Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

Enzymatic Desymmetrisation of Prochiral meso-1,2-Disubstituted-1,2-Diaminoethane for the Synthesis of Key Enantioenriched (-)-Nutlin-3 Precursor.

Herein we present the biocatalysed preparation of a mono-N-carbamate-protected precursor of antitumoral Nutlin-3a through enantioselective alkoxycarbonylation of meso-1,2-disubstituted-1,2-diaminoethane using enzyme lipases and dialkyl carbonates as acylating agents. A series of supported or free lipase enzymes were screened in combination with commercially available diallyl, diethyl and dimethyl carbonates. The reactions were conducted at different temperatures, for different reaction times and with variable co-solvent systems to evaluate the effects on the enzyme catalytic activity. The best results in terms of conversion, enantiomeric excess and yield were obtained when lipase from Candida antarctica B (CAL-B) was used with diallyl carbonate (DAC) when conducting the reaction solventless at 75 °C.

Advances in targeted therapy for osteosarcoma based on molecular classification.

Osteosarcoma, a highly malignant tumor, is characterized by widespread and recurrent chromosomal and genetic abnormalities. In recent years, a number of elaborated sequencing analyses have made it possible to cluster the osteosarcoma based on the identification of candidate driver genes and develop targeted therapy. Here, we reviewed recent next-generation genome sequencing studies and advances in targeted therapies for osteosarcoma based on molecular classification. First, we stratified osteosarcomas into ten molecular subtypes based on genetic changes. And we analyzed potential targeted therapies for osteosarcoma based on the identified molecular subtypes. Finally, the development of targeted therapies for osteosarcoma investigated in clinical trials were further summarized and discussed. Therefore, we indicated the importance of molecular classification on the targeted therapy for osteosarcoma. And the stratification of patients based on the genetic characteristics of osteosarcoma will help to obtain a better therapeutic response to targeted therapies, bringing us closer to the era of personalized medicine.

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

Combination of a p53-activating CP-31398 and an MDM2 or a FAK inhibitor produces growth suppressive effects in mesothelioma with wild-type p53 genotype.

A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.

p53 suppression is essential for oncogenic SPAG5 upregulation in lung adenocarcinoma.

Aberrant expression of sperm-associated antigen 5 (SPAG5) is implicated to play oncogenic roles in several types of cancers. However, the functions of SPAG5 in lung adenocarcinoma remain unclear. In this study, we investigated the role of SPAG5 in lung adenocarcinoma. We found that SPAG5 was upregulated in most of the lung adenocarcinoma cell lines as compared to normal lung epithelial cells. SPAG5 knockdown suppressed proliferation, colony forming, and migration of lung adenocarcinoma A549 cells in vitro and inhibited tumor growth in vivo. These suggest that upregulated SPAG5 promotes lung tumor progression. Importantly, treatment with MDM2 inhibitor, Nutlin-3a, restored p53 and p21 expression and suppressed SPAG5 expression in wild-type p53 lung adenocarcinoma cells, A549 and H460, but not in p53-null lung cancer cells, H1299. This suggests that the p53 signal pathway is essential for SPAG5 suppression. In addition, knocking-down p53 or p21 in A549 and H460 cells attenuated Nutlin-3a-induced repression of SPAG5, which further supports that the p53-p21 axis is required for SPAG5 repression. Thus, SPAG5 can serve as a prognostic marker, and therapeutic strategy targeting the p53-p21-SPAG5 axis may have important clinical implications.

Kelch-like proteins: Physiological functions and relationships with diseases.

Kelch-like gene family members (KLHLs) encode proteins with a bric-a-brac, tramtrack, broad complex (BTB)/poxvirus and zinc finger (POZ) domain, a BACK domain, and six Kelch motifs, which frequently interact with Cullin3 to form E3 ligase complexes that mediate the ubiquitination of substrate proteins. In recent years, studies have revealed that mutations and abnormal expression of KLHLs are closely related to the pathogenesis of various human diseases. Increasing evidence has shown that Kelch-like protein family members (KLHLs) exert important biological functions through the ubiquitination of specific substrates. This review provides an overview of the identified substrates of different KLHLs, summarizes the current knowledge of KLHLs and discusses the biological functions of KLHLs in different diseases.

Combination treatment with auranofin and nutlin-3a induces synergistic cytotoxicity in breast cancer cells.

