P53 Tumor Suppressor Pathway Research Articles

Novel bioassays based on 3D-printed device for sensing of hypoxia and p53 pathway in 3D cell models.

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine.

P53-dependent crosstalk between DNA replication integrity and redox metabolism mediated through a NRF2-PARP1 axis.

Mechanisms underlying p53-mediated protection of the replicating genome remain elusive, despite the quintessential role of p53 in maintaining genomic stability. Here, we uncover an unexpected function of p53 in curbing replication stress by limiting PARP1 activity and preventing the unscheduled degradation of deprotected stalled forks. We searched for p53-dependent factors and elucidated RRM2B as a prime factor. Deficiency in p53/RRM2B results in the activation of an NRF2 antioxidant transcriptional program, with a concomitant elevation in basal PARylation in cells. Dissecting the consequences of p53/RRM2B loss revealed a crosstalk between redox metabolism and genome integrity that is negotiated through a hitherto undescribed NRF2-PARP1 axis, and pinpoint G6PD as a primary oxidative stress-induced NRF2 target and activator of basal PARylation. This study elucidates how loss of p53 could be destabilizing for the replicating genome and, importantly, describes an unanticipated crosstalk between redox metabolism, PARP1 and p53 tumor suppressor pathway that is broadly relevant in cancers and can be leveraged therapeutically.

Computational-based drug design of novel small molecules targeting p53-MDMX interaction

The regulation of the p53 tumor suppressor pathway is critically dependent on the activity of Murine Double Minute 2 (MDM2) and Murine Double Minute X (MDMX) proteins. In certain types of cancer cells, excessive amount of MDMX can poly-ubiquitinate p53, which can result in its degradation, leading to a subsequent reduction in the levels of this protein. Therefore, the design of small-molecule inhibitors targeting the MDMX-p53 interaction has emerged as a promising strategy for cancer therapy. In this study, we employed computational techniques including pharmacophore modeling and molecular docking to identify three potential small molecule inhibitors (CID_25094615, CID_137634453, and CID_25094344) of the MDMX-p53 interaction from a PubChem database. Molecular dynamics of 100000 ps were conducted to assess the stability of the MDMX-inhibitor complexes. Our results showed that all three compounds exhibit stable binding with MDMX, with significantly lower root mean square deviation (RMSD) and fluctuation (RMSF) values than the control ligand, indicating superior stability. Additionally, the three compounds exhibit stronger intermolecular hydrogen bond (HBOND) interactions compared to the control, suggesting stronger stability. Overall, our findings highlight the potential of these compounds as lead candidates for the development of novel anticancer agents that target the MDMX-p53 interaction. Communicated by Ramaswamy H. Sarma

Silencing ribosomal protein L4 enhances the inhibitory effects of triptolide on non-small cell lung cancer cells by disrupting the mouse double minute 2 protein–P53 tumor suppressor pathway

Silencing ribosomal protein L4 enhances the inhibitory effects of triptolide on non-small cell lung cancer cells by disrupting the mouse double minute 2 protein–P53 tumor suppressor pathway

Characterizing DNA methylation signatures of retinoblastoma using aqueous humor liquid biopsy

Retinoblastoma (RB) is a cancer that forms in the developing retina of babies and toddlers. The goal of therapy is to cure the tumor, save the eye and maximize vision. However, it is difficult to predict which eyes are likely to respond to therapy. Predictive molecular biomarkers are needed to guide prognosis and optimize treatment decisions. Direct tumor biopsy is not an option for this cancer; however, the aqueous humor (AH) is an alternate source of tumor-derived cell-free DNA (cfDNA). Here we show that DNA methylation profiling of the AH is a valid method to identify the methylation status of RB tumors. We identify 294 genes directly regulated by methylation that are implicated in p53 tumor suppressor (RB1, p53, p21, and p16) and oncogenic (E2F) pathways. Finally, we use AH to characterize molecular subtypes that can potentially be used to predict the likelihood of treatment success for retinoblastoma patients.

