MDM2-mediated P53 Degradation Research Articles

ZBTB7A interferes with the RPL5-P53 feedback loop and reduces endoplasmic reticulum stress-induced apoptosis of pancreatic cancer cells.

Endoplasmic reticulum (ER) stress is a primary mechanism leading to cell apoptosis, making it of great research interests in cancer management. This study delves into the function of ribosomal protein L5 (RPL5) in ER stress within pancreatic cancer (PCa) cells and investigates its regulatory mechanisms. Bioinformatics predictions pinpointed RPL5 as an ER stress-related gene exhibiting diminished expression in PCa. Indeed, RPL5 was found to be poorly expressed in PCa tissues and cells, with this reduced expression correlating with an unfavorable prognosis. Moreover, RPL5 overexpression led to heightened levels of p-PERK, p-eIF2α, and CHOP, bolstering the proapoptotic effect of Tunicamycin, an ER stress activator, on PCa cells. Additionally, the RPL5 overexpression curbed cell proliferation, migration, and invasion. Tunicamycin enhanced the binding between RPL5 and murine double minute 2 (MDM2), thus suppressing MDM2-mediated ubiquitination and degradation of P53. Consequently, P53 augmentation intensified ER stress, which further enhanced the binding between RPL5 and MDM2 through PERK-dependent eIF2α phosphorylation, thereby establishing a positive feedback loop. Zinc finger and BTB domain containing 7A (ZBTB7A), conspicuously overexpressed in PCa samples, repressed RPL5 transcription, thereby reducing P53 expression. Silencing of ZBTB7A heightened ER stress and subdued the malignant attributes of PCa cells, effects counteracted upon RPL5 silencing. Analogous outcomes were recapitulated in vivo employing a xenograft tumor mouse model, where ZBTB7A silencing dampened the tumorigenic potential of PCa cells, an effect reversed by additional RPL5 silencing. In conclusion, this study suggests that ZBTB7A represses RPL5 transcription, thus impeding the RPL5-P53 feedback loop and mitigating ER-induced apoptosis in PCa cells.

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity, however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation, and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.

Hydrogen sulfide inhibits alveolar type II cell senescence and limits pulmonary fibrosis via promoting MDM2-mediated p53 degradation.

Senescence of alveolar type II (AT2) cells is an important driver of pulmonary fibrosis. This study aimed to investigate whether and how dysregulation of hydrogen sulfide (H2 S) production affected AT2 cell senescence, and then explored the effect of H2 S on the communication between AT2 and fibroblasts. ICR mice were intratracheally administered with bleomycin (3 mg/kg). Sodium hydrosulfide (NaHS, 28 μmol/kg/d) was intraperitoneally injected for 2 weeks. The H2 S-generating enzyme cystathionine-β-synthase (CBS) knockout heterozygous (CBS+/- ) mice were used as a low H2 S production model. Analysis of microarray datasets revealed downregulation of H2 S-generating enzymes in lung tissues of patients with pulmonary fibrosis. Decreased H2 S production was correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis. CBS+/- mice exhibited increased levels of p53 and p21. The numbers of AT2 cells positive for p53 and p21 were increased in CBS+/- mice as compared to control mice. H2 S donor NaHS attenuated bleomycin-induced AT2 cell senescence both invivo and invitro. H2 S donor suppressed bleomycin-induced senescence-associated secretory phenotype (SASP) of AT2 cells via inhibiting p53/p21 pathway, consequently suppressing proliferation and myofibroblast transdifferentiation of fibroblasts. Mechanically, H2 S suppressed p53 expression by enhancing the mouse double-minute 2 homologue (MDM2)-mediated ubiquitination and degradation of p53. H2 S inactivated p53-p21 pathway, consequently suppressing AT2 cell senescence as well as cell communication between senescent AT2 cells and fibroblasts. Aberrant H2 S synthesis may contribute to the development of pulmonary fibrosis through promoting the activation loop involving senescent AT2 cells and activated fibroblasts.

