Enhances P53 Activity Research Articles

ATF2/BAP1 Axis Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage via P53 Pathway.

Neuronal apoptosis plays an essential role in the pathogenesis of brain injury after subarachnoid hemorrhage (SAH). BAP1 (BRCA1-associated protein 1) is considered to exert pro-apoptotic effects in multiple diseases. However, evidence supporting the effect of BAP1 on the apoptotic response to SAH is lacking. Therefore, we aimed to confirm the role of BAP1 in SAH-induced apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to detect BAP1 expression in the cerebrospinal fluid. Endovascular perforation was performed in mice to induce SAH. Lentiviral short hairpin RNA targeting BAP1 mRNA was transduced into the ipsilateral cortex of mice with SAH to investigate the role of BAP1 in neuronal damage. Luciferase and coimmunoprecipitation assays were performed to investigate the mechanism through which BAP1 participates in hemin-induced SAH. First, BAP1 expression was upregulated in the cerebrospinal fluid of patients with SAH and positively associated with unfavorable outcomes. ATF2 (activating transcription factor-2) then regulated BAP1 expression by binding to the BAP1 promoter. In addition, BAP1 overexpression enhanced P53 activity and stability by reducing P53 proteasome-mediated degradation. Subsequently, elevated P53 promoted neuronal apoptosis via the P53 pathway. Inhibition of the neuronal BAP1/P53 axis significantly reduced neurological deficits and neuronal apoptosis and improved neurological dysfunction in mice after SAH. Our results suggest that the neuronal ATF2/BAP1 axis exerts a brain-damaging effect by modulating P53 activity and stability and may be a novel therapeutic target for SAH.

Abstract 5473: Combined inhibition of MDM2 and PARP lead to a synergistic anti-tumoral response in p53 wild-type rhabdomyosarcoma

Abstract Targeting MDM2-p53 interaction to enhance p53 activity represents an attractive antitumoral strategy for p53 wild-type tumors. However, early-phase clinical trials in hematological and solid cancers have shown limited efficacy of MDM2 inhibitors highlighting the necessity for exploring more effective combinations with additional agents. In this study, we provide results from a 22-drug combination screening demonstrating the therapeutic potential of combining Siremadlin (MDM2 inhibitor) and Olaparib (PARP inhibitor) for treating p53 wild-type rhabdomyosarcoma (RMS). Cell survival, cell death and apoptosis analysis revealed synergistic effects when combining Siremadlin and Olaparib in vitro. Combination of both drugs resulted in a significant increase of p53 activity as evidenced by p53 accumulation and K382 acetylation along with an increased expression of p53 targets. Finally, combination of both drugs resulted in significant reduction of tumor growth and increased overall survival outperforming individual treatments and control groups. In summary, our study demonstrates, for the first time, the synergistic effect of the combination between Siremadlin and Olaparib in the inhibition of p53 wild-type RMS tumor growth in vitro and in vivo. These findings advocate for the exploration of this drug combination in clinical trials and underscore the need for further investigation in tumors sharing similar molecular features. Citation Format: Guillem Pons, Gabriel Gallo-Oller, Lia García-Gilabert, Patricia Zarzosa, Júlia Sansa-Girona, Natalia Navarro, Paula Cabré-Fernández, Miguel F Segura, Josep Sánchez de Toledo, Soledad Gallego, Lucas Moreno, Josep Roma. Combined inhibition of MDM2 and PARP lead to a synergistic anti-tumoral response in p53 wild-type rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5473.

Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.