The Therapeutic Potential of the Restoration of the p53 Protein Family Members in the EGFR-Mutated Lung Cancer.

Despite the recent development of precision medicine and targeted therapies, lung cancer remains the top cause of cancer-related mortality worldwide. The patients diagnosed with metastatic disease have a five-year survival rate lower than 6%. In metastatic disease, EGFR is the most common driver of mutation, with the most common co-driver hitting TP53. EGFR-positive patients are offered the frontline treatment with tyrosine kinase inhibitors, yet the development of resistance and the lack of alternative therapies make this group of patients only fit for clinical trial participation. Since mutant p53 is the most common co-driver in the metastatic setting, therapies reactivating the p53 pathway might serve as a promising alternative therapeutic approach in patients who have developed a resistance to tyrosine kinase inhibitors. This review focuses on the molecular background of EGFR-mutated lung cancer and discusses novel therapeutic options converging on the reactivation of p53 tumor suppressor pathways.

Integrated virtual screening and molecular dynamics simulation revealed promising drug candidates of p53-MDM2 interaction.

In the vast majority of malignancies, the p53 tumor suppressor pathway is compromised. In some cancer cells, high levels of MDM2 polyubiquitinate p53 and mark it for destruction, thereby leading to a corresponding downregulation of the protein. MDM2 interacts with p53 via its hydrophobic pocket, and chemical entities that block the dimerization of the protein-protein complex can restore p53 activity. Thus far, only a few chemical compounds have been reported as potent arsenals against p53-MDM2. The Protein Data Bank has crystallogaphic structures of MDM2 in complex with certain compounds. Herein, we have exploited one of the complexes in the identification of new p53-MDM2 antagonists using a hierarchical virtual screening technique. The initial stage was to compile a targeted library of structurally appropriate compounds related to a known effective inhibitor, Nutlin 2, from the PubChem database. The identified 57 compounds were subjected to virtual screening using molecular docking to discover inhibitors with high binding affinity for MDM2. Consequently, five compounds with higher binding affinity than the standard emerged as the most promising therapeutic candidates. When compared to Nutlin 2, four of the drug candidates (CID_140017825, CID_69844501, CID_22721108, and CID_22720965) demonstrated satisfactory pharmacokinetic and pharmacodynamic profiles. Finally, MD simulation of the dynamic behavior of lead-protein complexes reveals the stability of the complexes after a 100,000ps simulation period. In particular, when compared to the other three leads, overall computational modeling found CID_140017825 to be the best pharmacological candidate. Following thorough experimental trials, it may emerge as a promising chemical entity for cancer therapy.

AIB1 is a novel target of the high-risk HPV E6 protein and a biomarker of cervical cancer progression.

The high‐risk human papillomaviruses (HPV‐16, ‐18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ‐containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high‐risk E6 but not low‐risk E6 expression led to increases in AIB1 transcript levels by real‐time RT‐PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV‐16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Enterobacteria impair host p53 tumor suppressor activity through mRNA destabilization

Increasing evidence highlights the role of bacteria in the physiopathology of cancer. However, the underlying molecular mechanisms remains poorly understood. Several cancer-associated bacteria have been shown to produce toxins which interfere with the host defense against tumorigenesis. Here, we show that lipopolysaccharides from Klebsiella pneumoniae and other Enterobacteria strongly inhibit the host tumor suppressor p53 pathway through a novel mechanism of p53 regulation. We found that lipopolysaccharides destabilize TP53 mRNA through a TLR4-NF-κB-mediated inhibition of the RNA-binding factor Wig-1. Importantly, we show that K. pneumoniae disables two major tumor barriers, oncogene-induced DNA damage signaling and senescence, by impairing p53 transcriptional activity upon DNA damage and oncogenic stress. Furthermore, we found an inverse correlation between the levels of TLR4 and p53 mutation in colorectal tumors. Hence, our data suggest that the repression of p53 by Enterobacteria via TLR4 alleviates the selection pressure for p53 oncogenic mutations and shapes the genomic evolution of cancer.

