Search

Somatic hypermutation in cancer has gained momentum with the increased use of tumour mutation burden as a biomarker for immune checkpoint inhibitors. Spontaneous deamination of 5-methylcytosine to thymine at CpG dinucleotides is one of the most ubiquitous endogenous mutational processes in normal and cancer cells. Here, we performed a systematic investigation of somatic CpG hypermutation at a pan-cancer level. We studied 30,191 cancer patients and 103 cancer types and developed an algorithm to identify somatic CpG hypermutation. Across cancer types, we observed the highest prevalence in paediatric leukaemia (3.5%), paediatric high-grade glioma (1.7%), and colorectal cancer (1%). We discovered germline variants and somatic mutations in the mismatch repair complex MutSα (MSH2-MSH6) as genetic drivers of somatic CpG hypermutation in cancer, which frequently converged on CpG sites and TP53 driver mutations. We further observe an association between somatic CpG hypermutation and response to immune checkpoint inhibitors. Overall, our study identified novel cancer types that display somatic CpG hypermutation, strong association with MutSα-deficiency, and potential utility in cancer immunotherapy.

p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is a significant focus for personalized cancer therapy. Prior research indicates that reactivating p53 suppresses cancer cell proliferation and tumor growth in animal models. Early clinical evidence with a compound selectively targeting p53 mutants with substitutions of tyrosine 220 suggests potential therapeutic benefits of reactivating p53 in patients. This study identifies and examines the UCI-1001 compound series as a potential corrector for several p53 mutations. The findings indicate that UCI-1001 treatment in p53 mutant cancer cell lines inhibits growth and reinstates wild-type p53 activities, including DNA binding, target gene activation, and induction of cell death. Cellular thermal shift assays, conformation-specific immunofluorescence staining, and differential scanning fluorometry suggest that UCI-1001 interacts with and alters the conformation of mutant p53 in cancer cells. These initial results identify pyrimidine trione derivatives of the UCI-1001 series as candidates for p53 corrector drug development.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the WIN site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of functional affinity in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Objective To investigate the relationship between expression levels of epidermal growth factor receptor (EGFR), proliferation marker protein Ki-67 (Ki-67), P53 and circulating tumor cell (CTC) and the prognosis of triple negative breast cancer. Methods A total of 95 patients with triple negative breast cancer were selected. The expression levels of EGFR, Ki-67 and P53 in pathological tissue specimens were detected by immunohistochemistry. All patients underwent 8 cycles of chemotherapy, and the expression of CTC was detected before and after chemotherapy by isolation epithelial tumor cells (ISET). The relationship between CTC expression before and after chemotherapy and efficacy of chemotherapy was analyzed. The correlation between CTC and expression levels of EGFR, Ki-67 and P53 was analyzed. Patients were followed up for progression free survival (PFS). Risk factors of progression for triple negative breast cancer were analyzed by COX regression. Results The positive detection rates of EGFR, Ki-67 and P53 were 44.21% (42/95), 63.16% (60/95) and 56.84% (54/95). The positive detection rate of CTC was lower in patients after chemotherapy than that before chemotherapy (14.74% vs. 61.05%, P＜0.05). The efficacy of chemotherapy was negatively correlated with the positive expression of CTC after chemotherapy (P＜0.001). COX regression analysis found that clinical stage Ⅲ, positive EGFR and CTC after chemotherapy were independent risk factors of progression of triple negatively breast cancer (P＜0.05). PFS of patients at clinical stage Ⅰ, stage Ⅱ, and stage Ⅲ decreased in order (P＜0.05). PFS of EGFR positive patients was shorter than that of EGFR negative patients (P＜0.05). PFS of CTC positive patients after chemotherapy was shorter than that of CTC negative patients after chemotherapy (P＜0.05). Conclusion The positive expression of EGFR before chemotherapy and the positive expression of CTC after chemotherapy are related to the poor survival prognosis of patients with triple negative breast cancer. The lower the positive detection rate of CTC after chemotherapy, the better the efficacy of chemotherapy. [ABSTRACT FROM AUTHOR]

Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest. [BMB Reports 2023; 56(10): 557-562].

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

Skeletal muscle loss and weakness are associated with bad prognosis and poorer quality of life in cancer patients. Tumor-derived factors have been implicated in muscle dysregulation by inducing cachexia and apoptosis. Here, we show that extracellular vesicles secreted by breast cancer cells impair mitochondrial homeostasis and function in skeletal muscle, leading to decreased mitochondrial content and energy production and increased oxidative stress. Mechanistically, miR-122-5p in cancer-cell-secreted EVs is transferred to myocytes, where it targets the tumor suppressor TP53 to decrease the expression of TP53 target genes involved in mitochondrial regulation, including Tfam, Pgc-1α, Sco2, and 16S rRNA. Restoration of Tp53 in muscle abolishes mitochondrial myopathology in mice carrying breast tumors and partially rescues their impaired running capacity without significantly affecting muscle mass. We conclude that extracellular vesicles from breast cancer cells mediate skeletal muscle mitochondrial dysfunction in cancer and may contribute to muscle weakness in some cancer patients.