Molecular Mechanisms of Chloroquine and Hydroxychloroquine Used in Cancer Therapy

Tumour relapse, chemotherapy resistance, and metastasis continue to be unsolved issues in cancer therapy. A recent approach has been to scrutinise drugs used in the clinic for other illnesses and modify their structure to increase selectivity to cancer cells. Chloroquine (CQ) and hydroxychloroquine (HCQ), known antimalarials, have successfully treated autoimmune and neoplastic diseases. CQ and HCQ, well-known lysosomotropic agents, induce apoptosis, downregulate autophagy, and modify the tumour microenvironment. Moreover, they affect the Toll 9/NF-κB receptor pathway, activate stress response pathways, enhance p53 activity and CXCR4-CXCL12 expression in cancer cells, which would help explain their effects in cancer treatment. These compounds can normalise the tumour associated vasculature, promote the activation of the immune system, change the phenotype of tumour-associated macrophages (from M2 to M1), and stimulate cancer-associated fibroblasts. We aim to review the historical aspects of CQ and its derivatives and the most relevant mechanisms that support the therapeutic use of CQ and HCQ for the treatment of cancer.

Autophagy inhibition impairs leukemia stem cell function in FLT3-ITD AML but has antagonistic interactions with tyrosine kinase inhibition.

The FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to target leukemia stem cells (LSC) and do not generate sustained responses. Autophagy is an important cellular stress response contributing to hematopoietic stem cells (HSC) maintenance and promoting leukemia development. Here we investigated the role of autophagy in regulating FLT3-ITD AML stem cell function and response to TKI treatment. We show that autophagy inhibition reduced quiescence and depleted repopulating potential of FLT3-ITD AML LSC, associated with mitochondrial accumulation and increased oxidative phosphorylation. However, TKI treatment reduced mitochondrial respiration and unexpectedly antagonized the effects of autophagy inhibition on LSC attrition. We further show that TKI-mediated targeting of AML LSC and committed progenitors was p53-dependent, and that autophagy inhibition enhanced p53 activity and increased TKI-mediated targeting of AML progenitors, but decreased p53 activity in LSC and reduced TKI-mediated LSC inhibition. These results provide new insights into the role of autophagy in differentially regulating AML stem and progenitor cells, reveal unexpected antagonistic effects of combined oncogenic tyrosine kinase inhibition and autophagy inhibition in AML LSC, and suggest an alternative approach to target AML LSC quiescence and regenerative potential.

Pgc-1α controls epidermal stem cell fate and skin repair by sustaining NAD+ homeostasis during aging

ObjectiveThe epidermal barrier is renewed by the activation, proliferation, and differentiation of keratinocyte stem cells after injury and aging impedes this repair process through undefined mechanisms. We previously identified a gene signature of metabolic dysfunction in aged murine epidermis, but the precise regulators of epidermal repair and age-related growth defects are not well established. Aged mouse models as well as mice with conditional epidermal loss of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1α) were used to explore the cellular pathways which control skin repair after injury and stress. MethodsAged mice or those with epidermal Pgc-1α deletion (epiPgc-1α KO) and young or Pgc1afl/fl controls were subjected to wound injury, UVB exposure or the inflammatory agent TPA. In vivo and ex vivo analyses of wound closure, skin structure, cell growth and stem cell differentiation were used to understand changes in epidermal re-growth and repair resulting from aging or Pgc-1α loss. ResultsAging impairs epidermal re-growth during wound healing and results in lower expression of Pgc-1α. Mice with conditional deletion of epidermal Pgc-1α exhibit greater inflammation- and UVB-induced cell differentiation, reduced proliferation, and slower wound healing. epiPgc-1α KO mice also displayed reduced keratinocyte NAD+ levels, shorter telomeres, and greater poly ADP-ribosylation, resulting in enhanced stress-stimulated p53 and p21 signaling. When NAD+ was reduced by Pgc-1α loss or pharmacologic inhibition of NAD+ synthesis, there was reduced stress-induced proliferation, increased differentiation, and protection against DNA damage via enhanced epidermal shedding. Similarly, aged mice exhibit disrupted epidermal NAD+ homeostasis and enhanced p53 activation, resulting in p21 growth arrest after wounding. NAD+ precursor treatment restores epidermal growth from old skin to that of young. ConclusionsOur studies identify a novel role for epidermal Pgc-1α in controlling epidermal repair via its regulation of cellular NAD+ and downstream effects on p53-driven growth arrest. We also establish that parallel mechanisms are evident in aged epidermis, showing that NAD+ signaling is an important controller of physiologic skin repair and that dysfunction of this pathway contributes to age-related wound repair defects.