Tumor suppressor p53 promotes ferroptosis in oxidative stress conditions independent of modulation of ferroptosis by p21, CDKs, RB, and E2F

p53 is a well-established critical cell cycle regulator. By inducing transcription of the gene encoding p21, p53 inhibits cyclin-dependent kinase (CDK)-mediated phosphorylation of cell cycle inhibitor retinoblastoma (RB) proteins. Phosphorylation of RB releases E2F transcription factor proteins that transactivate cell cycle-promoting genes. Here, we sought to uncover the contribution of p53, p21, CDK, RB, and E2F to the regulation of ferroptosis, an oxidative form of cell death. Our studies have uncovered unexpected complexity in this regulation. First, we showed that elevated levels of p53 enhance ferroptosis in multiple inducible and isogenic systems. On the other hand, we found that p21 suppresses ferroptosis. Elevation of CDK activity also suppressed ferroptosis under conditions where p21 suppressed ferroptosis, suggesting that the impact of p21 must extend beyond CDK inhibition. Furthermore, we showed that overexpression of E2F suppresses ferroptosis in part via a p21-dependent mechanism, consistent with reports that this transcription factor can induce transcription of p21. Finally, deletion of RB genes enhanced ferroptosis. Taken together, these results show that signals affecting ferroptotic sensitivity emanate from multiple points within the p53 tumor suppressor pathway.