BackgroundDespite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming of antitumor T lymphocytes due to poor antigen presentation as well as their exclusion and impairment by the immunosuppressive tumor microenvironment (TME). In a recent phase I trial in patients with NSCLC, in situ vaccination (ISV) with dendritic cells engineered to secrete CCL21 (CCL21-DC), a chemokine that facilitates the recruitment of T cells and DC, promoted T lymphocyte tumor infiltration and PD-L1 upregulation.MethodsMurine models of NSCLC with distinct driver mutations (KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)) and varying tumor mutational burden were used to evaluate the efficacy of combination therapy with CCL21-DC ISV plus ICI. Comprehensive analyses of longitudinal preclinical samples by flow cytometry, single cell RNA-sequencing (scRNA-seq) and whole-exome sequencing were performed to assess mechanisms of combination therapy.ResultsISV with CCL21-DC sensitized immune-resistant murine NSCLCs to ICI and led to the establishment of tumor-specific immune memory. Immunophenotyping revealed that CCL21-DC obliterated tumor-promoting neutrophils, promoted sustained infiltration of CD8 cytolytic and CD4 Th1 lymphocytes and enriched progenitor T cells in the TME. Addition of ICI to CCL21-DC further enhanced the expansion and effector function of T cells both locally and systemically. Longitudinal evaluation of tumor mutation profiles revealed that CCL21-DC plus ICI induced immunoediting of tumor subclones, consistent with the broadening of tumor-specific T cell responses.ConclusionsCCL21-DC ISV synergizes with anti-PD-1 to eradicate murine NSCLC. Our data support the clinical application of CCL21-DC ISV in combination with checkpoint inhibition for patients with NSCLC.

BackgroundPatients with acute myeloid leukemia (AML) who have tumor protein p53 (TP53) mutations or a complex karyotype have a poor prognosis, and hypomethylating agents are often used. The authors evaluated the efficacy of entospletinib, an oral inhibitor of spleen tyrosine kinase, combined with decitabine in this patient population.MethodsThis was a multicenter, open-label, phase 2 substudy of the Beat AML Master Trial (ClinicalTrials.gov identifier NCT03013998) using a Simon two-stage design. Eligible patients aged 60 years or older who had newly diagnosed AML with mutations in TP53 with or without a complex karyotype (cohort A; n = 45) or had a complex karyotype without TP53 mutation (cohort B; n = 13) received entospletinib 400 mg twice daily with decitabine 20 mg/m2 on days 1-10 every 28 days for up to three induction cycles, followed by up to 11 consolidation cycles, in which decitabine was reduced to days 1-5. Entospletinib maintenance was given for up to 2 years. The primary end point was complete remission (CR) and CR with hematologic improvement by up to six cycles of therapy.ResultsThe composite CR rates for cohorts A and B were 13.3% (95% confidence interval, 5.1%-26.8%) and 30.8% (95% confidence interval, 9.1%-61.4%), respectively. The median duration of response was 7.6 and 8.2 months, respectively, and the median overall survival was 6.5 and 11.5 months, respectively. The study was stopped because the futility boundary was crossed in both cohorts.ConclusionsThe combination of entospletinib and decitabine demonstrated activity and was acceptably tolerated in this patient population; however, the CR rates were low, and overall survival was short. Novel treatment strategies for older patients with TP53 mutations and complex karyotype remain an urgent need.

BACKGROUND: There is growing evidence that unfavorable neighborhood contexts may influence prostate cancer progression. Whether these associations may be explained in part by differences in tumor-level somatic alterations remain unclear. METHODS: Data on tumor markers (PTEN, p53, ERG, and SPINK1) were obtained from 1,157 participants with prostate cancer in the Health Professionals Follow-up Study. Neighborhood greenness, socioeconomic status, and the income Index of Concentration at Extremes were obtained from satellite and census data and linked to participants address at diagnosis and at study enrollment. Exposures were scaled to an interquartile range and modeled as tertiles. Bivariate associations between tertiles of neighborhood factors and tumor markers were assessed in covariate adjusted logistic regression models to estimate ORs and 95% confidence intervals. RESULTS: There was no association between any of the neighborhood contextual factors and PTEN, p53, ERG, or SPINK1 in bivariate or multivariable adjusted models. Results were generally consistent when modeling exposure using exposure at diagnosis or at study enrollment. CONCLUSIONS: In this multilevel study of men with prostate cancer, we found no evidence of associations between neighborhood context and tumor tissue markers. IMPACT: Our results provide some of the first empirical data in support of the hypothesis that prostate cancer risk conferred by tumor tissue markers may arise independently of underlying neighborhood context. Prospective studies in more diverse populations are needed to confirm these findings.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPβ. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

EIF4E, an mRNA cap-binding protein, is necessary for cap-dependent translation. Overexpression of EIF4E is known to promote cancer development by preferentially translating a group of oncogenic mRNAs. Thus, 4EGI-1, a disruptor of EIF4E-EIF4G1 interaction, was developed to inhibit oncoprotein expression for cancer therapy. Interestingly, RBM38, an RNA-binding protein, interacts with EIF4E on TP53 mRNA, prevents EIF4E from binding to TP53 mRNA cap, and inhibits TP53 expression. Thus, Pep8, an eight amino acid peptide derived from RBM38, was developed to disrupt the EIF4E-RBM38 complex, leading to increased TP53 expression and decreased tumor cell growth. Herein, we have developed a first-in-class small-molecule compound 094, which interacts with EIF4E via the same pocket as does Pep8, dissociates RBM38 from EIF4E, and enhances TP53 translation in RBM38- and EIF4E-dependent manners. Structure-activity relationship studies identified that both the fluorobenzene and ethyl benzamide are necessary for compound 094 to interact with EIF4E. Furthermore, we showed that compound 094 is capable of suppressing three-dimensional tumor spheroid growth in RBM38- and TP53-dependent manners. In addition, we found that compound 094 cooperates with the chemotherapeutic agent doxorubicin and EIF4E inhibitor 4EGI-1 to suppress tumor cell growth. Collectively, we showed that two distinct approaches can be used together to target EIF4E for cancer therapy by enhancing wild-type TP53 expression (094) and by suppressing oncoprotein expression (4EGI-1).

Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-β oligomer (AβO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AβO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AβO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AβO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AβO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AβO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.

