Reactivation Of P53 Research Articles

An unusual liver tumor with challenging morphologic features and immunophenotype: A case report of metastatic HPV-driven basaloid anal squamous cell carcinoma

Abstract Introduction/Objective Basaloid squamous cell carcinoma (BSCC), a rare subtype of squamous cell carcinoma, occurs most commonly in esophagus, lungs, and head and neck. Anal BSCC is extremely rare and has a 10-20% rate of metastasis. High-risk HPV (HR-HPV) is the main causative agent. BSCC occurrence in an atypical site without a known primary tumor, and with atypical immunoprofile can be a diagnostic challenge. Methods/Case Report We report an 83-year-old female with history of breast carcinoma who presented for worsening chest pain. Imaging studies showed pulmonary nodules, 1.8 cm hepatic mass, and pelvic nodules. Liver biopsy showed infiltrating nests of carcinoma with solid and cribriform patterns and luminal necrosis. Some tumor nests had basaloid features with peripheral palisading. Tumor was positive for CK7 and pankeratin; and negative for ER, SOX10, GATA3, GCDFP-15 (Breast markers), CDX2 (GI marker), P63 and P40 (squamous markers), INSM1, Synaptophysin (Neuroendocrine) and TTF1 (lung adenocarcinoma). There was microfocal staining with CK20 and SATB2. Tumor morphology and immunophenotype were not entirely specific to the site of origin. Further search into patient’s medical history showed a recent anal mucosal biopsy revealing a high-grade squamous epithelial lesion with diffuse P16 reactivity. The slides for the anal lesion were reviewed and showed morphologic features similar to the liver biopsy. Additional studies showed both tumors were positive for HR-HPV ISH, P16 (diffuse), and CK5/6 (patchy); and negative for P63. The morphologic features and immunoprofile were supportive of metastasis from HPV-driven BSCC of anus. Subsequent PET Scan revealed a rectal/anal mass. Results (if a Case Study enter NA) NA Conclusion For tumors with unusual morphology and immunophenotype, complete medical history review, and communication with clinician are important tools in achieving correct diagnosis. HR-HPV ISH is a new tool that can be very useful in reaching correct diagnosis in metastatic anogenital or oropharyngeal squamous carcinomas with basaloid features.

Antibacterial Peptide Nisin Activates p53 Gene Expression and Triggers Apoptosis in Human Colon Cancer Cells

ABSTRACTAntimicrobial peptides (AMPs) are renowned for their broad‐spectrum efficacy against viruses, bacteria, and fungi, thereby holding promise for medicinal applications. Nisin, a bacteriocin synthesized by gram‐positive bacteria such as Lactobacillus and Streptococcus, has been used as a food preservative owing to its antimicrobial characteristics for decades. However, recent developments prompt further research on its application as therapeutic interventions, mainly focusing on anticancer activity. In line with this, the present study portrays the effects of nisin on human colorectal cancer cells and the underlying molecular mechanisms. A dose‐dependent decrease in cell viability with increasing concentrations of nisin was determined by the Alamar blue viability assay, which was supported by significant morphological changes in cells. Subsequently, we probed the apoptotic effects of nisin using EtBr/AO staining, confirming the induction of apoptosis upon nisin treatment, which was further confirmed by ELISA analysis. Elevated levels of Caspase 7 indicated increased apoptosis in nisin‐treated cells. As pharmaceutical p53 reactivation is considered as an attractive strategy to combat chemoresistance and adverse effects, the effect of nisin on p53 expression was determined by mRNA analysis. An upregulation in p53 expression in nisin‐treated cells compared to untreated controls suggests that bacterial nisin reactivates mutated p53. To assess cell cycle dynamics, we utilized PI flow cytometry, unveiling a G0/G1 phase arrest in nisin‐treated cells in contrast to untreated controls. In summary, our results strongly advocate the potential of nisin as a compelling candidate for drug development against colorectal cancer.