UTP11 deficiency suppresses cancer development via nucleolar stress and ferroptosis

The eukaryotic ribosome is essential for cancer cell survival. Perturbation of ribosome biogenesis induces nucleolar stress or ribosomal stress, which restrains cancer growth, as rapidly proliferating cancer cells need more active ribosome biogenesis. In this study, we found that UTP11 plays an important role in the biosynthesis of 18S ribosomal RNAs (rRNA) by binding to the pre-rRNA processing factor, MPP10. UTP11 is overexpressed in human cancers and associated with poor prognoses. Interestingly, depletion of UTP11 inhibits cancer cell growth in vitro and in vivo through p53-depedednt and -independent mechanisms, whereas UTP11 overexpression promotes cancer cell growth and progression. On the one hand, the ablation of UTP11 impedes 18S rRNA biosynthesis to trigger nucleolar stress, thereby preventing MDM2-mediated p53 ubiquitination and degradation through ribosomal proteins, RPL5 and RPL11. On the other hand, UTP11 deficiency represses the expression of SLC7A11 by promoting the decay of NRF2 mRNA, resulting in reduced levels of glutathione (GSH) and enhanced ferroptosis. Altogether, our study uncovers a critical role for UTP11 in maintaining cancer cell survival and growth, as depleting UTP11 leads to p53-dependent cancer cell growth arrest and p53-independent ferroptosis.

The RNA binding protein RALY suppresses p53 activity and promotes lung tumorigenesis

The tumor suppressor p53 plays a pivotal role in tumor prevention. The activity of p53 is mainly restrained by the ubiquitin E3 ligase Mdm2. However, it is not well understood how the Mdm2-p53 pathway is intricately regulated. Here we report that the RNA binding protein RALY functions as an oncogenic factor in lung cancer. RALY simultaneously binds to Mdm2 and the deubiquitinating enzyme USP7. Via these interactions, RALY not only stabilizes Mdm2 by stimulating the deubiquitinating activity of USP7 toward Mdm2 but also increases the trans-E3 ligase activity of Mdm2 toward p53. Consequently, RALY enhances Mdm2-mediated ubiquitination and degradation of p53. Functionally, RALY promotes lung tumorigenesis, at least partially, via negative regulation of p53. These findings suggest that RALY destabilizes p53 by modulating the function of Mdm2 at multiple levels. Our study also indicates a critical role for RALY in promoting lung tumorigenesis via p53 inhibition.

Nonsense-mediated mRNA decay inhibition synergizes with MDM2 inhibition to suppress TP53 wild-type cancer cells in p53 isoform-dependent manner

The restoration of the normal function of the tumour suppressors, such as p53, is an important strategy in tumour therapeutics. Nonsense-mediated mRNA decay (NMD) inhibition by NMD inhibitor (NMDi) upregulates functional p53 isoforms, p53β and p53γ, and activates the p53 pathway. XR-2, a novel mouse double minute 2 homolog (MDM2) inhibitor, can disrupt the interaction between p53 and MDM2, thus decreasing the MDM2-mediated degradation of p53 and increasing the p53 protein levels. However, the combined effects of these two agents have not been thoroughly explored. This study combined XR-2 and NMDi in four TP53 wild-types and four TP53-mutated cancer cell lines. The combination of these two agents achieved significant synergistic effects on TP53 wild-type cancer cell lines by transactivating p53 target genes, inducing apoptosis, cell-cycle arrest and DNA damage repair. The p53β isoform induced by NMDi enhances the transactivation ability of p53α induced by XR-2, which partially explains the mechanism of the synergistic effects of XR-2 and NMDi. This study identified a combination treatment of NMDi and XR-2 which could serve as a novel cancer therapeutic approach for MDM2-overexpressed TP53 wild-type cancers and delineated a future therapy based on the further reactivation of p53.

MiRNA-103 downmodulates CCR5 expression reducing human immunodeficiency virus type-1 entry and impacting latency establishment in CD4+ T cells

SummaryActivated-to-memory transitioning CD4+ T cells display elevated expression of the HIV-1 co-receptor CCR5 and are more prone to HIV-1 latent infection. Here, we show that p53-regulated miRNA-103 downmodulates CCR5 levels in CD4+ T lymphocytes. We reveal that miRNA-103 mimics, as well as Nutlin-3, an inhibitor of Mdm2-mediated p53 degradation, decrease CCR5-dependent HIV-1 infection. Using a dual-reporter virus, we subsequently validate that in transitioning CD4+ T cells, Nutlin-3 treatment decreases the frequency of both productively and latently infected cells via upregulation of miRNA-103. Importantly, we provide evidence that CD4+ T cells from HIV-1 elite controllers express less CCR5 than those from antiretroviral therapy-naïve progressors, an effect linked to a significant increase in miRNA-103 levels. By contributing to the control of CCR5 expression in CD4+ T cells, miRNA-103 is likely to play a key role in countering the establishment of latent HIV-1 reservoirs in vivo.

