Blockade of deubiquitinase YOD1 degrades oncogenic PML/RARα and eradicates acute promyelocytic leukemia cells.

In most acute promyelocytic leukemia (APL) cells, promyelocytic leukemia (PML) fuses to retinoic acid receptor α (RARα) due to chromosomal translocation, thus generating PML/RARα oncoprotein, which is a relatively stable oncoprotein for degradation in APL. Elucidating the mechanism regulating the stability of PML/RARα may help to degrade PML/RARα and eradicate APL cells. Here, we describe a deubiquitinase (DUB)-involved regulatory mechanism for the maintenance of PML/RARα stability and develop a novel pharmacological approach to degrading PML/RARα by inhibiting DUB. We utilized a DUB siRNA library to identify the ovarian tumor protease (OTU) family member deubiquitinase YOD1 as a critical DUB of PML/RARα. Suppression of YOD1 promoted the degradation of PML/RARα, thus inhibiting APL cells and prolonging the survival time of APL cell-bearing mice. Subsequent phenotypic screening of small molecules allowed us to identify ubiquitin isopeptidase inhibitor I (G5) as the first YOD1 pharmacological inhibitor. As expected, G5 notably degraded PML/RARα protein and eradicated APL, particularly drug-resistant APL cells. Importantly, G5 also showed a strong killing effect on primary patient-derived APL blasts. Overall, our study not only reveals the DUB-involved regulatory mechanism on PML/RARα stability and validates YOD1 as a potential therapeutic target for APL, but also identifies G5 as a YOD1 inhibitor and a promising candidate for APL, particularly drug-resistant APL treatment.

RUNX3 derived hsa_circ_0005752 accelerates the osteogenic differentiation of adipose-derived stem cells via the miR-496/MDM2-p53 pathway.

BACKGROUND: Circular RNAs (circRNAs) are non-coding RNAs that play a pivotal role in bone diseases. RUNX3 was an essential transcriptional regulator during osteogenesis. However, it is unknown whether RUNX3 regulates hsa_circ_0005752 during osteogenic differentiation. METHODS: The levels of hsa_circ_0005752 and RUNX3 were measured by qRT-PCR after osteogenic differentiation of ADSCs. The osteogenic differentiation was analyzed by Alkaline phosphatase (ALP) staining and Alizarin red staining (ARS). qRT-PCR and western blot were used to assess the expressions of osteogenic differentiation-related molecules. RNA pull-down, RIP, and luciferase reporter assays determine the interactions between miR-496 and hsa_circ_0005752 or MDM2 mRNA. CHIP-PCR analyzed the interaction between RUNX3 and LPAR1. Finally, the potential roles of RUNX3 were investigated during osteogenic differentiation with or without hsa_circ_0005752 knockdown. RESULTS: Hsa_circ_0005752 and RUNX3 were significantly increased, and miR-496 was remarkably decreased in ADSCs after osteogenic differentiation. Hsa_circ_0005752 could promote osteogenic differentiation, as shown by enhancing ALP and ARS staining intensity. Hsa_circ_0005752 enhanced the expressions of Runx2, ALP, Osx, and OCN. Furthermore, hsa_circ_0005752 directly targeted miR-496, which can directly bind to MDM2. RUNX3 bound to the LPAR1 promoter and enhanced hsa_circ_0005752 expressions. Moreover, the enhanced expression of hsa_circ_0005752 by RUNX3 could promote osteogenic differentiation, whereas knockdown of hsa_circ_0005752 partially antagonized the effects of RUNX3. CONCLUSION: Our study demonstrated that RUNX3 promoted osteogenic differentiation via regulating the hsa_circ_0005752/miR-496/MDM2 axis and thus provided a new therapeutic strategy for osteoporosis.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression.

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

Abrogation of USP7 is an alternative strategy to downregulate PD-L1 and sensitize gastric cancer cells to T cells killing.

Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing in vitro and in vivo. Besides, USP7 inhibitor suppressed GC cells proliferation by stabilizing P53 in vitro and in vivo. Collectively, our findings indicate that in addition to inhibiting cancer cells proliferation, USP7 inhibitor can also downregulate PD-L1 expression to enhance anti-tumor immune response simultaneously. Hence, these data posit USP7 inhibitor as an anti-proliferation agent as well as a novel therapeutic agent in PD-L1/PD-1 blockade strategy that can promote the immune response of the tumor.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53

The RNA recognition motif of NIFK is required for rRNA maturation during cell cycle progression.

Ribosome biogenesis governs protein synthesis. NIFK is transactivated by c-Myc, the key regulator of ribosome biogenesis. The biological function of human NIFK is not well established, except that it has been shown to interact with Ki67 and NPM1. Here we report that NIFK is required for cell cycle progression and participates in the ribosome biogenesis via its RNA recognition motif (RRM). We show that silencing of NIFK inhibits cell proliferation through a reversible p53-dependent G1 arrest, possibly by induction of the RPL5/RPL11-mediated nucleolar stress. Mechanistically it is the consequence of impaired maturation of 28S and 5.8S rRNA resulting from inefficient cleavage of internal transcribed spacer (ITS) 1, a critical step in the separation of pre-ribosome to small and large subunits. Complementation of NIFK silencing by mutants shows that RNA-binding ability of RRM is essential for the pre-rRNA processing and G1 progression. More specifically, we validate that the RRM of NIFK preferentially binds to the 5'-region of ITS2 rRNA likely in both sequence specific and secondary structure dependent manners. Our results show how NIFK is involved in cell cycle progression through RRM-dependent pre-rRNA maturation, which could enhance our understanding of the function of NIFK in cell proliferation, and potentially also cancer and ribosomopathies.

The induction of thioredoxin-1 by epinephrine withdraws stress via interaction with ß-arrestin-1.

Stress regulates a panel of important physiological functions and disease states. Epinephrine is produced under stresses threaten to homeostasis. Thioredoxin-1(Trx-1) is a redox regulating protein which is induced to resist stresses and related with various diseases. Thus, it is important to examine whether Trx-1 is induced by epinephrine and to understand the underlying molecular mechanisms that Trx-1 modulates epinephrine stress. Here, we show that the expression of Trx-1 was induced by epinephrine via ß-adrenergic receptor/Cyclic AMP/protein kinase A (PKA) signaling pathway in PC12 cells. The down-regulation of Trx-1 by siRNA aggravated accumulation of γ-H2AX and further decreased expression of p53 by epinephrine. Accordingly, Trx-1 overexpression alleviated accumulation of γ-H2AX and restored the expressions of p53 and C/EBP homologous protein (CHOP) in the cortex, hippocampus and thymus of mice. Moreover, Trx-1 overexpression reduced the malondialdehyde concentration by epinephrine. We further explored the mechanism on p53 and γ-H2AX regulated by Trx-1. We found that overexpression of Trx-1 suppressed ß-arrestin-1 expression through interaction with ß-arrestin-1. Consequently, the downregulation of ß-arrestin-1 suppressed the cell viability and the expressions of γ-H2AX and cyclin D1, and increased p53 expression. Taken together, our data suggest that Trx-1/ß-arrestin-1 interaction may represent a novel endogenous mechanism on protecting against stress.

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.