PPM1D Mutations in MDS Patients with Deletion of Chromosome 5q

Background: Patients with myelodysplastic syndrome with deletion of chromosome 5q (MDS del(5q)) have a favorable prognosis, but TP53 mutations occur in 13-18% of these patients and are known as unfavorable prognostic marker. Recently, Protein Phosphatase Magnesium-Dependent 1 (PPM1D) mutations have been described in clonal hematopoiesis and were found more commonly in patients with therapy-related MDS than in patients with primary MDS (15% vs. 3%) (Lindsley et al. 2017). PPM1D encodes a serine-threonine protein phosphatase that inhibits the p53 tumor suppressor pathway. It is therefore of great interest to evaluate the frequency and potential co-occurrence of PPM1D mutations in patients with MDS del(5q).Aim: To determine the frequency and potential clinical impact of PPM1D mutations in a large cohort of MDS del(5q) patients.Methods: 219 patients with MDS or AML following MDS (sAML) and deletion of chromosome 5q were included in the analysis. Patients were classified following the 2008 WHO classification. Patients with MDS (n=196) or sAML (n=23) were cytogenetically characterized by chromosome banding analysis and molecularly analyzed for mutations in all exons (1 to 6) of PPM1D by Sanger or next-generation sequencing (NGS). Patients were also analyzed for mutations in TP53 and CSNK1A1 .Results: A total of 11 PPM1D mutations were found in 10 (4.6%) MDS patients with del(5q). The mutation frequency was similar in patients with MDS with isolated del5q (8 of 167, 4.8%) compared to patients with more advanced MDS or secondary AML (2 of 52, 3.8%, P=.78). Four patients had nonsense mutations, 3 patients had frameshift mutations (one patient with 2 frameshift mutations), and 3 patients had missense mutations. Nine mutations were found in the previously described hotspot region of PPM1D between amino acids 427 and 542, which is located downstream of the phosphatase domain of PPM1D. Three of the 10 PPM1D mutated patients had additional cytogenetic aberrations besides del(5q) (trisomy 8; complex karyotype in two patients). TP53 was also mutated in the two patients with complex karyotype and in one additional patient with a nonsense mutation of PPM1D . None of the PPM1D mutated patients had a mutation in CSNK1A1 .Patients with mutated PPM1D were slightly older than PPM1D wildtype patients (median age 77.7 vs 71 years, not significant). Eight of 10 PPM1D mutated patients had a diagnosis of MDS del(5q) and 2 patients had MDS RAEB2 with del(5q) accompanied by a complex karyotype. There were no significant differences in the distribution of sex, WHO subtypes, cytogenetic risk according to the international prognostic scoring system (IPSS), WBC count, hemoglobin, platelet count and IPSS risk score between PPM1D wildtype and mutated patients. Two of the 10 patients were transfusion dependent and none of 7 patients with available information transformed to AML.Survival information was available for 156 patients with MDS del(5q) (excluding other WHO subtypes of MDS/sAML) including 8 patients with mutated PPM1D and 15 patients with mutated TP53 . Median follow-up from the time of sample harvest was 2.5 years. The probability of survival at 2 years was 100% for PPM1D mutated and TP53 mutated patients, respectively, and 84% for PPM1D/TP53 wildtype patients (P=.18, log-rank test, Figure 1A). None of 7 PPM1D mutated patients and 1 of 13 TP53 mutated patients with available information underwent allogeneic hematopoietic cell transplantation.Survival information was available for 45 patients with del(5q) and more advanced MDS or secondary AML. The 14 patients with TP53 mutation (including 2 patients with additional PPM1D mutation) had a significantly shorter OS than TP53 wildtype patients (P<.001, 2-year OS 0% vs 51%, Figure 1B). One PPM1D mutated patient responded well to lenalidomide treatment for the duration of 3.2 years.Conclusion:PPM1D mutations are recurrently mutated in MDS patients with del(5q) with a frequency of 4.6% and may co-occur with TP53 mutations. PPM1D mutated patients were not specifically associated with patient characteristics or outcome. Interestingly, in our cohort of patients with MDS with isolated del(5q), TP53 mutations were not associated with unfavorable outcome.VP and MM contributed equally as first authors. TH and MH contributed equally as last authors. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Platzbecker:Novartis: Consultancy, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Thiede:Novartis: Consultancy, Speakers Bureau; Roche: Consultancy; Agendix: Employment; Bayer: Consultancy, Speakers Bureau. Germing:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Schroeder:Celgene: Consultancy, Honoraria, Other: travel support. Kobbe:Celgene: Other: Advisory Board, Research Funding. Haase:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Dohner:Novartis: Honoraria, Research Funding. Sperr:Novartis: Other: Register; Phadia: Research Funding; Meda: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Blueprint: Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Pfizer: Honoraria; Deciphera: Honoraria, Research Funding; Teva: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Emerging Roles of TRIM8 in Health and Disease.

The superfamily of TRIM (TRIpartite Motif-containing) proteins is one of the largest groups of E3 ubiquitin ligases. Among them, interest in TRIM8 has greatly increased in recent years. In this review, we analyze the regulation of TRIM8 gene expression and how it is involved in many cell reactions in response to different stimuli such as genotoxic stress and attacks by viruses or bacteria, playing a central role in the immune response and orchestrating various fundamental biological processes such as cell survival, carcinogenesis, autophagy, apoptosis, differentiation and inflammation. Moreover, we show how TRIM8 functions are not limited to ubiquitination, and contrasting data highlight its role either as an oncogene or as a tumor suppressor gene, acting as a “double-edged weapon”. This is linked to its involvement in the selective regulation of three pivotal cellular signaling pathways: the p53 tumor suppressor, NF-κB and JAK-STAT pathways. Lastly, we describe how TRIM8 dysfunctions are linked to inflammatory processes, autoimmune disorders, rare developmental and cardiovascular diseases, ischemia, intellectual disability and cancer.

HIV-1 Protease Inhibitors Slow HPV16-Driven Cell Proliferation through Targeted Depletion of Viral E6 and E7 Oncoproteins.