P53 Pathway Inactivation Drives SMARCB1-deficient p53-wildtype Epithelioid Sarcoma Onset Indicating Therapeutic Vulnerability Through MDM2 Inhibition.

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy.

Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function.

ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2∼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2∼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Downregulation of Long Noncoding RNA CRYBG3 Enhances Radiosensitivity in Non-Small Cell Lung Cancer Depending on p53 Status.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with high recurrence and metastasis rates, and more than half of the patients diagnosed with NSCLC receive local radiotherapy. However, the intrinsic radio-resistance of cancer cells is a major barrier to effective radiotherapy for NSCLC. CRYBG3 is a long noncoding RNA (lncRNA) that was originally identified to be upregulated in NSCLC and enhanced metastasis of NSCLC cells by interacting with eEF1A1 to promote murine double minute 2 (MDM2) expression. The aims of this study were to reveal the contribution of CRYBG3 to the radioresistance of NSCLC and determine whether that is associated with MDM2-p53 pathway. Therefore, CRYBG3 was stably downregulated in A549 (wild-type p53) and H1299 (deficient p53) cells by infecting short hairpin RNA (shRNA) lentiviral particles. The results showed that downregulation of CRYBG3 increased DNA damage, enhanced apoptosis and pro-apoptotic protein expression in A549 or p53-overexpressed H1299 cells but not in H1299 or p53-silenced A549 cells after X-ray irradiation. However, the contribution of CRYBG3 to radioresistance was abolished by eEF1A1 or MDM2 knockdown in A549 cells. Thus, we concluded that downregulation of CRYBG3 enhanced radiosensitivity by reducing MDM2 expression then leading to decreased MDM2-mediated degradation of p53 in wild-type p53 expressing NSCLC cells. These findings suggested that CRYBG3 can be a potential target for therapeutic intervention of certain lung cancer subtypes.

MicroRNA-101-3p Suppresses Cancer Cell Growth by Inhibiting the USP47-Induced Deubiquitination of RPL11.

Simple SummaryAn abnormal expression of microRNA is commonly observed in cancer. Since a single miRNA can target numerous genes, it is important to understand the exact mechanism for the regulation of cancer growth by miRNAs. Here, we show that miR-101-3p, which is downregulated in several cancers, regulates RPL11 ubiquitination by targeting USP47, thereby controlling p53 levels by affecting the localization of RPL11 and its interaction with MDM2. Our results provide a novel mechanism for the inhibition of cancer cell growth by miR-101-3p, and suggest that miR-101-3p could be a potential target as an anticancer agent.MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate a countless number of genes in the cell, and the aberrant expression of miRNA can lead to cancer. Here, we demonstrate that miR-101-3p regulates the RPL11–MDM2–p53 pathway by targeting ubiquitin-specific peptidase 47 (USP47), consequently inhibiting cancer cell proliferation. We confirm that miR-101-3p directly binds to the 3′-UTR region of the USP47 gene and inhibits USP47 expression. In addition, the overexpression of miR-101-3p suppresses cell proliferation in a p53-dependent manner. MiR-101-3p promotes interaction between RPL11 and MDM2 by inducing the translocation of RPL11 from the nucleolus to the nucleoplasm, thus preventing the MDM2-mediated proteasomal degradation of p53. However, these phenomena are restored by the overexpression of USP47, but not by its catalytically inactive form. Indeed, miR-101-3p regulates RPL11 localization and its interaction with MDM2 by inhibiting the USP47-induced deubiquitination of RPL11. Finally, the expression of miR-101-3p is downregulated in lung cancer patients, and the patients with low miR-101-3p expression exhibit a lower survival rate, indicating that miR-101-3p is associated with tumorigenesis. Together, our findings suggest that miR-101-3p functions as a tumor suppressor by targeting USP47 and could be a potential therapeutic target for cancers.

Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate.

