cancer biology and clinical oncology. A better understanding of cancer secretome is particularly relevant for pancreatic ductal adenocarcinoma (PDAC), whose extremely high mortality rate is mainly due to early metastasis, resistance to conventional treatments, lack of recognizable symptoms, and assays for early detection. TP53 gene is a master transcriptional regulator controlling several key cellular pathways and it is mutated in ~75% of PDACs. We report the functional effect of the hot-spot p53 mutant isoforms R175H and R273H on cancer cell secretome, showing their influence on proliferation, chemoresistance, apoptosis, and autophagy, as well as cell migration and epithelial-mesenchymal transition. We compared the secretome of p53-null AsPC-1 PDAC cells after ectopic over-expression of R175H-mutp53 or R273H-mutp53 to identify the differentially secreted proteins by mutant p53. By using high-resolution SWATH-MS technology, we found a great number of differentially secreted proteins by the two p53 mutants, 15 of which are common to both mutants. Most of these secreted proteins are reported to promote cancer progression and epithelial-mesenchymal transition and might constitute a biomarker secreted signature that is driven by the hot-spot p53 mutants in PDAC.

Statin as anti-cancer therapy in autochthonous T-lymphomas expressing stabilized gain-of-function mutant p53 proteins

An important component of missense mutant p53 gain-of-function (mutp53 GOF) activities is the ability of stabilized mutp53 proteins to upregulate the mevalonate pathway, providing a rationale for exploring the statin family of HMG-CoA reductase inhibitors as anticancer agents in mutp53 tumors. In this small exploratory study we report on the effects of statin treatment in autochthonous mouse models of clinically advanced T-cell lymphoma expressing two different GOF mutp53 alleles. We find that Rosuvastatin monotherapy shows a modest, p53 allele-selective and transient anti-tumor effect in autochthonous T-lymphomas expressing the p53 R248Q DNA contact mutant, but not in tumors expressing the p53 R172H conformational mutant. p53 null mice also do not benefit. In vitro statin sensitivity is not a strong predictor for in vivo sensitivity, while subcutaneous allografts are. Future explorations of statins in combination therapies are justified to improve its anti-tumor effects and to better define the most statin-sensitive alleles and tumor types among mutp53-stabilized cancers.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer.

Over 80% of triple-negative breast cancers (TNBC) express mutant p53 (mtp53) and some contain oncogenic gain-of-function (GOF) p53. We previously reported that GOF mtp53 R273H upregulates the chromatin association of mini chromosome maintenance (MCM) proteins MCM2-7 and PARP and named this the mtp53-PARP-MCM axis. In this study, we dissected the function and association between mtp53 and PARP using a number of different cell lines, patient-derived xenografts (PDX), tissue microarrays (TMA), and The Cancer Genome Atlas (TCGA) database. Endogenous mtp53 R273H and exogenously expressed R273H and R248W bound to nascent 5-ethynyl-2´-deoxyuridine-labeled replicating DNA. Increased mtp53 R273H enhanced the association of mtp53 and PARP on replicating DNA. Blocking poly-ADP-ribose gylcohydrolase also enhanced this association. Moreover, mtp53 R273H expression enhanced overall MCM2 levels, promoted cell proliferation, and improved the synergistic cytotoxicity of treatment with the alkylating agent temozolomide in combination with the PARP inhibitor (PARPi) talazoparib. Staining of p53 and PARP1 in breast cancer TMAs and comparison with the TCGA database indicated a higher double-positive signal in basal-like breast cancer than in luminal A or luminal B subtypes. Higher PARP1 protein levels and PAR proteins were detected in mtp53 R273H than in wild-type p53-expressing PDX samples. These results indicate that mtp53 R273H and PARP1 interact with replicating DNA and should be considered as dual biomarkers for identifying breast cancers that may respond to combination PARPi treatments. SIGNIFICANCE: p53 gain-of-function mutant 273H and PARP1 interact with replication forks and could serve as potential biomarkers for breast cancer sensitivity to PARP inhibitors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/394/F1.large.jpg.

Abstract C22: Loss of KDM6A promotes pancreatic cancer progression by upregulating epithelial-mesenchymal transition pathway