Abstract A026: Studying the synergistic potential of a p53 reactivator with ROS-mediated therapies for the treatment of pancreatic cancer

Abstract Introduction: Pancreatic cancer, with a dismal five-year survival rate of just 12%, demands innovative approaches due to its resistance to current therapies. TP53 mutations, present in 75% of patients, make mutant p53 an appealing target. Thus, p53 reactivation, restoring its tumour suppressor functions, represents a promising strategy to address this challenge. While the proposed reactivator has shown efficacy in other malignancies, its potential in pancreatic cancer therapy requires further exploration. Methods: In this project, we have assessed the potential of repurposing a reactivator of mutant p53 protein, to inactivate pancreatic cancer cells. We have performed RNA-sequencing analysis in patient-derived models available in the lab, to study the signalling pathways regulated by the p53 reactivator anticancer activity. We have also studied the possibility to synergistically combine this repurposing strategy with reactive oxygen species (ROS)-mediated therapeutic approaches, as we previously observed p53 reactivation inhibits the glutathione antioxidant pathway. Results: The combination treatment induced synergistic cell death (3-fold increase) compared to monotherapy as evaluated by different molecular assays (e.g. MTT and Live/Death Assay). It also increased ROS levels (Flow cytometry) and induced paraptosis and apoptosis (Western Blot, Tomography Electron Microscopy). The RNA-seq data revealed differential expression of more than 200 genes, confirming the activation of oxidative stress and a cellular response to unfolded proteins. Moreover, Sanger sequencing results demonstrated that the sensibility of each pancreatic model to treatment was dependent on the mutational status of TP53. Conclusion/discussion: The combination therapy of a TP53 reactivator and a ROS mediated therapeutic strategy works synergistically in patient derived models, inducing cell death and changes in the expression of certain RNAs directly involved in unfolded protein response. This pilot study shows that this combination is a feasible strategy to treat pancreatic cancer, setting the path for its testing on in vivo models. Citation Format: Daniel Parra Sánchez, Andrés García-Sampedro, Isabela Laughland, Sylvain Peuget, Galina Selivanova, Stephen P Pereira, Pilar Acedo. Studying the synergistic potential of a p53 reactivator with ROS-mediated therapies for the treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A026.

Astilbin Induces Apoptosis in Oral Squamous Cell Carcinoma through p53 Reactivation and Mdm-2 Inhibition.

Oral squamous cell carcinoma (OSCC) is a frequently occurring malignancy in the head and neck region. The most commonly mutated gene in OSCC is the tumor suppressor gene p53 (TP53), linked to lower survival and treatment resistance in OSCC patients. Astilbin is a flavonoid amongst several herbal treatments with a variety of pharmacological actions mainly including antioxidant, anti-inflammatory, and anti-cancer characteristics. This study evaluated the effects of astilbin on proliferation of OSCC cell lines SCC90 and SCC4 (bearing a p53 mutation) in relevance to p53 and Mdm-2 pathways. Astilbin inhibited the proliferation of SCC4 and SCC90 cells in a dose- and time-dependent manner. The IC50 values for both the cell lines were about 75 μM for astilbin. A p53 activator (RITA) was used to determine the effects of astilbin on p53 activity, and the results demonstrated synergistic reduction in cell growth. However, when combined with pifithrin-α (a p53 inhibitor), astilbin demonstrated a strong inhibition of its response. Astilbin reduced the mitochondrial membrane potential in SCC4 cells, which is a sign of apoptotic activity. Astilbin decreased the amounts of Mdm-2 (negative regulator of p53) and increased the expression of the p53 gene and protein. In a p53-dependent manner, astilbin suppressed the ability of SCC4 cells to form colonies and heal wounds. This was followed by the induction of mitochondrial intrinsic apoptosis via the activation of caspases 9 and 3, cleavage of PARP, and the suppression of pro-apoptotic Bid. Astilbin-induced p53-mediated apoptosis in OSCC cells as herbal medicinal ingredients.

An MDM2 degrader shows potent cytotoxicity to MDM2-overexpressing acute lymphoblastic leukemia cells with minimal toxicity to normal cells/tissues

The MDM2 oncogene is amplified and/or overexpressed in various human cancers and elevated expression of MDM2 protein acts as a survival factor promoting cancer progression through both p53-dependent and -independent pathways. Here, we report a novel small-molecule chemical compound (MX69-102) that we identified to induce MDM2 protein degradation, resulting in reactivation of p53, inhibition of XIAP, and potent cell growth inhibition and apoptosis in MDM2-overexpressing acute lymphoblastic leukemia (ALL) in vitro and in vivo. We have previously identified a compound (MX69) that binds to the MDM2 C-terminal RING domain and induces MDM2 protein degradation. In the present study, we performed structural modifications of MX69 and selected analog MX69-102, showing increased MDM2-targeting activity. MX69-102 exhibited significantly enhanced inhibitory and apoptotic effects on a group of MDM2-overexpressing ALL cell lines in vitro with IC50 values of about 0.2 μM, representing an approximately 38-fold increase in activity compared to MX69. MX69-102 also showed effective inhibition on xenografted human MDM2-overexpressing ALL in SCID mice. Importantly, MX69-102 had minimal or no inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in vivo in animal models. Based on the strong inhibitory and apoptotic activity against MDM2-overexpressing ALL, along with minimal or no toxicity to normal cells/tissues, MX69-102 is a candidate for further development as a novel MDM2-targeted therapeutic drug for refractory/MDM2-overexpressing ALL.