Abstract 250: Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy

Abstract Canonical p53-activated pathways can influence a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages (TAMs). We examined whether p53 activity in the tumor microenvironment (TME) influences antitumor immunity and show that p53 signaling induced pharmacologically with APR-246 (eprenetapopt) can augment the efficacy of immune checkpoint blockade (ICB) in preclinical models, a strategy that is also being tested in patients (NCT04383938). We first investigated the effects of combining APR-246 with ICB in wildtype C57BL6 (B6) mice bearing syngeneic p53 wildtype MC38 colon cancer and B16 melanoma tumors. The combination of an anti-PD-1 antibody (RMP1-14) with APR-246 in mice significantly delayed tumor growth (p < 0.001) and improved survival of tumor-bearing mice, compared to monotherapies (p < 0.01). To further dissect the effects of APR-246 on myeloid and T cells in the TME, we used a conditional knockout of p53 in CSF1R+myeloid cells (CSF1Rcre/p53fl mice), or T cells (CD8cre/p53fl mice). CSF1Rcre/p53fl had loss of tumor control and worse survival with APR-246+anti-PD-1. CD8cre/p53fl had intact tumor control. To study enhanced p53 activity in the TME, we performed flow cytometry, cytokine multiplex and global transcriptional profiling by RNA seq. We found enhanced p53-activity led to increased infiltration of T cells, increased MHC-II expression in TAMs and downregulation of M2-associated cytokines. This was associated with cellular senescence in TAMs and induction of canonical p53-induced senescence-associated secretory phenotype (SASP). Our preclinical findings informed the development of a phase I/II clinical trial using APR-246 with pembrolizumab for patients with advanced solid tumors (NCT04383938). We studied peripheral blood samples from two of the patients with tumor regression and two patients in whom tumors progressed on therapy. We analyzed peripheral blood mononuclear cells (PBMCs) and serum prior to therapy, and at the beginning of cycle 2 and 5 for the patients with tumor control, and at the end of therapy for patients who had progression. Single cell RNA-seq of PBMCs demonstrated a signature consistent with T cell activation and proliferation, and SASP-associated changes in the myeloid compartment as seen in mice. T cell profiling of PBMCs by flow cytometry demonstrated strong proliferation of T cells in patients with tumor control. Serum cytokine analysis demonstrated robust in IL-12, IFN-gamma and Eotaxin-1 in the two responders, which was not seen in the patients whose tumors progressed. Our study illustrates p53-induced SASP in TAMs as a mechanism to reprogram the TME and augment responses to ICB. Ongoing studies will help determine biomarkers that are predictive of response to APR-246+ICB therapy. Citation Format: Arnab Ghosh, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, Nathan Suek, Aliya Holland, Yu-Jui Ho, Mohsen Abu-Akeel, Luis Felipe Campesato, Levi Mark Bala Mangarin, Cailian Liu, Hong Zhong, Sadna Budhu, Andrew Chow, Roberta Zappasodi, Marcus Ruscetti, Scott W. Lowe, Taha Merghoub, Jedd D. Wolchok. Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 250.

Inhibition of STAT3Y705 phosphorylation by Stattic suppresses proliferation and induces mitochondrial-dependent apoptosis in pancreatic cancer cells