The tumor suppressor p53 is the most frequently mutated protein in human cancer and tops the list of high-value precision oncology targets. p53 prevents initiation and progression of cancer by inducing cell-cycle arrest and various forms of cell death. Tumors have thus evolved ways to inactivate p53, mainly by TP53 mutations or by hyperactive p53 degradation. This review focuses on two types of p53 targeting compounds, MDM2 antagonists and mutant p53 correctors. MDM2 inhibitors prevent p53 protein degradation, while correctors restore tumor suppressor activity of p53 mutants by enhancing thermodynamic stability. Herein we explore both novel and repurposed p53 targeting compounds, discuss their mode of action, and examine the challenges in advancing them to the clinic.

Our objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi-institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36-3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11-2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC.

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.

Objective(s): It is worthwhile to note that, some probiotics such as Lactobacilli and Bifidobacteria isolated from dairy products have significant therapeutic effects against cancer cells. Here, we evaluated anti-proliferation and the apoptotic effects of isolated Lactobacillus fermentum Ab.RS22 from traditional dairy products on the HeLa cervical cancer cells in vitro.Materials and Methods: The viability of treated HeLa cells with supernatant of Lactobacillus in 0.5, 0.75, 1, 1.5, and 2 ng/ml concentrations, and IC50 values were detected by tetrazolium bromide. The L. fermentum Ab.RS22-induced cell death by flow cytometry was confirmed through evaluation of the expression of caspase-3, P53, PTEN, and AKT genes by quantitative reverse transcription-polymerase chain reactions (qRT-PCR).Results: Most cytotoxicity effects of Lactobacillus on HeLa cells were detected in 2 ng/ml at 24 hr (P

Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

The transcription factor and tumor suppressor protein p53 is the most frequently mutated and inactivated gene in cancer. Mutations in p53 result in deregulated cell proliferation and genomic instability, both hallmarks of cancer. There are currently no therapies available that directly target mutant p53 to rescue wild-type function. In this study, we identify covalent compsounds that selectively react with the p53 somatic mutant cysteine Y220C and restore wild-type thermal stability.SignificanceThe tumor suppressor p53 is the most mutated gene in cancer, and yet no therapeutics to date directly target the mutated protein to rescue wild-type function. In this study, we identify the first allele-specific compound that selectively reacts with the cysteine p53 Y220C to rescue wild-type thermal stability and gene activation. See related commentary by Lane and Verma, p. 14. This article is highlighted in the In This Issue feature, p. 1.

INTRODUCTION: The expression of androgen receptor (AR) is not commonly tested or studied in uterine cancers, unlike estrogen receptor (ER) and progesterone receptor (PR) which are positive in most endometrial carcinomas. In this series, we evaluated the expression of AR and its comparison to ER and PR in different types of endometrial cancers and have reviewed the literature. MATERIALS AND METHODS: The status of AR, ER, and PR expression were evaluated in 71 cases which were categorized into endometrial endometrioid cancer (EEC), non-endometrioid endometrial cancers (NEEC), and metastatic carcinomas of endometrium. Expression of the receptors were compared to each other as well as to mismatch repair proteins (MMR), p53, and body mass index (BMI) using Fishers Exact test in the StatPlus software. RESULTS: In EECs, the positivity was 97% for all the three receptors. In NEEC, positivity rates were 68%, 48%, and 35% for AR, ER, and PR respectively. In Metastatic carcinomas, AR and ER positivity was seen in 100% while PR was positive in 75% of the cases. In all cancers, the rates were 17% (11/66) for MMR loss, 57% (30/53) for p53 aberrant expression, and 76% (54/71) for the patients with BMI of ≥ 25 (kg/m2). CONCLUSION: AR is expressed in a high percentage of endometrial cancers. Its significance is more evident in high-grade NEEC where ER and PR may not be expressed. These findings warrant further evaluation of AR expression and candidacy of this pathway as a potential therapeutic target in endometrial cancers.

Hepatocellular carcinoma (HCC) is a deadly malignancy with high genetic heterogeneity. TP53 mutation and c-MET activation are frequent events in human HCCs. Here, we discovered that the simultaneous mutations in TP53 and activation of c-MET occur in ~20% of human HCCs, and these patients show a poor prognosis. Importantly, we found that concomitant deletion of Trp53 and overexpression of c-MET (c-MET/sgp53) in the mouse liver led to HCC formation in vivo. Consistent with human HCCs, RNAseq showed that c-MET/sgp53 mouse HCCs were characterized by activated c-MET and Ras/MAPK cascades and increased tumor cell proliferation. Subsequently, a stably passaged cell line derived from a c-MET/sgp53 HCC and corresponding subcutaneous xenografts were generated. Also, in silico analysis suggested that the MEK inhibitor trametinib has a higher inhibition score in TP53 null human HCC cell lines, which was validated experimentally. We consistently found that trametinib effectively inhibited the growth of c-MET/sgp53 HCC cells and xenografts, supporting the possible usefulness of this drug for treating human HCCs with TP53-null mutations. Altogether, our study demonstrates that loss of TP53 cooperates with c-MET to drive hepatocarcinogenesis in vivo. The c-MET/sgp53 mouse model and derived HCC cell lines represent novel and useful preclinical tools to study hepatocarcinogenesis in the TP53 null background.

Acute kidney injury (AKI) is a pathological condition characterized by a rapid decrease in glomerular filtration rate and nitrogenous waste accumulation during hemodynamic regulation. Alisol B, from Alisma orientale, displays anti-tumor, anti-complement, and anti-inflammatory effects. However, its effect and action mechanism on AKI is still unclear. Herein, alisol B significantly attenuated cisplatin (Cis)-induced renal tubular apoptosis through decreasing expressions levels of cleaved-caspase 3 and cleaved-PARP and the ratio of Bax/Bcl-2 depended on the p53 pathway. Alisol B also alleviated Cis-induced inflammatory response (e.g. the increase of ICAM-1, MCP-1, COX-2, iNOS, IL-6, and TNF-α) and oxidative stress (e.g. the decrease of SOD and GSH, the decrease of HO-1, GCLC, GCLM, and NQO-1) through the NF-κB and Nrf2 pathways. In a target fishing experiment, alisol B bound to soluble epoxide hydrolase (sEH) as a direct cellular target through the hydrogen bond with Gln384, which was further supported by inhibition kinetics and surface plasmon resonance (equilibrium dissociation constant, K D = 1.32 μM). Notably, alisol B enhanced levels of epoxyeicosatrienoic acids and decreased levels of dihydroxyeicosatrienoic acids, indicating that alisol B reduced the sEH activity in vivo. In addition, sEH genetic deletion alleviated Cis-induced AKI and abolished the protective effect of alisol B in Cis-induced AKI as well. These findings indicated that alisol B targeted sEH to alleviate Cis-induced AKI via GSK3β-mediated p53, NF-κB, and Nrf2 signaling pathways and could be used as a potential therapeutic agent in the treatment of AKI.