DIPG-70. NQO1 AS A RESISTANCE DRIVER AND THERAPEUTIC TARGET IN DMG

Abstract p53 pathway reactivation through pharmacologic inhibition of negative regulators was identified as a promising therapeutic strategy for TP53 wild-type (WT) high-grade gliomas, including Diffuse Midline Gliomas (DMGs). Despite widespread theoretical promise, the therapeutic efficacy of MDM2 inhibitors and PPM1D inhibitors as single agents have each been limited by issues, including sub-lethal effects on cancer cells and innate or emerging resistance leading to tumor progression. We carried out a genome-scale CRISPR activating (CRISPRa) screen to identify genes that, when overexpressed, confer resistance to p53 reactivation therapies. NAD(P)H quinone dehydrogenase 1 (NQO1) emerged as a top hit and was confirmed through secondary CRISPRa screens and low-throughput validations as driving a resistance phenotype in TP53 WT DMGs. Our subsequent work has aimed to elucidate the mechanisms through which NQO1 mediates the effects of p53 reactivation therapies, and assess the feasibility and impact of exploiting NQO1 in combination therapies towards a cure.

Targeting mutant p53 with arsenic trioxide: A preclinical study focusing on triple negative breast cancer

New treatments are urgently required for triple-negative breast cancer (TNBC). As TP53 is mutated in approximately 80% of TNBC, it is theoretically an attractive target for new drugs for this disease. Arsenic trioxide (ATO), which is used to treat promyelocytic leukaemia, was recently shown to reactivate mutant p53 and restore wild-type functionality. The aim of this study was to evaluate ATO as a potential new treatment for TNBC. Using a panel of 20 cell lines, we found that TNBC cell lines were more sensitive to ATO than non-TNBC cell lines (P = 0.045). Consistent with its ability to reactivate mutant p53, ATO was a more potent inhibitor of proliferation in cell lines with mutant TP53 than the wildtype TP53 (P = 0.027). Direct evidence of mutant p53 reactivation was the induction of multiple wild-type p53 canonical target genes such as CDKN1A, SLC7A11, BBC3, PMAIP1, SESN2, SRXN1 and TXNRD1. Our findings support the activation of mutant p53 by ATO and, furthermore, the possible repurposing of ATO to treat TP53-mutated TNBC.

TP53 Mutations in Acute Leukemias and Myelodysplastic Syndromes: Insights and Treatment Updates.

TP53 mutations are found in 5%-10% of de novo myelodysplastic syndrome (MDS) and AML cases. By contrast, in therapy related MDS and AML, mutations in TP53 are found in up to 30%-40% of patients. The majority of inactivating mutations observed in MDS and AML are missense mutations localized in a few prevalent hotspots. TP53 missense mutations together with truncating mutations or chromosomal loss of TP53 determine a loss-of-function effect on normal p53 function. Clonal expansion of TP53-mutant clones is observed under the selection pressure of chemotherapy or MDM2 inhibitor therapy. TP53-mutant clones are resistant to current chemotherapy, and when responses to treatment have been observed, they have correlated poorly with overall survival. The most heavily investigated and targeted agent for patients with TP53-mutant MDS and AML has been APR-246 (eprenetapopt) a p53 reactivator, in combination with azacitidine, but also in triplets with venetoclax. Despite positive results in phase II trials, a phase III trial did not confirm superior response or improved survival. Other agents, like magrolimab (anti-CD47 antibody), failed to demonstrate improved activity in TP53-mutant MDS and AML. Agents whose activity is not dependent on a functional apoptosis system like anti-CD123 antibodies or cellular therapies are in development and may hold promises. Delivering prognostic information in a dismal disease like TP53-mutated MDS and AML is particularly challenging. The physician should balance hope and realism, describing the trajectory of possible treatments and at the same time indicating the poor outcome, together with promoting adaptive coping in patients and elaborating on the nature of the disease.