Patients with pancreatic cancer (PC) show dismal prognosis and high mortality. The development of PC is associated with the overactivation of STAT3. Here, we have determined that the non-peptide small molecule Stattic inhibits PC development by targeting STAT3. In vitro, Stattic treatment time- and dose-dependently inhibited proliferation of pancreatic cancer cells (PCCs) by reducing c-Myc expression and enhancing p53 activity. Consequently, p-Rb, cyclin D1, Chk1, and p21 (cell cycle proteins) were downregulated, and PCCs were arrested at the G1 phase, which was also confirmed by decreased Ki67 expression and unaltered PCNA expression. In addition, Stattic-induced mitochondrial-dependent apoptosis by elevating cleaved caspase-3, and Bax, cytochrome C levels, while reducing expression of Bcl-2, which may be regulated by reduced survivin expression. Further studies showed that Stattic exerts its anti-tumor effect via inhibition of STAT3Y705 phosphorylation and nuclear localization in PCCs. In a nude mouse tumorigenesis model, Stattic inhibited PC growth by antagonizing STAT3Y705 phosphorylation. Interleukin-6 used as a molecule agonist to activate STAT3, as well as overexpression of STAT3, could partially reverse Stattic-mediated anti-proliferation and pro-apoptotic effects of PCCs. Thus, these findings indicate that inhibition of STAT3Y705 phosphorylation by Stattic suppresses PCC proliferation and promotes mitochondrial-mediated apoptosis.

CYLD deficiency enhances metabolic reprogramming and tumor progression in nasopharyngeal carcinoma via PFKFB3

Aberrant cancer metabolism contributes to cell proliferation and tumor progression. However, the contribution of enhanced glycolysis, observed during cancer metabolism, to the pathogenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. CYLD, an NF-κB inhibitor, is frequently deficient in NPC. Here, we investigated the role of CYLD in the metabolic reprogramming of NPC and found that restoration of CYLD expression suppressed glycolysis in NPC cells. Mechanistic dissection showed that CYLD stabilized p53 and facilitated its nuclear translocation, thereby enhancing p53 activity by removing K63-linked and K48-linked ubiquitin chains of p53, which can bind to the PFKFB3 promoter and inhibit its transcription. Additionally, CYLD interacted with FZR1 to promote APC/C-FZR1 E3 ligase activity, which further ubiquitinated and degraded PFKFB3 via the 26S proteasomal system. Furthermore, clinical tissue array analysis indicated that low expression of CYLD was correlated with high expression of PFKFB3 and poor prognosis among patients with NPC. In conclusion, CYLD suppressed PFKFB3 expression via two factors, namely, p53 and FZR1, to inhibit glycolysis and delay tumor growth and progression in NPC. CYLD is a biomarker indicating poor prognosis of patients with NPC.

Alterations of the Mdm2 C-Terminus Differentially Impact Its Function In Vivo.

This in vivo study highlights that alterations to the C-terminus of Mdm2 perturb its regulation of the tumor suppressor p53.

Enhanced p53 Activation By Dual Inhibition of MDM2 and XPO1 Disrupts MYC Transcriptional Program and Restores Sensitivity to BCL-2 Inhibition in Ven/HMA Resistant AML

Enhanced p53 Activation By Dual Inhibition of MDM2 and XPO1 Disrupts MYC Transcriptional Program and Restores Sensitivity to BCL-2 Inhibition in Ven/HMA Resistant AML

ANP32B-mediated repression of p53 contributes to maintenance of normal and CML stem cells

AbstractProper regulation of p53 signaling is critical for the maintenance of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). The hematopoietic cell–specific mechanisms regulating p53 activity remain largely unknown. Here, we demonstrate that conditional deletion of acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) in hematopoietic cells impairs repopulation capacity and postinjury regeneration of HSCs. Mechanistically, ANP32B forms a repressive complex with p53 and thus inhibits the transcriptional activity of p53 in hematopoietic cells, and p53 deletion rescues the functional defect in Anp32b-deficient HSCs. Of great interest, ANP32B is highly expressed in leukemic cells from patients with chronic myelogenous leukemia (CML). Anp32b deletion enhances p53 transcriptional activity to impair LSC function in a murine CML model and exhibits synergistic therapeutic effects with tyrosine kinase inhibitors in inhibiting CML propagation. In summary, our findings provide a novel strategy to enhance p53 activity in LSCs by inhibiting ANP32B and identify ANP32B as a potential therapeutic target in treating CML.