Many types of cancer feature TP53 mutations with oncogenic properties. However, whether the oncogenic activity of mutant p53 is affected by the cellular metabolic state is unknown. Here we show that cancer-associated mutant p53 protein is stabilized by 2-hydroxyglutarate generated by malic enzyme 2. Mechanistically, malic enzyme 2 promotes the production of 2-hydroxyglutarate by adjusting glutaminolysis, as well as through a reaction that requires pyruvate and NADPH. Malic enzyme 2 depletion decreases cellular 2-hydroxyglutarate levels in vitro and in vivo, whereas elevated malic enzyme 2 expression increases 2-hydroxyglutarate production. We further show that 2-hydroxyglutarate binds directly to mutant p53, which reduces Mdm2-mediated mutant p53 ubiquitination and degradation. 2-Hydroxyglutarate supplementation is sufficient for maintaining mutant p53 protein stability in malic enzyme 2-depleted cells, and restores tumour growth of malic enzyme 2-ablated cells, but not of cells that lack mutant p53. Our findings reveal the previously unrecognized versatility of malic enzyme 2 catalytic functions, and uncover a role for mutant p53 in sensing cellular 2-hydroxyglutarate levels, which contribute to the stabilization of mutant p53 and tumour growth.

DNA Damage Triggers the Nuclear Accumulation of RASSF6 Tumor Suppressor Protein via CDK9 and BAF53 To Regulate p53 Target Gene Transcription.

RASSF6, a member of the tumor suppressor Ras-association domain family (RASSF) proteins, regulates cell cycle arrest and apoptosis via p53 and plays a tumor suppressor role. We previously reported that RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. In this study, we demonstrated that RASSF6 has nuclear localization and nuclear export signals and that DNA damage triggers the nuclear accumulation of RASSF6. We found that RASSF6 directly binds to BAF53, the component of SWI/SNF complex. DNA damage induces CDK9-mediated phosphorylation of BAF53, which enhances the interaction with RASSF6 and increases the amount of RASSF6 in the nucleus. Subsequently, RASSF6 augments the interaction between BAF53 and BAF60a, another component of the SWI/SNF complex, and further promotes the interaction of BAF53 and BAF60a with p53. BAF53 silencing or BAF60a silencing attenuates RASSF6-mediated p53 target gene transcription and apoptosis. Thus, RASSF6 is involved in the regulation of DNA damage-induced complex formation, including BAF53, BAF60a, and p53.

Loss of RPS27a expression regulates the cell cycle, apoptosis, and proliferation via the RPL11-MDM2-p53 pathway in lung adenocarcinoma cells

BackgroundDepletion of certain ribosomal proteins induces p53 activation, which is mediated mainly by ribosomal protein L5 (RPL5) and/or ribosomal protein L11 (RPL11). Therefore, RPL5 and RPL11 may link RPs and p53 activation. Thus, this study aimed to explore whether RPs interact with RPL11 and regulate p53 activation in lung adenocarcinoma (LUAD) cells.MethodsThe endogenous RPL11-binding proteins in A549 cells were pulled down through immunoprecipitation and identified with a proteomics approach. Docking analysis and GST-fusion protein assays were used to analyze the interaction of ribosomal protein S27a (RPS27a) and RPL11. Co-immunoprecipitation and in vitro ubiquitination assays were used to detect the effects of knockdown of RPS27a on the interaction between RPS27a and RPL11, and on p53 accumulation. Cell cycle, apoptosis, cell invasion and migration, cell viability and colony-formation assays were performed in the presence of knockdown of RPS27a. The RPS27a mRNA expression in LUAD was analyzed on the basis of the TCGA dataset, and RPS27a expression was detected through immunohistochemistry in LUAD samples. Finally, RPS27a and p53 expression was analyzed through immunohistochemistry in A549 cell xenografts with knockdown of RPS27a.ResultsRPS27a was identified as a novel RPL11 binding protein. GST pull-down assays revealed that RPS27a directly bound RPL11. Knockdown of RPS27a weakened the interaction between RPS27a and RPL11, but enhanced the binding of RPL11 and murine double minute 2 (MDM2), thereby inhibiting the ubiquitination and degradation of p53 by MDM2. Knockdown of RPS27a stabilized p53 in an RPL11-dependent manner and induced cell viability inhibition, cell cycle arrest and apoptosis in a p53-dependent manner in A549 cells. The expression of RPS27a was upregulated in LUAD and correlated with LUAD progression and poorer prognosis. Overexpression of RPS27a correlated with upregulation of p53, MDM2 and RPL11 in LUAD clinical specimens. Knockdown of RPS27a increased p53 activation, thus, suppressing the formation of A549 cell xenografts in nude mice.ConclusionsRPS27a interacts with RPL11, and RPS27a knockdown enhanced the binding of RPL11 and MDM2, thereby inhibiting MDM2-mediated p53 ubiquitination and degradation; in addition, RPS27a as important roles in LUAD progression and prognosis, and may be a therapeutic target for patients with LUAD.Graphical

Genomic gain of RRS1 promotes hepatocellular carcinoma through reducing the RPL11-MDM2-p53 signaling.

