MDM2-mediated P53 Ubiquitination Research Articles

HADHA promotes glioma progression by accelerating MDM2-mediated p53 ubiquitination.

Glioma represents a notoriously aggressive and malignant tumor that targets the central nervous system, with a poor prognosis for patients. In this research, we set out to examine the role of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA) in glioma, its clinical significance, as well as its potential biological mechanisms. In this study, we used immunohistochemistry staining to assess the expression level of HADHA in glioma tissues. We also evaluated the correlation between HADHA expression and patient survival using the Kaplan-Meier method. To determine the role of HADHA in glioma cells, we conducted loss-of-function assays in vitro and in vivo. Additionally, we utilized co-immunoprecipitation and protein stability assays to investigate the potential mechanisms involving HADHA, MDM2, and p53 in glioma. Our research findings indicate that gliomas exhibit high levels of HADHA. Clinically, high expression of HADHA suggests an increased risk of malignant tumors, recurrence, and reduced survival rates. Functionally, knocking down HADHA can lead to decreased proliferation, enhanced apoptosis, and inhibited migration of glioma cells. Mechanistically, HADHA accelerates MDM2-mediated p53 ubiquitination through interaction with MDM2. Consistently, MDM2 knockdown or overexpression of p53 can attenuate the promoting effect of HADHA overexpression on the malignant progression of glioma. We have discovered a novel role of HADHA in promoting MDM2-mediated p53 ubiquitination, which contributes to the progression of glioma. This finding provides a new perspective to understand the pathogenesis of glioma and offers a potential target for developing innovative therapeutic strategies.

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

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

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation

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

UTP11 deficiency suppresses cancer development via nucleolar stress and ferroptosis

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

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

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

Abstract 9474: Trim35-Mediated Ubiquitination and Degradation of Nuclear PKM2 is Sufficient to Promote Heart Failure

Introduction: Pyruvate Kinase M2 (PKM2) is a glycolytic enzyme that can translocate to the nucleus to regulate different transcription factors (TF) in different tissues or pathologic states. Although its function has been studied extensively in cancer, its biologic role in the heart and specifically terminally differentiated adult cardiomyocytes remains unresolved. Hypothesis: PKM2 may be critical in regulating cardiomyocyte(CM)-specific TF important for cardiac survival. Methods: Transverse aortic constriction (TAC) banding model was used to assess the levels and modifications of PKM2 during heart failure (HF). CM-specific PKM2-deficient and CM-specific Trim35 overexpressing(OE) mice were generated to evaluate the role of PKM2/Trim35 in adult CM. Human Dilated Left Ventricle biopsies were used to translate mechanistic findings into a clinical setting. Results: Here we show that nuclear PKM2 ( S37 P-PKM2) in CM interacts with several pro-survival or pro-apoptotic TF, including Gata-4, Gata-6, and p53. CM-specific PKM2-deficient mice developed age-dependent dilated cardiac dysfunction and had decreased levels of Gata-4/6, but increased levels of p53, compared to control Cre+ hearts. Mechanistically, we found that nuclear PKM2 can prevent caspase-1 dependent cleavage Gata-4/Gata-6, while also providing a platform for Mdm2-mediated ubiquitination of p53. In a preclinical HF model, nuclear PKM2, along with Gata-4/6 levels decreased, but p53 levels increased (compared to sham controls). Loss of nuclear PKM2 was ubiquitination-dependent and associated with the induction of the E3 ubiquitin-ligase TRIM35. CM-specific Trim35 OE mice had decreased S37 P-PKM2 and GATA-4/6, along with increased p53 levels (compared to littermate controls) and had similar cardiac dysfunction to CM-specific PKM2-deficient mice. In a well-characterized cohort of patients with dilated left ventricles, we found a significant increase in TRIM35 levels that was associated with a decrease in S37 P-PKM2, Gata-4/6 levels, but an increase in p53 levels, compared to non-failing ventricles. Conclusion: This study provides insight into a previously unrecognized biologic role for PKM2 in providing a molecular platform for TF essential for preserving cardiac survival.

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

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

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Anticancer and Antimicrobial Evaluations on Alternative Reading Frame (ARF) Peptides and their Derivatives.