Mutant p53 reactivators protect breast cancer cells from ferroptosis.

Ferroptosis is a novel nonapoptotic form of cell death characterized by iron-dependent reactive oxygen species-mediated lipid peroxidation. In several different cell systems, the tumor suppressor p53 can enhance sensitivity to ferroptotic inducers. At least half of all human cancers show loss of function of p53. Furthermore, many of those tumors express mutant forms of p53 that has lost its wild-type function. Several groups have designed small molecules that can reactivate the wild-type function of these missense p53 mutants. We reasoned that p53 reactivators may also enhance sensitivity of certain cancer cells to ferroptosis stimuli. To test this idea we combined a number of different p53 reactivators with small molecule inducers of ferroptosis. In contrast, we observed that several p53 reactivators protected cells from cell death induced by ferroptotic inducers. Surprisingly, this protection still occurred in p53-null cell lines. We observed that these reactivators were neither free radical scavengers nor ion chelators. One of these p53 reactivator molecules, NSC 59984, reduced expression of GPX4, which is unlikely to explain its ability to reduce sensitivity to ferroptosis. We suggest that these p53 reactivators function via an unknown, p53-independent manner to suppress ferroptosis.

Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Reconnaissance of Allostery via the Restoration of Native p53 DNA-Binding Domain Dynamics in Y220C Mutant p53 Tumor Suppressor Protein.

Allosteric regulation of protein dynamics infers a long-range deliberate propagation of information via micro- and macroscale interactions. The Y220C structural mutant is one of the most frequent cancerous p53 mutants. The mutation is distally located from the DNA-binding site of the p53 DNA-binding domain yet causes changes in DNA recognition. This system presents a unique opportunity to examine the allosteric control of mutated proteins under a drug design paradigm. We focus on the key case study of p53 Y220C mutation restoration by a series of new compounds suggested to have Y220C reactivation properties in comparison to our previous findings on the restorative potential of PK11000, a compound studied extensively for reactivation in vitro and in vivo. Previously, we implemented all-atom molecular dynamics (MD) simulations and our lab's techniques of MD-Sectors and MD-Markov state models on the wild type, the Y220C mutant, and Y220C with PK11000 to characterize the effector's restorative properties in terms of conformational dynamics and hydrogen bonding. In this study, we turn to probing the effects made by docking the battery of a new but less well-tested set of aminobenzothiazole derivative compounds reported by Baud et al., which show promise of Y220C rescue. We find that while complete and precise reconstitution of p53 WT molecular dynamics may not be observed as was the case with PK11000, dispersed local reconstitution of loop dynamics provides evidence of rescuing effects by aminobenzothiazole derivative N,2-dihydroxy-3,5-diiodo-4-(1H-pyrrol-1-yl)benzamide, Effector 22, like what we observed for PK11000. Generalizable insights into the mutation and allosteric reactivation of p53 by various effectors by reconstitution of WT dynamics observed in statistical conformational ensemble analysis and network inference are discussed, considering the development of allosteric drug design rooted in first principles.

Combination p53 activation and BCL-xL/BCL-2 inhibition as a therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

Due to the rarity of TP53 mutations in acute lymphoblastic leukemia (ALL), p53 re-activation by antagonism of the p53-MDM2 interaction represents a potential therapeutic strategy for the majority of ALL. Here, we demonstrate the potent antileukemic activity of the MDM2 antagonist idasanutlin in high-risk and relapsed ex vivo coculture models of TP53 wildtype ALL (n = 40). Insufficient clinical responses to monotherapy MDM2 inhibitors in other cancers prompted us to explore optimal drugs for combination therapy. Utilizing high-throughput combination screening of 1971 FDA-approved and clinically advanced compounds, we identified BCL-xL/BCL-2 inhibitor navitoclax as the most promising idasanutlin combination partner. The idasanutlin-navitoclax combination was synergistically lethal to prognostically-poor, primary-derived and primary patient blasts in ex vivo coculture, and reduced leukemia burden in two very high-risk ALL xenograft models at drug concentrations safely attained in patients; in fact, the navitoclax plasma concentrations were equivalent to those attained in contemporary “low-dose” navitoclax clinical trials. We demonstrate a preferential engagement of cell death over G1 cell cycle arrest, mechanistically implicating MCL-1-binding pro-apoptotic sensitizer NOXA. The proposed combination of two clinical-stage compounds independently under clinical evaluation for ALL is of high clinical relevance and warrants consideration for the treatment of patients with high-risk and relapsed ALL.