Simple SummaryHigh-risk strains of human papillomavirus (HPV) such as HPV16 cause anogenital and oropharyngeal cancers, with HPV-associated cervical cancer being the fourth most common cancer worldwide and a major cause of death for women. Anti-HPV vaccines are available but coverage is low, and while the vaccines prevent infection they are not therapeutic for treating cancers in individuals already infected with high-risk HPV. Instead, current treatments for HPV-associated cancers include surgery, chemotherapy, and radiation, and affect diseased and healthy tissues alike. Accordingly, there is a dire need for better, molecularly targeted strategies. Herein, we report the novel finding that at least four protease inhibitor drugs currently used to treat HIV/AIDS—lopinavir, ritonavir, nelfinavir, and saquinavir—cause marked and selective depletion of HPV16 E6 and E7 oncoproteins in HPV-positive cancer cells and a 3-D cell culture model for HPV16-positive epithelial pre-cancer (NIKS16 cells). E6 and E7 depletion correlated with anticancer phenotypes including increased p53 protein levels and induction of cell growth arrest in CaSki cells as well as slowed epithelium growth and reversal of HPV16-driven phenotypes consistent with inhibition of the cellular autophagy pathway in the NIKS16 model. Based on this progress in vitro, we propose that HIV protease inhibitors, already known to have anticancer properties, be studied further in the context of developing a targeted therapy for HPV16-driven cancers and pre-cancers.High-risk human papillomavirus strain 16 (HPV16) causes oral and anogenital cancers through the activities of two viral oncoproteins, E6 and E7, that dysregulate the host p53 and pRb tumor suppressor pathways, respectively. The maintenance of HPV16-positive cancers requires constitutive expression of E6 and E7. Therefore, inactivating these proteins could provide the basis for an anticancer therapy. Herein we demonstrate that a subset of aspartyl protease inhibitor drugs currently used to treat HIV/AIDS cause marked reductions in HPV16 E6 and E7 protein levels using two independent cell culture models: HPV16-transformed CaSki cervical cancer cells and NIKS16 organotypic raft cultures (a 3-D HPV16-positive model of epithelial pre-cancer). Treatment of CaSki cells with some (lopinavir, ritonavir, nelfinavir, and saquinavir) but not other (indinavir and atazanavir) protease inhibitors reduced E6 and E7 protein levels, correlating with increased p53 protein levels and decreased cell viability. Long-term (>7 day) treatment of HPV16-positive NIKS16 raft cultures with saquinavir caused epithelial atrophy with no discernible effects on HPV-negative rafts, demonstrating selectivity. Saquinavir also reduced HPV16′s effects on markers of the cellular autophagy pathway in NIKS16 rafts, a hallmark of HPV-driven pre-cancers. Taken together, these data suggest HIV-1 protease inhibitors be studied further in the context of treating or preventing HPV16-positive cancers.

Germline and Somatic Genetic Variants in the p53 Pathway Interact to Affect Cancer Risk, Progression, and Drug Response.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

Cross Talk between the Circadian Clock Proteins and TP53 in Cancer and Therapeutic Significance.

The circadian rhythms regulate physiological and cellular processes that maintain normal homeostasis, keeping our cells synchronized to the dark-light cycle. The disruption of circadian clock genes has been associated with various diseases, including cancer. The clock genes impact several cancer-related signaling trajectories, including the tumor suppressor's p53 pathway involvement in the regulation of the cell cycle and apoptosis. In most human cancers, p53 loses its normal functions and tumor-suppressive activity through function-inactivating mutations. Herein, we review the roles of each of the clock genes (PER1-3, CRY1/2, BMAL1, CLOCK, REV-ERBα/β, RORα, and SIRT1) in their association and regulation of p53. We analyzed by bioinformatics the expression of several clock genes (CLOCK, CRY1-2, PER1-3), p53, and apoptotic and metabolic genes across all tumors in the TCGA platform, and found deregulated patterns in many cases. Our findings support the development of new therapies targeted against some clock genes to restore p53 activities and the inhibition of tumor development. Such therapies will complement a large number of currently tested treatments targeting the restoration of wild-type p53 to prevent tumor growth and lead cancer cells to apoptosis.