Loss of function in the tumor suppressor gene TP53 is the most common alteration seen in human cancer. In mice, P53 deletion in all cells leads predominantly to the development of T-cell lymphomas, followed by B-cell lymphomas, sarcomas and teratomas. In order to dissect the role of P53 in the hematopoietic system, we generated and analyzed two different mouse models deficient for P53. A pan-hematopoietic P53 deletion mouse was created using Vav1-Cre based deletion; and a B-cell-specific deletion mouse was created using a CD19-Cre based deletion. The Vav1-P53CKO mice predominantly developed T-cell malignancies in younger mice, and myeloid malignancies in older mice. In T-cell malignancies, there was accelerated thymic cell maturation with overexpression of Notch1 and its downstream effectors. CD19-P53CKO mice developed marginal zone expansion in the spleen, followed by marginal zone lymphoma, some of which progressed to diffuse large B-cell lymphomas. Interestingly, marginal zone and diffuse large B-cell lymphomas had a unique gene expression signature characterized by activation of the PI3K pathway, compared with wild type marginal zone or follicular cells of the spleen. This study demonstrates lineage specific P53 deletion leading to distinct phenotypes secondary to unique gene expression programs set in motion.

PurposeTo evaluate efficacy and safety of venetoclax + azacitidine in treatment-naïve patients with acute myeloid leukemia harboring poor-risk cytogenetics and TP53mut or TP53wt.Patients and methodsWe analyzed data from a phase III study (NCT02993523) comparing venetoclax (400 mg orally days 1-28) + azacitidine (75 mg/m2 days 1-7) or placebo + azacitidine, and from a phase Ib study (NCT02203773) of venetoclax + azacitidine. Patients were ineligible for intensive therapy. TP53 status was analyzed centrally; cytogenetic studies were performed locally.ResultsPatients (n = 127) with poor-risk cytogenetics receiving venetoclax + azacitidine (TP53wt = 50; TP53mut = 54) were compared with patients with poor-risk cytogenetics (n = 56) receiving azacitidine alone (TP53wt = 22; TP53mut = 18).For poor-risk cytogenetics + TP53wt patients, venetoclax + azacitidine versus azacitidine alone resulted in composite remission rates (CRc) of 70% versus 23%, median duration of remission (DoR) of 18.4 versus 8.5 months, and median overall survival (OS) of 23.4 versus 11.3 months, respectively. Outcomes with venetoclax + azacitidine were comparable with similarly treated patients with intermediate-risk cytogenetics and TP53wt.For poor-risk cytogenetics + TP53mut patients, venetoclax + azacitidine versus azacitidine alone resulted in CRc of 41% versus 17%, median DoR of 6.5 versus 6.7 months, and median OS of 5.2 versus 4.9 months, respectively.For poor-risk cytogenetics + TP53mut patients, predominant grade ≥3 adverse events (AE) for venetoclax + azacitidine versus azacitidine were febrile neutropenia (55%/39%), thrombocytopenia (28%/28%), neutropenia (26%/17%), anemia (13%/6%), and pneumonia (28%/33%). AEs were comparable between TP53mut and TP53wt patients.ConclusionsIn poor-risk cytogenetics + TP53mut patients, venetoclax + azacitidine improved remission rates but not DoR or OS compared with azacitidine alone. However, in poor-risk cytogenetics + TP53wt patients, venetoclax + azacitidine resulted in higher remission rates and longer DoR and OS than azacitidine alone, with outcomes comparable with similarly treated patients with intermediate-risk cytogenetics. Toxicities were similar in TP53mut and TP53wt patients. See related commentary by Green and Zeidner, p. 5235.

TP53 mutation is the most frequent genetic event in head and neck squamous cell carcinoma (HNSCC), found in more than 80% of patients with human papillomavirus-negative disease. As mutations in the TP53 gene are associated with worse outcomes in HNSCC, novel therapeutic approaches are needed for patients with TP53-mutated tumors. The National Cancer Institute sponsored a Clinical Trials Planning Meeting to address the issues of identifying and developing clinical trials for patients with TP53 mutations. Subcommittees, or breakout groups, were tasked with developing clinical studies in both the locally advanced and recurrent and/or metastatic (R/M) disease settings as well as considering signal-seeking trial designs. A fourth breakout group was focused on identifying and standardizing biomarker integration into trial design; this information was provided to the other breakout groups prior to the meeting to aid in study development. A total of 4 concepts were prioritized to move forward for further development and implementation. This article summarizes the proceedings of the Clinical Trials Planning Meeting with the goal of developing clinical trials for patients with TP53-mutant HNSCC that can be conducted within the National Clinical Trials Network.