Abstract 261: Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells

Abstract Background. Stem-like cells contribute to the development and progression of human cancers. Genes that control symmetric cell divisions have an evolutionarily conserved role in the regulation of cell polarity and in tumorigenesis. Two cells generated by asymmetric divisions have reduced proliferative potential compared with symmetric divisions. The cell survival oncoprotein Akt (Protein Kinase B) is encoded by three isoforms that are altered in human cancers. The Akt isoform-specific impact on breast cancer stem cell function and polarity is controversial, with Akt1 either reducing (1) or promoting (2) cell polarity and migration in tissue culture. Deletion of Akt1 reduced mammary tumor progression and metastasis in transgenic mice (2,3). Methods. Analysis of Akt1, cyclin D1 and Notch signaling was conducted from public databases, and human as well as murine breast cancer samples. Cells derived from Akt1 gene deletion MMTV-ErbB2 breast oncomice (MMTV-ErbB2/Akt1+/+ and MMTV-ErbB2/Akt1−/−) and from the human MCF-7 breast cancer cell line with regulated expression of cyclin D1 and p53 were analyzed for the symmetry of cell division, stem cell function, and signaling pathways. Results. In breast tumors derived from MMTV-ErbB2 transgenics, associated with reduced metastasis, Akt1 genetic deletion reduced the frequency of symmetric cell divisions and the production of mammospheres. Akt1 deletion increased p53 and reduced cyclin D1. Pharmacological reactivation of p53 with Nutlin correlated with the reinduction of asymmetric cellular divisions and reduction in mammospheres in murine and human BCa cells. Akt1 deletion abrogated Nutlin-mediated induction of asymmetric cell divisions. Cyclin D1 induced symmetrical cell divisions, mammosphere formation and reduced p53 and Numb. Conclusions. Akt1 promoted symmetrical cell division at least in part via cyclin D1 suppression of Numb, and induction of γ secretase activity and thereby Notch signaling. Reference. (1) Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol 2005;171:1023-34. (2) Ju X, Katiyar S, Wang C, Liu M, Jiao X, Li S, et al. Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci U S A 2007;104:7438-43. (3) Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, et al. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer. Mol Cell 2020;80:87-101 e5 Citation Format: Xuanmao Jiao, Xin Sun, Zhiping Li, Xiaoming Ju, Danni Li, Ritika Harish, Gordon Robertson, Anthony W. Ashton, Richard G. Pestell. Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 261.

Abstract A008: Disruption of SLFN5 enhances vulnerability to APR-246, a mutant p53 reactivator in glioblastoma

Abstract Glioblastoma multiforme (GBM) or grade 4 IDHWT glioma is the most malignant brain tumor with a median survival of ~15 months. Current treatment options are limited to a combination of surgery, radiation, and the alkylating agent temozolomide (TMZ). However, despite this aggressive treatment regimen, patients inevitably develop recurrent tumors, which depict a high degree of resistance to DNA damage induced by TMZ and irradiation. Therefore, it is imperative to better understand cellular signals contributing to GBM tumorigenesis and therapy resistance. We have previously established potential roles for members of the protein family of schlafens (SLFNs) in GBM. SLFNs have established roles in cell differentiation, cell proliferation, immune responses, and an expanding role in cancer biology. Several human SLFNs have been linked to chemosensitivity in various cancer types. In particular, SLFN5 mRNA levels progressively increased from grade 2 to grade 4 glioma suggesting a possible therapeutic target for GBM. Therefore, in an effort to better understand the role of SLFN5 in GBM tumorigenesis, CRISPR/Cas9-mediated SLFN5 knockout glioblastoma cell lines and PDX lines were generated. Cells lacking expression of SLFN5 showed increased DNA damage signaling, as judged by elevated basal levels of the DNA damage marker γH2AX and persistent phosphorylation of Chk2. Gene ontology analysis indicated increased expression of p53 pathway components in SLFN5 knockout cells. In GBM, p53 activity is commonly deregulated due to high mutations in the p53 pathway. In line with this, SLFN5 knockout cells depicted increased sensitivity to the mutant p53 reactivator APR-246 correlating with p53 mutational status. APR-246 induced PARP cleavage in SLFN5 knockout cells was rescued by the ROS scavenger N-acetyl-D-cysteine (NAC). Our results suggest increased DNA damage signaling in SLFN5 deficient cells. Furthermore, mutant p53 reactivator APR-246 induced apoptosis is dependent on ROS signaling. In summary, we provide evidence for a protective role of SLFN5 to limit DNA damage and ROS signaling in GBM. Citation Format: Ricardo E. Perez, Frank Eckerdt, Leonidas C. Platanias. Disruption of SLFN5 enhances vulnerability to APR-246, a mutant p53 reactivator in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A008.