Auranofin is a gold complex categorized as an anti-rheumatic agent. Recently, several investigators suggested that auranofin may act as a potent anti-cancer drug for breast tumors. Nutlin-3a is a cis-imidazoline analog which prevents interaction between mouse double minute 2 homolog (MDM2) and the tumor suppressor p53. The aim of this study was to examine cell growth inhibition mediated by auranofin or nutlin-3a individually as well as in combination with MCF-7 and MDA-MB-231 cells. To assess any potential synergistic effects between auranofin and nutlin-3a, low concentrations of auranofin and nutlin-3a were simultaneously incubated with MCF-7 and MDA-MB-231 cells. Cell viability assay, caspase-3/7 assay, and poly (ADP-ribose) polymerase cleavage revealed that auranofin and nutlin-3a exerted a synergistic effect on cancer cell apoptosis. Isobologram analysis of MCF-7 and MDA-MB-231 cells noted evident synergism between auranofin and nutlin-3a. The combined treatment increased the expression of mitochondrial pro-apoptotic factors such as Bcl-2 associated X protein and Bcl-2 homologous antagonist/killer. Further, combination treatment significantly enhanced reactive oxygen species (ROS) generation in MCF-7 and MDA-MB-231 cells. In conclusion, data demonstrated that combined treatment with auranofin and nutlin-3a exhibited a synergistic action on breast cancer cells and this combination may be considered for use as a novel therapeutic strategy for breast cancer.

TRIAD1 Is a Novel Transcriptional Target of p53 and Regulates Nutlin-3a-Induced Cell Death.

Nutlin-3a is a non-genotoxic, p53-activating, MDM2 inhibitor being investigated as an anticancer agent. Although Nutlin-3a selectively antagonizes the ubiquitin E3 ligase activity of MDM2, its efficacy is not entirely regulated by MDM2 levels in cancer cells. Here, we report that the cytotoxic effects of Nutlin-3a are regulated by TRIAD1 via a positive feedback loop with p53. We found that Nutlin-3a enhanced TRIAD1 transcription in a p53-dependent manner. Using in silico analysis and promoter luciferase assays, we demonstrated that p53-mediated transcription of TRIAD1 is mediated by a p53 consensus sequence in the TRIAD1 promoter region. Silencing TRIAD1 expression in wild-type p53 (p53WT ) cancer cells suppressed Nutlin-3a-mediated p53 activation and p53 target gene expression. These effects were enhanced in TRIAD1-overexpressing p53WT cancer cells, but not in p53-deficient cancer cells. Furthermore, TRIAD1 knockdown significantly reduced the growth inhibitory and cytotoxic effects of Nutlin-3a in p53WT cancer cells, as demonstrated by cell viability assays, cell cycle analysis, clonogenic growth, and soft-agar colony forming assays. Together, these data indicate that TRIAD1 regulates Nutlin-3a-mediated p53 activation and the cytotoxic activity of Nutlin-3a. J. Cell. Biochem. 118: 1733-1740, 2017. © 2016 Wiley Periodicals, Inc.

Nutlin-3a selects for cells harbouring TP53 mutations.

TP53 mutations occur in half of all human tumours. Mutagen-induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock-in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen-treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2-5 months) and much effort is expended maintaining TP53-WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin-3a, an MDM2 inhibitor that leads to stabilisation and activation of wild-type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin-3a to examine the effect on cell growth and p53 activation. Nutlin-3a induced the p53 pathway in TP53-WT HUFs and inhibited cell growth, whereas most TP53-mutated HUFs were resistant to Nutlin-3a. We then assessed whether Nutlin-3a treatment could discriminate between TP53-WT and TP53-mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin-3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin-3a-resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin-3a-sensitive clones were TP53-WT. These data suggest that including a Nutlin-3a counter-screen significantly improves the specificity and efficiency of the HIMA, whereby TP53-mutated clones are selected prior to sequencing and TP53-WT clones can be discarded.

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways.

BACKGROUND: Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. METHODS: We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. RESULTS: Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. CONCLUSION: These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

A tryptophanol-derived oxazolopiperidone lactam is cytotoxic against tumors via inhibition of p53 interaction with murine double minute proteins.