Retraction for Bischof et al., "Human Papillomavirus Oncoprotein E7 Targets the Promyelocytic Leukemia Protein and Circumvents Cellular Senescence via the Rb and p53 Tumor Suppressor Pathways".

This article refers to:Human Papillomavirus Oncoprotein E7 Targets the Promyelocytic Leukemia Protein and Circumvents Cellular Senescence via the Rb and p53 Tumor Suppressor Pathways

Reduction of Squalene Epoxidase by Cholesterol Accumulation Accelerates Colorectal Cancer Progression and Metastasis

Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is suggested as a proto-oncogene. Paradoxically, SQLE is degraded by excess cholesterol, and low SQLE is associated with aggressive colorectal cancer (CRC). Therefore, we studied the functional consequences of SQLE reduction in CRC progression. Gene and protein expression data and clinical features of CRCs were obtained from public databases and 293 human tissues, analyzed by immunohistochemistry. Invitro studies showed underlying mechanisms of CRC progression mediated by SQLE reduction. Mice were fed a 2% high-cholesterol or a control diet before and after cecum implantation of SQLE genetic knockdown/control CRC cells. Metastatic dissemination and circulating cancer stem cells were demonstrated by invivo tracking and flow cytometry analysis, respectively. Invitro studies showed that SQLE reduction helped cancer cells overcome constraints by inducing the epithelial-mesenchymal transition required to generate cancer stem cells. Surprisingly, SQLE interacted with GSK3β and p53. Active GSK3β contributes to the stability of SQLE, thereby increasing cell cholesterol content, whereas SQLE depletion disrupted the GSK3β/p53 complex, resulting in a metastatic phenotype. This was confirmed in a spontaneous CRC metastasis mice model, where SQLE reduction, by a high-cholesterol regimen or genetic knockdown, strikingly promoted CRC aggressiveness through the production of migratory cancer stem cells. We showed that SQLE reduction caused by cholesterol accumulation aggravates CRC progression via the activation of the β-catenin oncogenic pathway and deactivation of the p53 tumor suppressor pathway. Our findings provide new insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC aggressiveness and a prognostic biomarker.

Abstract 3427: Finding critical cancer driving and cancer suppressing genes using functional genomics screening in vivo

Abstract P53 is one of the most potent tumour suppressors and its activity is also required for cancer cells to respond to diverse chemotherapeutic drugs. Focused functional genetic screens in haematopoietic cells in vivo revealed DNA repair to be one of the most critical downstream pathways for p53's tumour suppressive function. Recently, we extended our search for novel tumour suppressor genes acting potentially outside the p53 tumour suppressor pathway. To this end, we performed whole genome CRISPR/Cas9 knockout screening in vivo, using the Eµ-Myc transgenic mouse model. This identified previously unknown genes and hence pathways, which upon deletion/mutation lead to the acceleration of tumour onset. Amongst these novel tumour suppressor genes, we identified the GATOR1 members DEPDC5 and NPRL3, which are known inhibitors of the mTOR pathway. Interestingly, drug mediated inhibition of mTOR activity with rapamycin or Torin1 in GATOR1 deficient Eµ-Myc cells led to efficient killing of these malignant cells, which was in striking contrast to control Eµ-Myc lymphoma cells that were resistant to this treatment. In parallel we conducted CRISPR/Cas9 screens for identifying p53 bound promoter and enhancer elements in haematopoietic cells in vivo. Excitingly, we observed that targeting the NPRL3 promoter led to accelerated tumour onset in the Eµ-Myc transgenic model, suggesting that NPRL3 is under direct transcriptional control of p53. These results indicate that p53 mediated shutdown of the mTOR pathway through upregulation of the GATOR1 member NPRL3 is a critical tumour suppressive function of p53. Citation Format: Marco Josef Herold, Shinsuke Mizutani, Yexuan Deng, Ana Janic, Andrew Kueh, Martin Pal, Stephen Wilcox, Lin Tai, Gemma L. Kelly, Andreas Strasser. Finding critical cancer driving and cancer suppressing genes using functional genomics screening in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3427.