APR-246 enhances the anticancer effect of doxorubicin against p53-mutant AsPC-1 pancreatic cancer cells

Pancreatic cancer (PC) is a disease with a high mortality rate, and the early diagnosis of PC is still challenging. The 5-year relative survival rate remains below 8%, and therapeutic strategies are ineffective at increasing patient survival rates. In PC cells, resistance to treatment has been associated with genetic changes that initiate aberrant developmental signaling pathways; therefore, new strategies for treating PC are warranted. Here, we sought to explore the small molecule p53 reactivator, APR-246, either as a mono-drug or combined with doxorubicin (Doxo) in the p53 mutant (p53-mut) AsPC-1 PC cell line. Cytotoxicity of APR-246 and Doxo was studied alone or in combination with the AsPC-1 cell line with MTT assay. CalcuSyn software was used to calculate the combination index (CI), which was based on the Chou and Talalay technique. Apoptosis was assessed after staining with Acridine orange/ethidium bromide (AO/EB). Morphological alterations were examined under a 200x magnification by an inverted microscope. Gene expression was quantified using quantitative reverse transcription PCR assay (RT-qPCR). APR-246 alone exhibited modest anti-proliferative effects, while Doxo combined with APR-246 showed synergistic effects associated with morphological change; no more enhanced apoptosis was observed, as supported by NOXA gene levels. In conclusion, APR-246, whether monotherapy or combined with Doxo, was hindering the proliferation of p53-mut AsPC-1 PC cells, and further in vitro and in vivo research is warranted.

Inherently targeted estradiol-derived carbon dots for selective killing of ER (+) breast cancer cells via oridonin-triggered p53 pathway activation.

One of the most prevalent cancers globally is breast cancer and approximately two thirds of the breast cancers are hormone receptor positive with estrogen receptors (ER) being a prominent target. Notably, p53 that controls several cellular functions and prevents tumor formation, gets suppressed in breast cancers. Reactivation of p53 can lead to cell cycle arrest as well as apoptosis. Therefore, targeting the estrogen receptor for selective delivery of anticancer drugs that can reactivate p53 in ER (+) breast cancers can be a crucial method in breast cancer therapy. Herein, we have designed and developed estradiol-derived inherently targeted specific carbon dots (E2-CA-CD) from 17β-estradiol and citric acid following a solvothermal method. The synthesized carbon dots were characterized using spectroscopic and microscopic techniques. The water soluble, intrinsically fluorescent E2-CA-CD showed excellent biocompatibility in MCF-7, MDA-MB-231 as well as NIH3T3 cells and demonstrated target specific bioimaging in ER (+) MCF-7 cells due to the overexpressed ER receptors. Furthermore, oridonin, a well-known hydrophobic anticancer drug capable of upregulating the p53 pathway, was loaded on the carbon dots to increase its bioavailability. E2-CA-CD-Ori caused ∼2.2 times higher killing in ER (+) MCF-7 cells compared to ER (-) MDA-MB-231 cells and normal cells NIH3T3. Also, E2-CA-CD-Ori showed ∼3 fold better killing in MCF-7 cells compared to native oridonin. E2-CA-CD-Ori-induced killing of MCF-7 cells took place through the early to late apoptotic pathway along with the elevation of the intracellular ROS level. Importantly, E2-CA-CD-Ori triggered the activation of the p53 pathway in MCF-7 cells, which in turn induced apoptosis involving the upregulation of Bax and downregulation of Bcl-2 leading to the selective and efficient killing of ER (+) MCF-7 cells.