Alternative reading frame (ARF) protein up-regulates the intracellular level of a tumour suppressor protein, p53, by blocking MDM2 mediated p53 ubiquitination. The two homologous forms of ARF proteins are p19ARF in mice and p14ARF in humans. In our study, p19ARF-derived peptide ARF (26-44) and its cell-penetrating peptide conjugate Tat-ARF (26-44), p14ARF-derived peptide ARF (1-22), and its NrLS conjugate ARF (1-22)-NrLS were designed, and their anticancer properties were investigated. Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. Peptides synthesized using solid-phase peptide synthesis (SPPS) were purified using RPHPLC and characterized using Bruker MALDI-TOF mass spectrometry. Cytotoxicity was evaluated on HeLa and BE(2)-C cells by cell viability IC50 determination. Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Morphological studies were carried out using SEM and TEM techniques, live/dead staining, ROS and Hoest staining. Peptides Tat-ARF (1-22) and ARF (1-22)-NrLS exhibited potent cytotoxic effects, comparable to the known standard cisplatin. Cellular morphological studies showed signs of apoptosis which were confirmed by reactive oxygen species (ROS) generation and Hoechst nuclear staining. ARF peptides showed potent antimicrobial activities at low micromolar concentrations without haemolysis. Tat modification improved the activity of ARF (26-44) by 9 folds against HeLa and 5 folds against BE(2)-C cells. NrLS modification of ARF (1-22) imparted 12 fold potency against HeLa and 2-fold potency against BE(2)-C cells. This study helps to further understand the effect of these peptides on MDM2 proteins and their role in the apoptosis signalling pathway.

LncRNA PVT1 contributes to invasion and doxorubicin resistance of bladder cancer cells through promoting MDM2 expression and AURKB-mediated p53 ubiquitination.

In most bladder cancer (BC) patients, cancer cells will eventually develop chemical resistance causing increased mortality. This study aimed to explore the mechanism of lncRNA plasmacytoma variant translocation 1 (PVT1) in regulating doxorubicin (ADM) resistance of BC cells. We observed that PVT1 expression was upregulated in ADM-resistant BC cells compared with ADM-sensitive BC cells. Downregulation of PVT1 suppressed ADM-resistant BC cell proliferation and invasion, promoted apoptosis, and increased sensitivity to ADM, while PVT1 overexpression promoted ADM-sensitive BC cell growth and their resistance to ADM. Further study uncovered that PVT1 could interact with and promote mouse double minute 2 (MDM2) expression, and upregulated MDM2-mediated Aurora kinase B (AURKB). Furthermore, Nutlin-3, an inhibitor of MDM2, could counteract the promotive effects of PVT1 overexpression on ADM resistance of ADM-sensitive BC cell, the expression of multidrug-resistance-related proteins, and the inhibition of p53-mediated tumor suppressor genes. And, overexpression of MDM2 or AURKB reversed the promotive effects of PVT1 silence on the ADM sensitivity of ADM-resistant BC cell, and the inhibitory effect on expression multidrug resistance proteins. Mechanically, AURKB increased MDM2-mediated p53 ubiquitination. Taken together, PVT1 promoted BC cell proliferation and drug resistance via elevating MDM2 expression and AURKB-mediated p53 ubiquitination.

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

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

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

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

Long noncoding RNA Linc01612 represses hepatocellular carcinoma progression by regulating miR-494/ATF3/p53 axis and promoting ubiquitination of YBX1.

Long noncoding RNAs (lncRNAs) play an important role in the progression of hepatocellular carcinoma (HCC). Linc01612 is a novel lncRNA that function remains unknown in the progression of cancers, including HCC. In this study, we discovered that Linc01612 is significantly down-regulated in HCC tissues than in non-tumor tissues and correlated with poor prognosis. Linc01612 mainly localizes in the cytoplasm and functions as a tumor suppressor by repressing the growth and metastasis of hepatoma cells in vitro and in vivo. Mechanistically, in p53-expressing hepatoma cells, Linc01612 acts as a competitive endogenous RNA and promotes the expression of activation transcription factor 3 (ATF3) by sponging microRNA-494 (miR-494), which in turn inhibits MDM2-mediated ubiquitination of p53 and activates the p53 pathway. Furthermore, in p53-null hepatoma cells, Linc01612 exerts its biological functions by physically interacting with Y-box binding protein 1 protein (YBX1) and promoting the ubiquitin-mediated degradation of YBX1. Interestingly, the Linc01612-YBX1 signaling pathway is also present in p53-expressing hepatoma cells. In conclusion, our study indicated that Linc01612 is a functional lncRNA in HCC and Linc01612 may serve as a potential diagnostic biomarker and therapeutic target for HCC.