Sinonasal malignancies constitute 3% of head and neck cancers, with squamous cell carcinoma (SCC) the most common histology. Neuroendocrine carcinomas (NEC) are rare, with a subset showing neuroendocrine carcinoma and a non-neuroendocrine component. The pathogenesis of these combined tumors is largely unknown, and TP53 driver mutations may play a role. A database search for combined NEC was performed across two institutions (UNM and UCSF) spanning 15 years. Excluding NUT midline carcinoma, 3 cases met inclusion criteria. All were morphologically NEC + SCC and underwent a comprehensive immunohistochemical evaluation. Tumors demonstrated two components histologically: moderately to poorly differentiated SCC and high-grade NEC. Divergent differentiation was confirmed with lineage-specific markers. Only one patient received neoadjuvant chemotherapy prior to surgery, with a remarkable response (a marked decrease in the size of the primary lesion and resolution of liver metastases). Immunohistochemical staining for p53 was increased in 2 of 3 cases (both components), suggesting a role in the carcinogenesis of these tumors. Aberrant expression of beta-catenin was not identified. One case tested positive for p16, which can be seen in high grade NECs due to inactivation of Rb gene. Additionally, both cases with a small cell NEC component expressed PD-L1, suggesting that immunotherapy may be an effective treatment. Findings in this study support the role of p53 mutation in a subset of combined NEC + SCC of the sinonasal tract. Recognition of this rare entity is essential for optimal management of these aggressive neoplasms.

Background & Objective: The regulator of chromosome condensation 2 (RCC2) and RAS-related C3 botulinum toxin substrate 1 (Rac1) have been implicated in the promotion of breast cancer cell proliferation and migration. The signaling pathway involving p53/RCC2/Rac1 has been proposed to contribute to the regulation of colon cancer metastasis. However, until now, this pathway has not been thoroughly investigated in breast cancer. This study seeks to explore the influence of immunohistochemical expression and the correlation among RCC2, Rac1, and p53 in breast infiltrating ductal carcinoma (IDC). Methods: Immunostaining was performed on 120 breast IDC specimens using RCC2, Rac1, and p53 antibodies. Statistical analyses were conducted to examine the correlations between these antibodies. Results: A Positive expression of RCC2, Rac1, and p53 was observed in 116 (96.7%), 120 (100%), and 33 (27.5%) of the breast cancer cases, respectively. RCC2, Rac1, and p53 demonstrated association with poor prognostic parameters such as frequent mitoses, high Ki-67 status, positive lymphovascular invasion (LVI), and advanced tumor stage. A highly significant direct correlation was found between each immunohistochemical marker and the other two markers. Shorter overall survival was linked to multifocal tumors (P=0.017), advanced tumor stage (T3) (P=0.010), Luminal B subtype (P=0.015), progressive disease (P=0.003), positive Her2neu status (P=0.008), and metastasis to distant organs (P<0.001). However, RCC2, Rac1, and p53 did not exhibit a significant association with overall survival. Conclusion: The high expression levels of RCC2, Rac1, and p53 in breast IDC suggest their potential role in tumor behavior. The association of RCC2 and Rac1 with poor prognostic parameters may serve as predictive indicators for aggressive tumors, thus implying that targeted therapy could be beneficial in the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Objective(s): It is worthwhile to note that, some probiotics such as Lactobacilli and Bifidobacteria isolated from dairy products have significant therapeutic effects against cancer cells. Here, we evaluated anti-proliferation and the apoptotic effects of isolated Lactobacillus fermentum Ab.RS22 from traditional dairy products on the HeLa cervical cancer cells in vitro. Materials and Methods: The viability of treated HeLa cells with supernatant of Lactobacillus in 0.5, 0.75, 1, 1.5, and 2 ng/ml concentrations, and IC50 values were detected by tetrazolium bromide. The L. fermentum Ab.RS22-induced cell death by flow cytometry was confirmed through evaluation of the expression of caspase-3, P53, PTEN, and AKT genes by quantitative reverse transcriptionpolymerase chain reactions (qRT-PCR). Results: Most cytotoxicity effects of Lactobacillus on HeLa cells were detected in 2 ng/ml at 24 hr (P<0.01); also, the IC50 value was measured as 1.5 ng/ml. The findings of the flow cytometry assay showed that L. fermentum Ab.RS22 in 1.5 ng/ml concentration at 24 hr increased the percentage of both apoptosis and necrosis cells. Lactobacillus-induced cell death was verified through results of Real-time PCR; where expression of caspase-3, P53, and PTEN genes was increased (P<0.01), and also expression of AKT gene (anti-apoptotic) was decreased (P<0.05). Conclusion: Our findings showed that L. fermentum Ab.RS22 could dose-dependently inhibit the proliferation of the HeLa cells. Its apoptotic effect was confirmed via modulating PTEN/p53/Akt gene expression and activation of the caspase-3 mediated apoptosis pathway. Therefore, L. fermentum Ab.RS22 can be considered a valuable anticancer candidate against cervical cancer progression in subsequent studies. [ABSTRACT FROM AUTHOR]

PURPOSE: The status of p53 in a tumor can be inferred by next-generation sequencing (NGS) or by immunohistochemistry (IHC). We examined the association between p53 IHC and sequence and whether p53 IHC alone, or integrated with TP53 NGS, predicts the outcome. METHODS: From GOG-86P, a randomized phase II study of chemotherapy combined with either bevacizumab or temsirolimus in advanced endometrial cancer, 213 cases had p53 protein expression data measured by IHC and TP53 NGS data. An analysis was designed to integrate p53 expression by IHC with the presence or absence of a TP53 mutation. These variables were further correlated with progression-free survival (PFS) and overall survival (OS) in the chemotherapy plus bevacizumab arms versus the chemotherapy plus temsirolimus arm. RESULTS: In the analysis of p53 IHC, the most striking treatment effect favoring bevacizumab was in cases where p53 was overexpressed (PFS hazard ratio [HR]: 0.46, 95% CI, 0.26 to 0.88; OS HR: 0.31, 95% CI, 0.16 to 0.62). On integrated analysis, patients with TP53 missense mutations and p53 protein overexpression had a similar treatment effect on PFS (HR: 0.41, 95% CI, 0.22 to 0.83) and OS (HR: 0.28, 95% CI, 0.14 to 0.59) favoring bevacizumab plus chemotherapy relative to temsirolimus plus chemotherapy. Concordance between TP53 NGS and p53 IHC was 88%. Concordance was 92% when cases with TP53 mutations and POLE mutations or mismatch repair deficiency were removed. CONCLUSION: IHC for p53 alone or when integrated with sequencing for TP53 identifies a specific, high-risk tumor genotype/phenotype for which bevacizumab is particularly beneficial in improving outcomes when combined with chemotherapy.