Investigating the potential of zinc oxide nanoparticles and zinc ions as promising approaches to lung cancer

Lung cancer is the leading cause of cancer casualties globally, with adverse side effects and drug resistance making current therapeutic options inadequate. Therefore, novel therapeutics require development. A major genetic contributor to these cancers is the mutant TP53 gene, whose mutated protein fails to transactivate p21, a cell cycle control protein in p53-p21-RB signaling, resulting in uncontrolled cell growth and cisplatin resistance. Recent studies suggest zinc oxide nanoparticles (ZnO NPs) show promise as a targeted, cytotoxic therapy; however, their mechanisms and efficacy are strongly debated as they aggregate in solution, reducing bioavailability while demonstrating poor aqueous solubility. As such, we hypothesized that a combined treatment using either ZnO NPs and cisplatin or zinc ions and cisplatin would induce cancer cell apoptosis and reduce cisplatin resistance. We treated mutant p53 lung cancer cells with ZnO NPs, zinc ion solution isolated from the NP suspension, and/or cisplatin; all significantly reduced proliferation individually and in combination. Increased reactive oxygen species (ROS) and p21 upregulation from ZnO NP treatment suggests mechanisms involving p21, perhaps through a p53-independent mechanism or via p53 reactivation. Zinc ion solution rendered a reduction in p21 in the presence of significantly lower ROS induced by ZnO NP, suggesting a mechanism needing further elucidation. We subjected a control cell line, HaCaT cells, to the same treatments and demonstrated no significant reduction in proliferation in the presence of ZnO NPs or zinc ions. Our research suggests the use of ZnO NPs and zinc ions as potential strategies for lung cancer treatment regimens.

Selectively targeting BCL6 using a small molecule inhibitor is a potential therapeutic strategy for ovarian cancer.

Ovarian cancer is one of the tumors with the highest fatality rate among gynecological tumors. The current 5-year survival rate of ovarian cancer is <35%. Therefore, more novel alternative strategies and drugs are needed to treat ovarian cancer. The transcription factor B-cell lymphoma 6 (BCL6) is critically associated with poor prognosis and cisplatin resistance in ovarian cancer treatment. Therefore, BCL6 may be an attractive therapeutic target for ovarian cancer. However, the role of targeting BCL6 in ovarian cancer remains elusive. Here, we developed a novel BCL6 small molecule inhibitor, WK369, which exhibits excellent anti-ovarian cancer bioactivity, induces cell cycle arrest and causes apoptosis. WK369 effectively inhibits the growth and metastasis of ovarian cancer without obvious toxicity in vitro and in vivo. meanwhile, WK369 can prolong the survival of ovarian cancer-bearing mice. It is worth noting that WK369 also has significant anti-tumor effects on cisplatin-resistant ovarian cancer cell lines. Mechanistic studies have shown that WK369 can directly bind to the BCL6-BTB domain and block the interaction between BCL6 and SMRT, leading to the reactivation of p53, ATR and CDKN1A. BCL6-AKT, BCL6-MEK/ERK crosstalk is suppressed. As a first attempt, our study demonstrates that targeting BCL6 may be an effective approach to treat ovarian cancer and that WK369 has the potential to be used as a candidate therapeutic agent for ovarian cancer.

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

Pharmacological reactivation of p53 in the era of precision anticancer medicine.

p53, which is encoded by the most frequently mutated gene in cancer, TP53, is an attractive target for novel cancer therapies. Despite major challenges associated with this approach, several compounds that either augment the activity of wild-type p53 or restore all, or some, of the wild-type functions to p53 mutants are currently being explored. In wild-type TP53 cancer cells, p53 function is often abrogated by overexpression of the negative regulator MDM2, and agents that disrupt p53-MDM2 binding can trigger a robust p53 response, albeit potentially with induction of p53 activity in non-malignant cells. In TP53-mutant cancer cells, compounds that promote the refolding of missense mutant p53 or the translational readthrough of nonsense mutant TP53 might elicit potent cell death. Some of these compounds have been, or are being, tested in clinical trials involving patients with various types of cancer. Nonetheless, no p53-targeting drug has so far been approved for clinical use. Advances in our understanding of p53 biology provide some clues as to the underlying reasons for the variable clinical activity of p53-restoring therapies seen thus far. In this Review, we discuss the intricate interactions between p53 and its cellular and microenvironmental contexts and factors that can influence p53's activity. We also propose several strategies for improving the clinical efficacy of these agents through the complex perspective of p53 functionality.