WIP1 Inhibition by GSK2830371 Potentiates HDM201 through Enhanced p53 Phosphorylation and Activation in Liver Adenocarcinoma Cells.

Simple SummaryPatients with advanced intrahepatic cholangiocarcinoma (iCCA) have a very poor prognosis, and no targeted therapy is approved for advanced iCCA. A therapeutic strategy for wild-type p53 cancers is the reactivation of p53 by inhibition of its the negative regulators, MDM2, and WIP1. In the present study, we used HDM201 (an MDM2-p53 binding antagonist) to increase p53 stabilization and upregulate the expression of downstream targets (p21 and MDM2) in RBE and SK-Hep-1 liver adenocarcinoma cell lines. The survival rate and clonogenicity decreased after HDM201 treatment in a dose-dependent manner. Combined treatment with HDM201 and GSK2830371 (WIP1 inhibitor) increased p53 phosphorylation, leading to sustained p53 activation. This combination treatment resulted in G2/M phase arrest and promoted cytotoxicity compared with MDM2 inhibitor monotherapy. Furthermore, increased expression of p53 signaling pathway target genes were identified following combination treatment with HDM201 and GSK2830371, suggesting potential roles for this combination strategy in iCCA therapy.Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA.

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

Preferentially released miR-122 from cyclodextrin-based star copolymer nanoparticle enhances hepatoma chemotherapy by apoptosis induction and cytotoxics efflux inhibition

Chemotherapy, as one of the most commonly used treatment modalities for cancer therapy, provides limited benefits to hepatoma patients, owing to its inefficient delivery as well as the intrinsic chemo-resistance of hepatoma. Bioinformatic analysis identified the therapeutic role of a liver-specific microRNA — miR-122 for enhancing chemo-therapeutic efficacy in hepatoma. Herein, a cyclodextrin-cored star copolymer nanoparticle system (sCDP/DOX/miR-122) is constructed to co-deliver miR-122 with doxorubicin (DOX) for hepatoma therapy. In this nanosystem, miR-122 is condensed by the outer cationic poly (2-(dimethylamino) ethyl methacrylate) chains of sCDP while DOX is accommodated in the inner hydrophobic cyclodextrin cavities, endowing a sequential release manner of miR-122 and DOX. The preferentially released miR-122 not only directly induces cell apoptosis by down regulation of Bcl-w and enhanced p53 activity, but also increases DOX accumulation through inhibiting cytotoxic efflux transporter expression, which realizes synergistic performance on cell inhibition. Moreover, sCDP/DOX/miR-122 displays remarkably increased anti-tumor efficacy in vivo compared to free DOX and sCDP/DOX alone, indicating its great promising in hepatoma therapy.

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs – enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

Smad3 promotes AKI sensitivity in diabetic mice via interaction with p53 and induction of NOX4-dependent ROS production

The incidence and severity of acute kidney injury (AKI) is increased yearly in diabetic patients. Although the mechanisms for this remain unclear, the prevention of AKI in diabetic nephropathy is feasible and of value. As we detected highly activation of TGF-β/Smad3 signaling in both human biopsy and mouse model of diabetic nephropathy, we hypothesized that Smad3 activation in diabetic kidneys may increase AKI sensitivity. We tested our hypothesis in vitro using TGF-β type II receptor (TGF-βRII) disrupted tubular epithelial cells (TECs) and in vivo in mice with streptozotocin (STZ)-induced diabetic nephropathy before the induction of ischemia/reperfusion (I/R) injury. We found that high glucose (HG)-cultured TECs showed increased inflammation, apoptosis and oxidative stress following hypoxia/reoxygenation (H/R) injury. Disruption of TGF-βRII attenuated cell injury induced by H/R in HG-treated TECs. Consistently, Smad3 knockdown in diabetic kidney attenuated I/R-induced AKI. Mechanistically, Smad3 binds to p53 and enhances p53 activity in cells treated with HG and H/R, which may lead to TECs apoptosis. Additionally, ChIP assay showed that Smad3 bound with the promoter region of NOX4 and induced ROS production and inflammation. In conclusion, our results demonstrate that Smad3 promotes AKI susceptibility in diabetic mice by interacting with p53 and NOX4.