Hepatocellular carcinomas (HCCs) are characterized by frequent somatic genomic copy number alterations (CNAs), with most of them biologically unexplored. Here, we performed integrative analyses combining CNAs with the transcriptomic data to reveal the cis- and trans-effects of CNAs in HCC. We identified recurrent genomic gains of chromosome 8q, which exhibit strong trans-effects and are broadly associated with ribosome biogenesis activity. Furthermore, 8q gain-driven overexpression of ribosome biogenesis regulator (RRS1) promotes growth of HCC cells in vitro and in vivo. Mechanistically, RRS1 attenuates ribosomal stress through retaining RPL11 in the nucleolus, which, in turn, potentiates MDM2-mediated ubiquitination and degradation of p53. Clinically, higher RRS1 expression levels predict poor clinical outcomes for patients with HCC, especially in those with intact p53 Our findings established that the chromosome 8q oncogene RRS1 promotes HCC development through attenuating the RPL11-MDM2-p53 pathway and provided new potential targets for treatment of this malignancy.

New insight into the role of MDMX in MDM2-mediated p53 degradation and anti-cancer drug development.

Inactivation of the tumor suppressor p53 has been generally accepted as a hallmark of tumor. MDM2 and MDMX, the two closely related proteins are considered to be critical for negatively regulating p53 activity through inhibitory binding to and post-translational modification of the p53 protein. We have demonstrated that MDMX facilitates MDM2-mediated p53 ubiquitination and degradation via recruitment of the ubiquitin-conjugating enzyme UbcH5c to the MDM2-MDMX heterooligomers. Here, we discuss our new findings from genetically engineered mouse models and a potential therapeutic strategy.

Burkitt Lymphoma Genome Sequencing Project (BLGSP): Integrative Genomic and Transcriptomic Characterization of Burkitt Lymphoma