Inactivation of the p53 tumor suppressor protein by interaction with murine double minute (MDM) proteins, MDM2 and MDMX, is a common event in human tumors expressing wild-type p53. In these tumors, the simultaneous inhibition of these interactions with MDMs, for a full p53 reactivation, represents a promising anticancer strategy. Herein, we report the identification of a dual inhibitor of the p53 interaction with MDM2 and MDMX, the (S)-tryptophanol derivative OXAZ-1, from the screening of a small library of enantiopure tryptophanol-derived oxazolopiperidone lactams, using a yeast-based assay. With human colon adenocarcinoma HCT116 cell lines expressing wild-type p53 (HCT116 p53(+/+)) and its p53-null isogenic derivative (HCT116 p53(-/-)), it was shown that OXAZ-1 induced a p53-dependent tumor growth-inhibitory effect. In fact, OXAZ-1 induced p53 stabilization, up-regulated p53 transcription targets, such as MDM2, MDMX, p21, Puma and Bax, and led to PARP cleavage, in p53(+/+), but not in p53(-/-), HCT116 cells. In addition, similar tumor cytotoxic effects were observed for OXAZ-1 against MDMX-overexpressing breast adenocarcinoma MCF-7 tumor cells, commonly described as highly resistant to MDM2-only inhibitors. In HCT116 p53(+/+) cells, the disruption of the p53 interaction with MDMs by OXAZ-1 was further confirmed by co-immunoprecipitation. It was also shown that OXAZ-1 potently triggered a p53-dependent mitochondria-mediated apoptosis, characterized by reactive oxygen species generation, mitochondrial membrane potential dissipation, Bax translocation to mitochondria, and cytochrome c release, and exhibited a p53-dependent synergistic effect with conventional chemotherapeutic drugs. Collectively, in this work, a novel selective activator of the p53 pathway is reported with promising antitumor properties to be explored either alone or combined with conventional chemotherapeutic drugs. Moreover, OXAZ-1 may represent a promising starting scaffold to search for new dual inhibitors of the p53-MDMs interaction.

Multifunctional nanoparticle-EpCAM aptamer bioconjugates: a paradigm for targeted drug delivery and imaging in cancer therapy.

The promising proposition of multifunctional nanoparticles for cancer diagnostics and therapeutics has inspired the development of theranostic approach for improved cancer therapy. Moreover, active targeting of drug carrier to specific target site is crucial for providing efficient delivery of therapeutics and imaging agents. In this regard, the present study investigates the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles, functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. A wide spectrum of in vitro analysis (cellular uptake study, cytotoxicity assay, cell cycle and apoptosis analysis, apoptosis associated proteins study) revealed superior therapeutic potentiality of targeted NPs over other formulations in EpCAM expressing cells. Moreover, our nanotheranostic system served as a superlative bio-imaging modality both in 2D monolayer culture and tumor spheroid model. Our result suggests that, these aptamer-guided multifunctional NPs may act as indispensable nanotheranostic approach toward cancer therapy. FROM THE CLINICAL EDITOR: This study investigated the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. It was concluded that the studied multifunctional targeted nanoparticle may become a viable and efficient approach in cancer therapy.

Microfluidic Fabricated Liposomes for Nutlin-3a Ocular Delivery as Potential Candidate for Proliferative Vitreoretinal Diseases Treatment.

Purpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.

New Insights into Chemoresistance Mediated by Mdm2 Inhibitors: The Benefits of Targeted Therapy over Common Cytostatics.

The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.

Enhanced Anti-Melanoma Activity of Nutlin-3a Delivered via Ethosomes: Targeting p53-Mediated Apoptosis in HT144 Cells.