The tumor suppressor p53 oscillates in response to DNA double-strand breaks, a behavior that has been suggested to be essential to its anti-cancer function. Nearly all human cancers have genetic alterations in the p53 pathway; a number of these alterations have been shown to be oncogenic by experiment. These alterations include somatic mutations and copy number variations as well as germline polymorphisms. Intriguingly, they exhibit a mixed pattern of interactions in tumors, such as co-occurrence, mutual exclusivity, and paradoxically, mutual antagonism. Using a differential equation model of p53-Mdm2 dynamics, we employ Hopf bifurcation analysis to show that these alterations have a common mode of action, to abolish the oscillatory competence of p53, thereby, we suggest, impairing its tumor suppressive function. In this analysis, diverse genetic alterations, widely associated with human cancers clinically, have a unified mechanistic explanation of their role in oncogenesis.

Neuroendocrine carcinomas (NEC) of the breast are exceedingly rare tumors, which are classified in the WHO system as small cell (SCNEC) and large cell (LCNEC) carcinoma based on indistinguishable features from their lung counterparts. In contrast to lung and enteropancreatic NEC, the genomics of breast NEC have not been well-characterized. In this study, we examined the clinicopathologic, immunohistochemical, and genetic features of 13 breast NEC (7 SCNEC, 4 LCNEC, 2 NEC with ambiguous small versus large cell morphology [ANEC]). Co-alterations of TP53 and RB1 were identified in 86% (6/7) SCNEC, 100% (2/2) ANEC, and 50% (2/4) LCNEC. The one SCNEC without TP53/RB1 alteration had other p53 pathway aberrations (MDM2 and MDM4 amplification) and was immunohistochemically RB negative. PIK3CA/PTEN pathway alterations and ZNF703 amplifications were each identified in 46% (6/13) NEC. Two tumors (1 SCNEC, 1 LCNEC) were CDH1 mutated. By immunohistochemistry, 100% SCNEC (6/6) and ANEC (2/2) and 50% (2/4) LCNEC (83% NEC) showed RB loss, compared to 0% (0/8) grade 3 neuroendocrine tumors (NET) (p

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

PurposePrognostically favorable IDH-mutant gliomas are known to produce oncometabolite D-2-hydroxyglutarate (2HG). In this study, we investigated metabolite-based features of patients with grade 2 and 3 glioma using 2HG-specific in vivo MR spectroscopy, to determine their relationship with image-guided tissue pathology and predictive role in progression-free survival (PFS).MethodsForty-five patients received pre-operative MRIs that included 3-D spectroscopy optimized for 2HG detection. Spectral data were reconstructed and quantified to compare metabolite levels according to molecular pathology (IDH1R132H, 1p/19q, and p53); glioma grade; histological subtype; and T2 lesion versus normal-appearing white matter (NAWM) ROIs. Levels of 2HG were correlated with other metabolites and pathological parameters (cellularity, MIB-1) from image-guided tissue samples using Pearson's correlation test. Metabolites predictive of PFS were evaluated with Cox proportional hazards models.ResultsQuantifiable levels of 2HG in 39/42 (93%) IDH+ and 1/3 (33%) IDH- patients indicated a 91.1% apparent detection accuracy. Myo-inositol/total choline (tCho) showed reduced values in astrocytic (1p/19q-wildtype), p53-mutant, and grade 3 (vs. 2) IDH-mutant gliomas (p

Introduction: This study investigates the impact of two high-intensity interval training (HIIT) programs on PGC-1α, p53, and citrate synthase (CS) proteins within cardiomyocytes of male type 2 diabetic rats, aiming to discern potential molecular mechanisms influencing cardiac health. Material & Methods: Twenty-four male Wistar rats were randomly assigned to control (NC), diabetic control (DC), diabetic with type 1 HIIT (HIIT-1), and diabetic with type 2 HIIT (HIIT-2) groups. Streptozotocin (STZ) induced type 2 diabetes, excluding the NC group. A four-week HIIT intervention, six sessions per week, preceded the analysis of heart tissue for PGC-1α, p53, and CS protein levels. Statistical analysis employed GraphPad Prism software version 8 and one-way ANOVA (P < 0.05). Results: Both HIIT-1 (p=0.004) and HIIT-2 (p=0.007) groups exhibited significantly elevated cardiac PGC-1α levels compared to DC. CS levels increased notably in HIIT-1 (p=0.001) and HIIT-2 (p

CHD8 is an ATP-dependent chromatin-remodeling factor whose monoallelic mutation defines a subtype of autism spectrum disorders (ASDs). Previous work found that CHD8 is required for the maintenance of hematopoiesis by integrating ATM-P53-mediated survival of hematopoietic stem/progenitor cells (HSPCs). Here, by using Chd8F/FMx1-Cre combined with a Trp53F/F mouse model that suppresses apoptosis of Chd8-/- HSPCs, we identify CHD8 as an essential regulator of erythroid differentiation. Chd8-/-P53-/- mice exhibited severe anemia conforming to congenital dyserythropoietic anemia (CDA) phenotypes. Loss of CHD8 leads to drastically decreased numbers of orthochromatic erythroblasts and increased binucleated and multinucleated basophilic erythroblasts with a cytokinesis failure in erythroblasts. CHD8 binds directly to the gene bodies of multiple Rho GTPase signaling genes in erythroblasts, and loss of CHD8 results in their dysregulated expression, leading to decreased RhoA and increased Rac1 and Cdc42 activities. Our study shows that autism-associated CHD8 is essential for erythroblast cytokinesis.

Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here, we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Furthermore, we show that ketone bodies, specifically β-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that β-hydroxybutyrate functions as an HDAC inhibitor within MuSCs, leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting.

At high altitude Andean region, hypoxia-induced excessive erythrocytosis (EE) is the defining feature of Monge's disease or chronic mountain sickness (CMS). At the same altitude, resides a population that has developed adaptive mechanism(s) to constrain this hypoxic response (non-CMS). In this study, we utilized an in vitro induced pluripotent stem cell model system to study both populations using genomic and molecular approaches. Our whole genome analysis of the two groups identified differential SNPs between the CMS and non-CMS subjects in the ARID1B region. Under hypoxia, the expression levels of ARID1B significantly increased in the non-CMS cells but decreased in the CMS cells. At the molecular level, ARID1B knockdown (KD) in non-CMS cells increased the levels of the transcriptional regulator GATA1 by 3-fold and RBC levels by 100-fold under hypoxia. ARID1B KD in non-CMS cells led to increased proliferation and EPO sensitivity by lowering p53 levels and decreasing apoptosis through GATA1 mediation. Interestingly, under hypoxia ARID1B showed an epigenetic role, altering the chromatin states of erythroid genes. Indeed, combined Real-time PCR and ATAC-Seq results showed that ARID1B modulates the expression of GATA1 and p53 and chromatin accessibility at GATA1/p53 target genes. We conclude that ARID1B is a novel erythroid regulator under hypoxia that controls various aspects of erythropoiesis in high-altitude dwellers.

BackgroundTelomere dysfunction is associated with idiopathic pulmonary fibrosis (IPF) and worse outcomes following lung transplantation. Telomere dysfunction may impair immunity by upregulating p53 and arresting proliferation, but its influence on allograft-specific immune responses is unknown. We hypothesized that subjects undergoing lung transplantation for IPF would have impaired T cell proliferation to donor antigens.MethodsWe analyzed peripheral blood mononuclear cells (PBMC) from 14 IPF lung transplant recipients and 12 age-matched non-IPF subjects, before and 2 years after transplantation, as well as PBMC from 9 non-transplant controls. We quantified T cell proliferation and cytokine secretion to donor antigens. Associations between PBMC telomere length, measured by quantitative PCR, and T cell proliferation to alloantigens were evaluated with generalized estimating equation models.ResultsIPF subjects demonstrated impaired CD8+ T cell proliferation to donor antigens pre-transplant (p < 0.05). IL-2, IL-7, and IL-15 cytokine stimulation restored T cell proliferation, while p53 upregulation blocked proliferation. IPF subjects had shorter PBMC telomere lengths than non-IPF subjects (p < 0.001), and short PBMC telomere length was associated with impaired CD8+ T cell proliferation to alloantigens (p = 0.002).ConclusionsIPF as an indication for lung transplant is associated with short PBMC telomere length and impaired T cell responses to donor antigens. However, the rescue of proliferation following cytokine exposure suggests that alloimmune anergy could be overcome. Telomere length may inform immunosuppression strategies for IPF recipients.

Background: C-phycocyanin, a biliprotein from Spirulina platensis, is a future candidate for cancer management. This agent is originated from edible blue-green algae, and numerous in vivo and in vitro research have reported on its anti-cancer properties. The effects of Cphycocyanin have been investigated on caspases 3, 8, 9, and p53 pathways in the human colorectal adenocarcinoma cell line (HT-29) and human umbilical vein endothelial cells (HUVECs). Methods: In the current study, we investigated the effect of C-phycocyanin on caspase 3, 8, 9, and p53-mediated apoptosis pathways in two cell lines (HT-29 & HUVEC), using quantitative real-time PCR and flow cytometry. The cytotoxicity of phycocyanin on HT-29 cells was compared with HUVEC normal cells via colorimetric assays. Results: Based on our findings at molecular level, the expression of caspases 3, 8, 9, and p53 genes were increased in colorectal cancer cells treated with C-phycocyanin. The results were confirmed by an increase in the number of colorectal cancer cells in the early and late stages of apoptosis as compared to the control, untreated cells. In addition, the results of colorimetric assay showed that C-phycocyanin has no cytotoxic effects on normal HUVECs cells. Conclusions: Based on our experimental data, it is evident that C-phycocyanin has measurable effects on cell apoptosis. Since tumorigenesis is halted by apoptosis, C-phycocyanin can be a hopeful candidate for the treatment of human colorectal cancer in the future. [ABSTRACT FROM AUTHOR]

Background: Autoantibody detection is a promising approach to cancer screening. Serum p53 antibodies have been time tested in various cancers, including oral squamous cell carcinoma (OSCC). This study is aimed to detect and determine the level of p53 autoantibodies (p53-AAbs) in saliva. The association of clinicopathological features among patients with and without OSCC was also explored as a novel method for the detection of autoantibodies. Methods: One hundred preoperative saliva samples from patients with histologically confirmed OSCC and a hundred from normal healthy individuals were collected. Anti p53 detection kit assessed levels of salivary p53-AAbs. The cut-off value was 1.3 U/mL by Enzyme-linked immunosorbent assay (ELISA). The p53-AAb levels were expressed in terms of the median and interquartile range (IQR). Fischer's exact test and Chi-square test were used to determine the association with clinicopathological features among patients with OSCC and healthy controls with tobacco consumption habits. Results: Median level of p53-AAb is 0.234 U/mL (IQR 0.18-0.37U/mL) in healthy controls and 0.285U/mL (IQR 0.16-0.58U/mL) in OSCC. p53-AAbs was positive in 15% of 100 patients with OSCC, which was statistically higher (P < 0.001) among OSCC, and controls were negative for p53-AAb. No significant correlation of p53-AAbs with the patient's age, gender, site, clinical staging (TNM), and pathologic grade was observed. However, a significant association was seen between the node involvement and salivary p53-AAbs. Conclusion: Salivary p53-AAb positivity was seen in a higher proportion in OSCC patients than in healthy controls with tobacco consumption, and the levels did differ significantly among OSCC and healthy controls. [ABSTRACT FROM AUTHOR]