IntroductionBurkitt lymphoma (BL) is an aggressive B-cell non-Hodgkin lymphoma with a hallmark translocation involving MYC and an immunoglobulin enhancer. It is most common in children and has three clinical variants: endemic, sporadic, and HIV-associated. The Epstein-Barr virus (EBV)-associated endemic subtype (eBL) is highly prevalent in equatorial Africa, where it is the most common pediatric cancer. Survival is poor among African children with eBL due to late stage at presentation and limitations in the ability to support intensive chemotherapeutic regimens necessary to induce durable remissions. Previous genomic studies of smaller BL cohorts revealed that eBL harbors mutation patterns similar but not identical to sporadic cases from high-resource countries. The BLGSP aims at conducting an integrative molecular characterization of a large comprehensive BL cohort including an unprecedented representation of endemic cases. The objective is to define molecular features that drive lymphomagenesis, which can be translated to new therapeutic strategies deployable worldwide.MethodsTumor biopsies and matched normal tissue have been collected from patients with both endemic and sporadic BL along with treatment, outcome, and other clinical information. Molecular characterization includes whole genome sequencing of tumor and constitutional DNA (80X and 40X, respectively), and strand-specific ribo-depleted sequencing of tumor RNA and microRNA. Six centroblast and 6 centrocyte samples flow-sorted from healthy donor tissue underwent RNA and miRNA sequencing as normal comparators for gene expression. These data enable the identification of somatic mutations, significantly mutated genes (SMGs) and non-coding loci with potential regulatory function; the quantification of human and EBV gene expression; and the characterization of miRNA-mediated regulation of transcripts. SMGs were supported by two or more of the following methods with a 10% false discovery rate: OncodriveFM, OncodriveFML, OncodriveCLUST, and MutSigCV. We also re-analyzed the genome data for 17 sporadic cases from the ICGC MALY-DE project (Nat. Genet. 2015; 47: 1316-1325).ResultsA total of 101 cases (71 endemic and 30 sporadic) were included after quality control. We describe novel mutation features affecting known BL genes. Notably, DDX3X is the target of focal deletions and splicing branch point mutations, and in-frame deletions recurrently affect a putative ubiquitination site (K272) in GNAI2 . Our meta-analysis reveals 22 SMGs, including many that have not yet been linked to the disease (see figure). We associate recurrent mutations in the following genes with BL for the first time: SIN3A, HIST1H1E, KMT2D, CHD8, USP7, and RFX7 . Several SMGs are differentially mutated between endemic and sporadic BL, suggesting distinct underlying biology (see highlighted genes in figure). For instance, ARID1A is preferentially mutated in endemic cases whereas SMARCA4 shows the opposite trend. Multiple non-coding loci are also significantly mutated: a PAX5 enhancer, BCL6 super-enhancers, the PVT1 non-coding locus, and the miR-142 gene, among others. Three mutation signatures are detected across all tumors: one is associated with defective DNA mismatch repair, another has been linked to polymerase η and AID activity, and the last signature has an unknown etiology. Gene expression analysis reveals high correlation between tumors (Pearson coefficient > 0.9). This is consistent with the absence of robust clusters according to unsupervised clustering of expression profiles.DiscussionBLGSP is an ongoing international collaborative project aimed at providing a comprehensive molecular portrait of BL across all subtypes. We describe previously unreported mutation features affecting established BL genes and identify novel candidate coding and non-coding driver mutations, nominating compelling targets for therapy. For example, one of the novel SMGs, USP7, encodes a deubiquitinase that counteracts Mdm2-mediated ubiquitination and degradation of p53. We hypothesize that Mdm2 inhibitors might trigger synthetic lethality in USP7 -mutated, TP53 -wildtype tumors. In summary, this effort has identified candidate molecular targets for therapy that can potentially lead to more effective treatments that are less toxic than the current regimens.BMG and DSG contributed equally. RDM and LMS contributed equally. [Display omitted] DisclosuresCasper:TempTime: Consultancy, Other: Travel, Accommodation, Expenses; GSK: Other: Travel, Accommodation, Expenses; Janssen: Consultancy, Research Funding; Up to Date: Patents & Royalties; Roche: Consultancy, Other: Travel, Accommodation, Expenses. Mullighan:Loxo Oncology: Research Funding; Amgen: Consultancy. Abramson:Abbvie: Consultancy; Genentech: Consultancy; LAM Therapeutics: Research Funding; Gilead: Consultancy; Kite Pharma: Consultancy; Seattle Genetics: Consultancy; Celgene: Consultancy; Novartis: Consultancy. Noy:Pharmacyclics LLC, an AbbVie Company: Honoraria, Other: Travel, Accommodation, Expenses, Research Funding, Speakers Bureau. Morin:Epizyme, Inc: Consultancy.

Bibenzyl analogue DS-1 inhibits MDM2-mediated p53 degradation and sensitizes apoptosis in lung cancer cells

BackgroundLung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function. PurposeThe present study aimed to investigate the effect of 3,4-dihydroxy-5,4′-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells. MethodsThe efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed. ResultsDS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon–hydrogen bond interaction at Lys27, π–alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, and Phe31. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity. ConclusionsOur findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.

Design, synthesis, and biological evaluation of nitroisoxazole-containing spiro[pyrrolidin-oxindole] derivatives as novel glutathione peroxidase 4/mouse double minute 2 dual inhibitors that inhibit breast adenocarcinoma cell proliferation.

A series of highly active CF3-containing 3'-(nitroisoxazole)spiro[pyrrolidin-3,2'-oxindoles] were synthesized and found to be novel glutathione peroxidase 4 (GPX4)/mouse double minute 2 (MDM2) dual inhibitors. Bioactive spirooxindole and isoxazole skeletons were combined, and the resulting compounds exhibited strong activities against both targets. In particular, compound 3d displayed excellent activity in the suppression of MDM2-mediated degradation of p53, as well as levels of GPX4, in MCF-7 breast cancer cells. Moreover, 3d also exhibited inhibitory effects on MDM2 and GPX4 in MCF-7 xenograft model to trigger ferroptotic and apoptotic cell death in invivo experiments, which was consistent with the results of invitro experiments.