Galectin-7 Induction by EHMT2 Inhibition Enhances Immunity in Microsatellite Stability Colorectal Cancer.

BACKGROUND & AIMS: Although immunotherapy shows substantial advancement in colorectal cancer (CRC) with microsatellite instability high, it has limited efficacy for CRC with microsatellite stability (MSS). Identifying combinations that reverse immune suppression and prime MSS tumors for current immunotherapy approaches remains an urgent need. METHODS: An in vitro CRISPR screen was performed using coculture models of primary tumor cells and autologous immune cells from MSS CRC patients to identify epigenetic targets that could enhance immunotherapy efficacy in MSS tumors. RESULTS: We revealed EHMT2, a histone methyltransferase, as a potential target for MSS CRC. EHMT2 inhibition transformed the immunosuppressive microenvironment of MSS tumors into an immunomodulatory one by altering cytokine expression, leading to T-cell-mediated cytotoxicity activation and improved responsiveness to anti-PD1 treatment. We observed galectin-7 up-regulation upon EHMT2 inhibition, which converted a "cold" MSS tumor environment into a T-cell-inflamed one. Mechanistically, CHD4 repressed galectin-7 expression by recruiting EHMT2 to form a cotranscriptional silencing complex. Galectin-7 administration enhanced anti-PD1 efficacy in MSS CRC, serving as a potent adjunct cytokine therapy. CONCLUSIONS: Our findings suggest that targeting the EHMT2/galectin-7 axis could provide a novel combination strategy for immunotherapy in MSS CRC.

RNA m6 A methylation regulates sorafenib resistance in liver cancer through FOXO3-mediated autophagy.

N6-methyladenosine (m6 A) is an abundant nucleotide modification in mRNA, known to regulate mRNA stability, splicing, and translation, but it is unclear whether it is also has a physiological role in the intratumoral microenvironment and cancer drug resistance. Here, we find that METTL3, a primary m6 A methyltransferase, is significantly down-regulated in human sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promotes sorafenib resistance and expression of angiogenesis genes in cultured HCC cells and activates autophagy-associated pathways. Mechanistically, we have identified FOXO3 as a key downstream target of METTL3, with m6 A modification of the FOXO3 mRNA 3'-untranslated region increasing its stability through a YTHDF1-dependent mechanism. Analysis of clinical samples furthermore showed that METTL3 and FOXO3 levels are tightly correlated in HCC patients. In mouse xenograft models, METTL3 depletion significantly enhances sorafenib resistance of HCC by abolishing the identified METTL3-mediated FOXO3 mRNA stabilization, and overexpression of FOXO3 restores m6 A-dependent sorafenib sensitivity. Collectively, our work reveals a critical function for METTL3-mediated m6 A modification in the hypoxic tumor microenvironment and identifies FOXO3 as an important target of m6 A modification in the resistance of HCC to sorafenib therapy.

N6-methyladenosine demethylase FTO enhances chemo-resistance in colorectal cancer through SIVA1-mediated apoptosis.

N6-methyladenosine (m6A) is the most pervasive RNA modification and is recognized as a novel epigenetic regulation in RNA metabolism. Although the m6A modification involves various physiological processes, its roles in drug resistance in colorectal cancer (CRC) still remain unknown. We analyzed the RNA expression profile of m6A/A (%) with MRM mass spectrometry in human 5-fluorouracil (5-FU)-resistant CRC tissues, and used the m6A RNA immunoprecipitation assay to validate the m6A-regulated target. Our results have shown that the m6A demethylase FTO was up-regulated in human primary and 5-FU-resistant CRC. Depletion of FTO decreased cell growth, colony formation and metastasis in 5-FU-resistant CRC cells in vitro and in vivo. Mechanistically, we identified SIVA1, a critical apoptotic gene, as a key downstream target of the FTO-mediated m6A demethylation. The m6A demethylation of SIVA1 at the CDS region induced its mRNA degradation via a YTHDF2-dependent mechanism. The SIVA1 levels were negatively correlated with the FTO levels in clinical CRC tissues. Notably, inhibition of FTO significantly reduced the tolerance of 5-FU in 5-FU-resistant CRC cells via the FTO-SIVA1 axis, whereas SIVA1-depletion could restore the m6A-dependent 5-FU sensitivity in CRC cells. In summary, our findings demonstrate a critical role of FTO as an m6A demethylase enhancing chemo-resistance in CRC cells, and suggest that FTO inhibition may restore the sensitivity of chemo-resistant CRC cells to 5-FU.