Transient enhancement of p53 activity protects from radiation-induced gastrointestinal toxicity

Gastrointestinal (GI) syndrome is a serious side effect and dose-limiting toxicity observed in patients undergoing lower-abdominal radiotherapy. Previous mouse studies show that p53 gene dosage determines susceptibility to GI syndrome development. However, the translational relevance of p53 activity has not been addressed. Here, we used a knock-in mouse in which the p53-Mdm2 negative feedback loop is genetically disrupted. These mice retain biallelic p53 and thus, normal basal p53 levels and activity. However, due to the lack of p53-mediated Mdm2 transcription, irradiated Mdm2P2/P2 mice exhibit enhanced acute p53 activity, which protects them from GI failure. Intestinal crypt cells residing in the +4 and higher positions exhibit decreased apoptosis, increased p21 expression, and hyperproliferation to reinstate intestinal integrity. Correspondingly, pharmacological augmentation of p53 activity in wild-type mice with an Mdm2 inhibitor protects against GI toxicity without affecting therapeutic outcome. Our results suggest that transient disruption of the p53-Mdm2 interaction to enhance p53 activity could be a viable prophylactic strategy for alleviating GI syndrome in patients undergoing radiotherapy.

ROS -mediated p53 activation by juglone enhances apoptosis and autophagy in vivo and in vitro

Juglone (JG) exhibits a broad-spectrum of cytotoxicity against some cancer cells. However, its molecular mechanisms have not been investigated well. Here, the present results showed that JG significantly inhibited tumor growth in vivo. CCK-8 assays, flow cytometric analysis, western blotting and immunohistochemistry revealed that JG effectively inhibited cell proliferation and induced apoptosis through extrinsic pathways. We also observed that JG treatment induced autophagy flux via activiting the AMPK-mTOR signaling pathway. In addition, we found that JG enhanced p53 activation by increasing down-regulation of ubiquitin-mediated degradation. Inhibition of p53 by siRNA attenuated JG-induced cell death and autophagy. Moreover, JG enhanced the generation of hydrogen peroxide (H2O2) and superoxide anion radical (O2• −). Further experiments proved that H2O2 was a major factor since the H2O2 scavenger catalase (CAT) reduced both autophagy and cell death to a greater extent than the O2• − scavenger SOD. Overall, our results illustrated that JG caused apoptosis and autophagy via activating the ROS-mediated p53 pathway in human liver cancer cells in vitro and in vivo, which provided basic scientific evidence that JG serves as a potential anti-cancer agent.

Correction to: LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis, and Atherosclerosis by Enhancing p53 Activity.

HomeCirculationVol. 139, No. 17Correction to: LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis, and Atherosclerosis by Enhancing p53 Activity Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis, and Atherosclerosis by Enhancing p53 Activity Originally published23 Apr 2019https://doi.org/10.1161/CIR.0000000000000690Circulation. 2019;139:e887This article corrects the followingLincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis, and Atherosclerosis by Enhancing p53 ActivityIn the article by Wu et al, “LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis, and Atherosclerosis by Enhancing p53 Activity,” which published in the October 21, 2014, issue of Circulation (Circulation. 2014;130:1452–1465. doi: 10.1161/CIRCULATIONAHA.114.011675), a correction is needed.Both Drs Wang and Zeng were corresponding authors, however only Dr Wang was indicated as corresponding author on the journal website.The correction has been made to the current online version of the article, which is available at https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.114.011675.Footnoteshttps://www.ahajournals.org/journal/circ Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesLincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis, and Atherosclerosis by Enhancing p53 ActivityGengze Wu, et al. Circulation. 2014;130:1452-1465 April 23, 2019Vol 139, Issue 17 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIR.0000000000000690PMID: 31013140 Originally publishedApril 23, 2019 PDF download Advertisement