This study evaluated ethosomes as a novel nanodelivery system for nutlin-3a, a known MDM2 inhibitor and activator of the p53 pathway, to improve nutlin-3a's poor solubility, limiting its bio-distribution and therapeutic efficacy. The potential of nutlin-3a-loaded ethosomes was investigated on two in vitro models of melanoma: the HT144 cell line p53wild-type and the SK-MEL-28 cell line p53mutated. Nutlin-3a-loaded ethosomes were characterized for their physicochemical properties and used to treat melanoma cells at different concentrations, considering nutlin-3a solution and empty ethosomes as controls. The biological effects on cells were evaluated 24 and 48 h after treatment by analyzing the cell morphology and viability, cell cycle, and apoptosis rate using flow cytometry and the p53 pathway's activation via Western blotting. The results indicate that ethosomes are delivery systems able to maintain nutlin-3a's functionality and specific biological action, as evidenced by the molecular activation of the p53 pathway and the biological events leading to cell cycle block and apoptosis in p53wild-type cells. Nutlin-3a-loaded ethosomes induced morphological changes in the HT144 cell line, with evident apoptotic cells and a reduction in the number of viable cells of over 80%. Furthermore, nutlin-3a-loaded ethosomes successfully modulated two p53-regulated proteins involved in survival/apoptosis, with up to a 2.5-fold increase in membrane TRAIL-R2 and up to an 8.2-fold decrease in Notch-1 (Notch intracellular domain, NICD) protein expression. The expression of these molecules is known to be altered or dysfunctional in a large percentage of melanoma tumors. Notably, ethosomes, regardless of their nutlin-3a loading, exhibited the ability to reduce HT144 melanoma cellular migration, as assessed in real time using xCELLigence, likely due to the modification of lipid rafts, suggesting their potential antimetastatic properties. Overall, nutlin-3a delivery using ethosomes appears to be a significantly effective means for upregulating the p53 pathway and downregulating active Notch-1, while also taking advantage of their unexpected ability to reduce cellular migration. The findings of this study could pave the way for the development of specific nutlin-3a-loaded ethosome-based medicinal products for cutaneous use.

CLCA2: A Potential Guardian against Premature Senescence and Skin Aging.

Cellular senescence, a state of irreversible growth arrest, is implicated in various age-related pathologies, including skin aging. In this study, we investigated the role of CLCA2, a calcium-activated chloride channel accessory protein, in cellular senescence and its implications for skin aging. Utilizing UVB and Nutlin3a-induced senescence models, we observed the upregulation of CLCA2 at both transcriptomic and proteomic levels, suggesting its involvement in senescence pathways. Further analysis revealed that the depletion of CLCA2 led to accelerated senescence onset, characterized by classic senescence markers and a unique secretome profile. In 3D skin equivalent models, SEs constructed with CLCA2 knockdown fibroblasts exhibited features reminiscent of aged skin, underscoring the importance of CLCA2 in maintaining skin homeostasis. Our findings highlight CLCA2 as a novel regulator of cellular senescence and its potential implications for skin aging mechanisms.

The structure of an MDM2-Nutlin-3a complex solved by the use of a validated MDM2 surface-entropy reduction mutant.

The p53-binding site of MDM2 holds great promise as a target for therapeutic intervention in MDM2-amplified p53 wild-type forms of cancer. Despite the extensive validation of this strategy, there are relatively few crystallographically determined co-complex structures for small-molecular inhibitors of the MDM2-p53 interaction available in the PDB. Here, a surface-entropy reduction mutant of the N-terminal domain of MDM2 that has been designed to enhance crystallogenesis is presented. This mutant has been validated by comparative ligand-binding studies using differential scanning fluorimetry and fluorescence polarization anisotropy and by cocrystallization with a peptide derived from p53. Using this mutant, the cocrystal structure of MDM2 with the benchmark inhibitor Nutlin-3a has been determined, revealing subtle differences from the previously described co-complex of MDM2 with Nutlin-2.

Nutlin-3a induces KRAS mutant/p53 wild type lung cancer specific methuosis-like cell death that is dependent on GFPT2.

BACKGROUND: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. METHODS: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. RESULTS: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. CONCLUSION: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.

Modulation of anti-angiogenic activity using ultrasound-activated nutlin-loaded piezoelectric nanovectors.

Angiogenesis plays a fundamental role in tumor development, as it is crucial for tumor progression, metastasis development, and invasion. In this view, anti-angiogenic therapy has received considerable attention in several cancer types in order to inhibit tumor vascularization, and the progress of nanotechnology offers opportunities to target and release anti-angiogenic agents in specific diseased areas. In this work, we showed that the angiogenic behavior of human cerebral microvascular endothelial cells can be inhibited by using nutlin-3a-loaded ApoE-functionalized polymeric piezoelectric nanoparticles, which can remotely respond to ultrasound stimulation. The anti-angiogenic effect, derived from the use of chemotherapy and chronic piezoelectric stimulation, leads to disruption of tubular vessel formation, decreased cell migration and invasion, and inhibition of angiogenic growth factors in the presence of migratory cues released by the tumor cells. Overall, the proposed use of remotely activated piezoelectric nanoparticles could provide a promising approach to hinder tumor-induced angiogenesis.