Abstract 4190: A promising blood-brain-barrier penetrant MDM2-p53 antagonist, BI-MDM2, increases survival in orthotopic, glioblastoma patient-derived xenograft models

Abstract Robust function of the p53 tumor suppressor pathway is critical when treating with DNA-damage inducing agents such as radiation therapy (RT), which is a key component of standard care for GBM. MDM2 is an important negative regulator of p53 stability and MDM2 is amplified in approximately 14% of GBM. Based on the concept that suppression of MDM2 can reactivate p53 function and potentially have single agent or combinatorial effects, multiple MDM2 inhibitors have been developed. Here we report in vitro and in vivo efficacy and pharmacodynamic (PD) effects of a BBB-penetrant MDM2-p53 antagonist, BI-MDM2, in GBM patient-derived xenograft (PDX) models. In vitro studies in p53 wild-type (WT) lines with or without MDM2 amplification demonstrate IC50 values in cell viability assays of 2-12 nM in serum-free culture and 5-35 nM in serum-containing culture after seven days of treatment. In vivo studies were performed in p53 WT lines: the MDM2-amplified GBM108, and the non-amplified GBM14, grown as orthotopic tumors in nude mice. Weekly oral treatment at 2 mg/kg of BI-MDM2 doubled median survival (placebo, 28 days (d) vs 2 mg/kg BI-MDM2, 57 d. p&amp;lt;0.0001) while weekly dosing at 10 mg/kg extended the median over 5 fold (176 d. p&amp;lt;0.0001) with half of the mice still living at 223 days post-inoculation. To assess the PD properties of this compound, mice bearing orthotopic GBM108 were treated three weeks after inoculation with a single dose of 2 or 10 mg/kg BI-MDM2. Normal brain, tumor, and plasma were collected at 24 and 48 hours (h) after dosing and downstream p53 transcriptional targets, p21 and PUMA were evaluated by qRT-PCR. p21 mRNA relative quantification detection at 24h increased by 1.5-10.5-fold in the 2 mg/kg group of animals compared to vehicle while the 10 mg/kg dose led to an increase by 2-25 fold compared to vehicle. PUMA was minimally affected by the 2 mg/kg dose but increased 1.2-17-fold over vehicle in the 10 mg/kg group at 24h. The observed increases in p21 and PUMA varied little between the 24 and 48h timepoints. In an orthotopic GBM14 PDX efficacy study, BI-MDM2 was combined with 20 Gy RT delivered in 10 fractions over 2 weeks with dosing limited to 2 weeks of therapy or until mice reached a moribund state. While two doses of 10 mg/kg BI-MDM2 alone had a modest effect on survival (41 d vs 31 d with placebo; p=0.002), continued dosing until moribund further extended median survival (82 d with BI-MDM2; p=0.001). The combination of two doses of BI-MDM2 with 2 weeks of RT extended survival as compared to RT alone (107 d vs. 69 d, respectively; p=0.019), while with extended drug dosing combined with 2 weeks of RT, median survival has not yet been reached (&amp;gt;125 d). Taken together, these results suggest that BI-MDM2 is a promising therapeutic agent that may provide significant anti-tumor efficacy either alone or in combination with RT in both MDM2 amplified and non-amplified p53 WT patients. Citation Format: Ann C. Mladek Tuma, Shiv Gupta, Surabhi Talele, Afroz Shareef Mohammad, Katrina K. Bakken, Helen He, Zeng Hu, Margaret A. Connors, Danielle M. Burgenske, Brett L. Carlson, William F. Elmquist, Ulrike Weyer-Czernilofsky, Jann N. Sarkaria. A promising blood-brain-barrier penetrant MDM2-p53 antagonist, BI-MDM2, increases survival in orthotopic, glioblastoma patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4190.