Multi-combination therapy for temozolomide-resistant GBM: identification of temozolomide/small molecule inhibitor combinations that target the MDM2/p53 and PI3K-AKT/mTOR networks

Introduction: Glioblastoma (GBM) is the most aggressive malignant brain cancer in adults. Induction of the DNA damage response pathway by Temozolomide (TMZ), a DNA alkylating agent, activates p53 resulting in apoptosis. GBM can adapt by upregulatingthe pro-survival pathway regulator Protein Kinase B (AKT), which phosphorylates murine double minute 2 (MDM2) resulting in increased MDM2-mediated p53 ubiquitination. We hypothesize that a combination treatment of blood-brain-barrier penetrant small molecule inhibitors (SMIs) to AKT (GDC-0068) and MDM2 protein-protein interaction inhibitor (RG7388), will stabilize p53 expression and potentiate TMZ-mediated effects in a recurrent p53wt GBM xenoline.
 Methods: Dose response assays followed by Calcusyn statistical analysis determined optimal combination dose ratios. Incucyte imaging analyzedconfluence throughout a treatment cycle. Cellular response was characterized by: 1) Western blotting 2) Flow cytometry with SPiDER β-Gal and FITC Annexin V/ Propidium Iodide to quantify senescent and apoptotic cells, respectively 3) p53 siRNA knockdown to examine p53 dependency in treated cells.
 Results: Combination index identified synergismof TMZ in the presence of AKT and MDM2 inhibitors at clinically achievable concentrations. Incucyte confirmed a low-dose triple combination significantly inhibited tumor growth. Western blots detected low expression of cleaved PARP and elevated expression of p53 and p21 in RG7388-treated cells compared to vehicle, suggesting senescence-related growth inhibition. SPiDER β-Gal and FITC Annexin V/PI assays confirmed a high percentage of senescent cells and minimal apoptosis following combination treatment compared to vehicle or single SMI-treated cells. P53 siRNA knockdown confirmed that cell growth inhibition is p53 dependent in treated cells.
 Conclusion: This study provides rationale for targeting p53 in recurrent p53wt GBM and reveals that senescence could function as a potential therapeutic resistance mechanism. In future studies, targeting of the MDM2-p53 network in the presence of a SM senescence inhibitor will be evaluated to determine if this increases GBM cell death.

PHF6 and JAK3 Mutations Cooperatively Drive T-Cell Acute Lymphoblastic Leukemia Progression

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent hematologic malignancies resulted from gene mutations and/or genomic rearrangements that occur in T cell progenitors. The 5-year survival rate of T-ALL patients is less than 50%. Much efforts have been dedicated to decipher the molecular events underlying TALL transformation, with the goals to identify specific therapeutic targets and develop new and more effective drugs. As a member of JAK kinase family, JAK3 mutations can be identified in 16.1% of T-ALL cases, and JAK3 M511I mutation is the most common one within all JAK3 mutations. Activating JAK3 M511I mutation induced a lympho-proliferative disorder, that followed by a T-ALL-like disease. PHF6 mutation is one of the most common co-existing gene mutations with JAK3 in T-ALL patients. Co-mutation events of JAK3 and PHF6 account for 1.89%-10.0% in T-ALL cases. However, the role(s) of PHF6 and JAK3 co-mutations in tumorigenesis is unknown.Here in this study, we first analyzed the genetic data of 449 T-ALL cases from multiple clinical centers in which JAK/STAT mutations is about 21% of the total cases. Interestingly, we found that PHF6 mutations were significantly associated with JAK3 mutations in these T-ALL cases (P<0.05), and the JAK3 and PHF6 co-mutation occurred in 7.2% of the cohort. Significantly, the average survival time of PHF6 and JAK/STAT co-mutated group was much shorter than that of the single JAK/STAT mutated group (P<0.05) or none-PHF6/JAK/STAT mutated group (P<0.001). We generated Phf6 KO+JAK3 M511I mice by transplanting JAK3M511I infected Phf6 Lin - cells into wild-type mice. All Phf6 KO+JAK3M511Imice succumbed to leukemia from 74 to 101 days after transplantation with significantly shorter survival time than that of Phf6WT+JAK3M511Imice. The Phf6 KO+JAK3M511I mice showed more aggressive phenotypes of T-ALL than Phf6WT+JAK3M511I mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked increase in leukemia-initiating cell activity in Phf6 KO +JAK3M511Icells when compared with Phf6WT +JAK3M511Icells, supporting a role for loss of Phf6 in promoting leukemia blast self-renewal and proliferation.To investigate the underlying molecular mechanisms of Phf6 in accelerating T-ALL development,we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 in isogenic Phf6WT +JAK3M511I or Phf6KO +JAK3M511IT-ALL cells. Gene set enrichment analysis (GSEA) showed up-regulated cell cycle in Phf6 KO +JAK3 M511I leukemia cells. We then performed Chromatin immunoprecipitation sequencing (ChIP-seq) and found that PHF6 associated with BAI1 gene. qPCR and Western blot showed that the mRNA and protein expression of Bai1/BAI1 were significantly decreased in Phf6KO +JAK3M511Icells in comparison with Phf6 WT+JAK3M511I cells. BAI1 has been reported to prevent MDM2-mediated P53 ubiquitination, and loss of BAI1 reduces P53 level. We found that the protein expression of BAI1 and P53 was decreased, and the ubiquitination of P53 was significantly increased in PHF6 knockdown (KD) MOLT-4 cells (T-ALL) when compared with the control cells. BAI1 overexpression in PHF6 KD MOLT-4 cells significantly increased P53 expression in PHF6 KD+BAI1 OE MOLT-4 cells when compared with that of PHF6 KD cells. It suggested that Phf6 deficiency increased BAI1-mediated P53 degradation. To confirm this, we treated Phf6 KO + JAK3M511I mice with a JAK3 inhibitor (ruxolitinib) in combination with a MDM2 inhibitor (idasanutlin), and found a synergistic response of the treatment within a significant attenuation in leukemia burden in vivo.In conclusion, we found that PHF6 mutation frequently co-existed with JAK3 mutation in T-ALLs, and together they can drive an aggressive leukemia in mice model. Notably, PHF6 deficiency promoted JAK3-induced T-ALL progression by inhibiting BAI1-MDM2-P53 signaling pathway that is independent of JAK3-STAT signaling pathway. We further demonstrated that combination therapy by tofacitinib and idasanutlin can reduce the Phf6 KO and JAK3 M511I leukemia burden in vivo. Our study suggested that combinational usage of JAK3 inhibitors and MDM2 inhibitors may increase the drug benefit for T-ALL patients with PHF6 and JAK3 co-mutations. DisclosuresNo relevant conflicts of interest to declare.