Abstract Metastasis is one of the main reasons for poor survival in patients with pancreatic ductal adenocarcinoma (PDA). However, the molecular mechanism of PDA metastasis remains elusive. Recent studies failed to find metastasis-specific genetic mutations, and thus suggested that aberrant epigenetic regulation may be one of the crucial mechanisms. Many epigenetic regulator genes are altered in PDA, including KDM6A, a histone H3K27 demethylase, which has been identified by whole-genomic mutational analyses as a driver of PDA. Recent studies have shown that loss of KDM6A induces aggressive squamous-like PDA by activing superenhancers and promotes metastasis. However, the underlying mechanism by which KDM6A promotes metastasis remains elusive. Using stable KDM6A knockout PDA cell lines established by CRISPR/Cas9 system and siRNA, we knocked out or knocked down the expression of KDM6A in PDA cells. We also established an inducible KDM6A re-expressed PDA cell line that has homozygous KDM6A deletion. A novel Bru-seq technology was used to determine global changes of nascent RNA synthesis in KDM6A-null cells. Cell migration and invasion was assessed in vitro. To study the impact of KDM6A loss in vivo, we crossed KDM6Afl/fl mice with the well-established Ptf1aCre; Kras G12D; p53 R172H (KPC) PDA mouse model. Bru-seq data have shown that epithelial-mesenchymal transition (EMT) pathway is highly upregulated in KDM6A knockout cells and downregulated in KDM6A re-expressed PDA cells. EMT is a well-known process that is involved in tumor metastasis. KDM6A knockout cells demonstrated decreased cell proliferation but increased cell migration and invasion with mesenchymal cell morphology changes in vitro. Hallmark epithelial marker E-cadherin was significantly downregulated in KDM6A knockout cells, supporting the EMT process. Furthermore, genetic mouse model showed that mice that lose KDM6A expression developed PDA and metastasis much earlier than KPC mice. The morphology of the PDA developed in KDM6A-null KPC mice is more sarcomatoid or mesenchymal compared to PDA in KPC mice. Immunohistochemistry analysis also demonstrated loss of epithelial marker E-cadherin and gain of mesenchymal marker vimentin in tumor cells. In summary, these results suggest that loss of KDM6A promotes PDA development and metastasis by upregulating EMT signaling. These data linked epigenetic regulation to EMT signaling and provided insights into the role of KDM6A in PDA development. These novel findings also provided foundation for developing new therapeutic strategies. Citation Format: Sivakumar Jeyarajan, Zhujun Yi, Shanqiao Wei, Shuang Lu, Hong Kim, Michelle Paulsen, Karan Bedi, Mats Ljungman, Jiaqi Shi. Loss of KDM6A promotes pancreatic cancer progression by upregulating epithelial-mesenchymal transition pathway [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C22.

Ubiquitination of the HPV Oncoprotein E6 Is Critical for E6/E6AP-Mediated p53 Degradation.

High-risk Human papillomaviruses (HPVs) types are associated with more than 90% of premalignant and malignant squamous lesions of the uterine cervix. The E6 oncoprotein of high-risk HPVs is a key determinant in cell transformation because it induces the degradation of the host pro-apoptotic tumor suppressor p53. E6 recruits the intracellular ubiquitin ligase E6AP and subsequently induces proteasome-dependent p53 degradation. Neither E6 nor E6AP alone interact with p53; however, the precise mechanism of the functional regulation of the E6/E6AP/p53 complex is unclear. Here, we showed that the high-risk HPV E6 proteins are ubiquitinated during E6/E6AP/p53 complex assembly and degraded by the proteasome system. Increasing p53 expression enhanced E6/E6AP/p53 assembly and facilitated E6 ubiquitination and degradation. The dominant negative mutant of p53 R175H, which does not efficiently bind E6, decreased E6 ubiquitination and increased stability. Furthermore, we showed that the ubiquitin ligase E6AP is essential for E6 ubiquitination, and downregulation of E6AP expression increased E6 stability. We also showed that p53 R175H inhibited E6-mediated p53 degradation. Consistently, the host deubiquitinating enzyme USP15 removed ubiquitin chains from E6 proteins and inhibited E6-mediated p53 degradation. Crucially, ectopic expression of either p53 R175H or USP15 promoted p53-triggered apoptosis in human cervical cancer cells. Considering the importance of ubiquitinated E6 on p53 degradation, the disruption of E6 ubiquitination represents an attractive pharmacological intervention against HPV-positive human cervical cancer.ImportanceVirtually 100% of cervical cancers are linked to HPV infection. Commercial HPV vaccines are estimated to prevent up to 90% of HPV-associated cancers, while they do not eliminate persistent HPV infections and have no effect on the progression to malignancy. Hence, the development of novel therapeutic interventions against HPV is urgently required. The HPV oncoprotein E6 binds to the intracellular E3 ubiquitin ligase E6AP and p53 resulting in the degradation of p53. In this study, we demonstrate that HPV E6 is ubiquitinated by E6AP in presence of p53. Crucially, ubiquitination of E6 is important for p53 degradation and blockage of E6 ubiquitination negatively interferes with E6-mediated p53 degradation and enhances the apoptotic effects of p53 and the cytotoxicity of DNA damage in HPV-positive cervical cancer cells. Importantly, our data suggest that the HPV oncogene E6 might be an optimal pharmacologic.