The p53 protein is a transcriptional regulatory factor and many of its functions require that it forms a tetrameric structure. Although the tetramerization domain of mammalian p53 proteins (p53TD) share significant sequence similarities, it was recently shown that the tree shrew p53TD is considerably more thermostable than the human p53TD. To determine whether other mammalian species display differences in this domain, we used biophysical, functional, and structural studies to compare the properties of the p53TDs from six mammalian model organisms (human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum). The results indicate that the p53TD from the opossum and tree shrew are significantly more stable than the human p53TD, and there is a correlation between the thermostability of the p53TDs and their ability to activate transcription. Structural analysis of the tree shrew and opossum p53TDs indicated that amino acid substitutions within two distinct regions of their p53TDs can dramatically alter hydrophobic packing of the tetramer, and in particular substitutions at positions corresponding to F341 and Q354 of the human p53TD. Together, the results suggest that subtle changes in the sequence of the p53TD can dramatically alter the stability, and potentially lead to important changes in the functional activity, of the p53 protein. [ABSTRACT FROM AUTHOR]

Objectives: Ameloblastoma, comprising approximately 11% of all odontogenic tumors, is a locally aggressive tumor with a high recurrence rate. This study aimed to assess the immunohistochemical expression of Ki-67 and p53 and their association with clinical and pathological factors among patients with ameloblastoma. Methods: Retrospective follow-up data of patients histologically confirmed with ameloblastoma at Makerere College of Health Sciences in Kampala, Uganda from January 2012 to December 2018 were retrieved. Factors associated with Ki-67 and p53 immunohistochemical expression were determined using one-way one-way analysis of variance. Chi-square and Fisher’s exact statistical tests were used to assess factors associated with recurrence. A two-tailed p < 0.05 was considered statistically significant. Results: A total of 40 patients confirmed histologically with ameloblastoma were included in the analysis. The majority (62.5%) of cases were of the conventional type of ameloblastoma. The expressions of Ki-67 and p53 were 52.5% and 85.0%, respectively. Recurrence was found in 47.5% of patients and it was associated with conventional histological type (p=0.042), segmental resection (p < 0.001), tumor size (p < 0.001), and high p53 expression (p=0.041). Conclusions: Almost half the cases in this study had recurrence. The immunohistochemical expression of p53 was significantly higher than that of Ki-67.

Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discover that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which we name Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. We find that TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation.

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this "bivalent" peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs.

p53 is known to modulate metabolism and FDXR is required for steroidogenesis. Given that FDXR is a target/regulator of p53, the FDXR-p53 axis may play a unique role in lipid metabolism. Here, we found that expression of ABCA1, a cholesterol-efflux pump, was suppressed by loss of FDXR and/or p53, leading to activation of master lipogenic regulators SREBP1/2. Accordingly, lipid droplets, cholesterol, and triglycerides were increased by loss of FDXR or p53, which were further increased by loss of both FDXR and p53. To explore the biological significance of the FDXR-p53 axis, we generated a cohort of mice deficient in Fdxr and/or Trp53. We found that Fdxr+/-, Trp53+/-, and Fdxr+/-;Trp53+/- mice had a short life span and were prone to spontaneous tumors and liver steatosis. Moreover, the levels of serum cholesterol and triglycerides were significantly increased in Fdxr+/- and Trp53+/- mice, which were further increased in Fdxr+/-;Trp53+/- mice. Interestingly, loss of Fdxr but not p53 led to accumulation of serum low-density lipoprotein. Together, our findings reveal that the FDXR-p53 axis plays a critical role in lipid homeostasis and tumor suppression.

PurposeThere is an unmet need for identifying novel biomarkers in Barrett's esophagus that could stratify patients with regards to neoplastic progression. We investigate the expression patterns of extracellular matrix (ECM) molecules in Barrett's esophagus and Barrett's esophagus-related neoplasia, and assess their value as biomarkers for the diagnosis of Barrett's esophagus-related neoplasia and to predict neoplastic progression.Experimental designGene-expression analyses of ECM matrisome gene sets were performed using publicly available data on human Barrett's esophagus, Barrett's esophagus-related dysplasia, esophageal adenocarcinoma (ADCA) and normal esophagus. Immunohistochemical expression of basement membrane (BM) marker agrin (AGRN) and p53 was analyzed in biopsies of Barrett's esophagus-related neoplasia from 321 patients in three independent cohorts.ResultsDifferential gene-expression analysis revealed significant enrichment of ECM matrisome gene sets in dysplastic Barrett's esophagus and ADCA compared with controls. Loss of BM AGRN expression was observed in both Barrett's esophagus-related dysplasia and ADCA. The mean AGRN loss in Barrett's esophagus glands was significantly higher in Barrett's esophagus-related dysplasia and ADCA compared with non-dysplastic Barrett's esophagus (NDBE; P < 0.001; specificity = 82.2% and sensitivity = 96.4%). Loss of AGRN was significantly higher in NDBE samples from progressors compared with non-progressors (P < 0.001) and identified patients who progressed to advanced neoplasia with a specificity of 80.2% and sensitivity of 54.8%. Moreover, the combination of AGRN loss and abnormal p53 staining identified progression to Barrett's esophagus-related advanced neoplasia with a specificity and sensitivity of 86.5% and 58.7%.ConclusionsWe highlight ECM changes during Barrett's esophagus progression to neoplasia. BM AGRN loss is a novel diagnostic biomarker that can identify patients with NDBE at increased risk of developing advanced neoplasia.