Loss of TRIM31 promotes breast cancer progression through regulating K48- and K63-linked ubiquitination of p53

Breast cancer is the most common cancer in the world. Relapse and metastasis are important factors endangering the life of breast cancer patients, but the mechanism is still unclear. The stabilization of p53 is essential for preventing carcinogenesis, and ubiquitination is one of the main ways to regulate the stability of p53. Tripartite motif-containing 31 (TRIM31) is a new member of the TRIM family and functions as an E3 ubiquitin ligase. It acts as a cancer promoter or suppressor in the malignant processes of multiple cancers. However, the function of TRIM31 in breast cancer progression remains unknown. In this study, we showed that TRIM31 is downregulated in breast cancer tissues and negatively correlated with breast cancer progression. Both gain- and loss-of-function assays indicated that TRIM31 inhibits the proliferation, colony formation, migration, and invasion of breast cancer cells. Further investigation demonstrated that TRIM31 directly interacts with p53, and inducing the K63-linked ubiquitination of p53 via its RING domain, Meanwhile, TRIM31 suppresses the MDM2-mediated K48-linked ubiquitination of p53 through competitive inhibiting the interaction of MDM2 and p53, leading to the p53 stabilization and activation. Knockdown of p53 reversed the inhibitory effects of TRIM31 on the growth and metastasis of breast cancer cells. Moreover, we found that the RING and coiled-coil (C–C) domains of TRIM31 were essential for its tumor suppressor function. Taken together, our findings reveal a novel mechanism by which TRIM31 suppresses breast cancer development through the stabilization and activation of p53 and define a promising therapeutic strategy for restoring TRIM31 to treat breast cancer.

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

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

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

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

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

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

PICT1 is critical for regulating the Rps27a-Mdm2-p53 pathway by microtubule polymerization inhibitor against cervical cancer

In our previous study, it showed that P-3F, a podophyllotoxin derivative, causes the increased level of p53 expression by enhancing p53 stability, resulting from blockage of the Mdm2-p53 feedback loop via nucleolus-to-nucleoplasm translocation of Rps27a in human cervical cancer HeLa cell line. However, the mechanism of regulating Rps27a localization remains to be studied. In the current study, it has been demonstrated that the level of protein interacting with carboxyl terminus 1 (PICT1), originally identified as a tumor suppressor, was decreased in a concentration-dependent manner in response to P-3F, leading to inhibition of human cervical cancer cell lines proliferation. Also remarkably, reduction of serine phosphorylation of STMN1 at position 16 induced by P-3F was required in the downregulation of PICT1, in which p53 activity was likely to be directly involved. Note as well that, PICT1 also played an important role in p53 stability enhancement by inhibiting Mdm2-mediated p53 ubiquitination due to Rps27a translocation from the nucleolus to the nucleoplasm to interact with Mdm2 following treatment with P-3F. Collectively, these findings indicated that P-3F, a microtubule polymerization inhibitor, promotes the decreased level of PICT1 expression, which is critical for regulating the Rps27a-Mdm2-p53 pathway against cervical cancer.