Caged Luciferase Inhibitor-Based Bioluminescence Switching Strategy Enables Efficient Detection of Serum APN Activity and the Identification of Its Roles in Metastasis of Non-Small Cell Lung Cancer.

Bioluminogenic probes emerged as powerful tools for imaging and analysis of various bioanalyses, but traditional approaches would be limited to the low sensitivity during determine the low activity of protease in clinical specimens. Herein, we proposed a caged luciferase inhibitor-based bioluminescence-switching strategy (CLIBS) by using a cleavable luciferase inhibitor to modulate the activity of luciferase reporter to amplify the detective signals, which led to the enhancement of detection sensitivity, and enabled the determination of circulating Aminopeptidase N (APN) activity in thousands of times diluted serum. By applying the CLIBS to serum samples in non-small cell lung cancer (NSCLC) patients from two clinical cohorts, we revealed that, for the first time, higher circulating APN activities but not its concentration, were associated with more NSCLC metastasis or higher metastasis stages by subsequent clinical analysis, and can serve as an independent factor for forecasting NSCLC patients' risk of metastasis.

Loss-of-Function Genetic Screening Identifies Aldolase A as an Essential Driver for Liver Cancer Cell Growth Under Hypoxia.

BACKGROUND AND AIMS: Hypoxia is a common feature of the tumor microenvironment (TME), which promotes tumor progression, metastasis, and therapeutic drug resistance through a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of HCC. APPROACH AND RESULTS: From a genome-wide CRISPR/CRISPR-associated 9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion and consequently inhibits tumor growth. Supplementation with lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by hypoxia-inducible factor-1α and fat mass and obesity-associated protein-mediated N6 -methyladenosine modification through transcriptional and posttranscriptional regulation, respectively. Analysis of The Cancer Genome Atlas shows that elevated levels of ALDOA are significantly correlated with poor prognosis of patients with HCC. In a screen of Food and Drug Administration-approved drugs based on structured hierarchical virtual platforms, we identified the sulfamonomethoxine derivative compound 5 (cpd-5) as a potential inhibitor to target ALDOA, evidenced by the antitumor activity of cpd-5 in preclinical patient-derived xenograft models of HCC. CONCLUSIONS: Our work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in the hypoxic TME is a promising therapeutic strategy for treating HCC.

RNA-binding protein RALY reprogrammes mitochondrial metabolism via mediating miRNA processing in colorectal cancer.

OBJECTIVE: Dysregulated cellular metabolism is a distinct hallmark of human colorectal cancer (CRC). However, metabolic programme rewiring during tumour progression has yet to be fully understood. DESIGN: We analysed altered gene signatures during colorectal tumour progression, and used a complex of molecular and metabolic assays to study the regulation of metabolism in CRC cell lines, human patient-derived xenograft mouse models and tumour organoid models. RESULTS: We identified a novel RNA-binding protein, RALY (also known as hnRNPCL2), that is highly associated with colorectal tumour aggressiveness. RALY acts as a key regulatory component in the Drosha complex, and promotes the post-transcriptional processing of a specific subset of miRNAs (miR-483, miR-676 and miR-877). These miRNAs systematically downregulate the expression of the metabolism-associated genes (ATP5I, ATP5G1, ATP5G3 and CYC1) and thereby reprogramme mitochondrial metabolism in the cancer cell. Analysis of The Cancer Genome Atlas (TCGA) reveals that increased levels of RALY are associated with poor prognosis in the patients with CRC expressing low levels of mitochondrion-associated genes. Mechanistically, induced processing of these miRNAs is facilitated by their N6-methyladenosine switch under reactive oxygen species (ROS) stress. Inhibition of the m6A methylation abolishes the RALY recognition of the terminal loop of the pri-miRNAs. Knockdown of RALY inhibits colorectal tumour growth and progression in vivo and in organoid models. CONCLUSIONS: Collectively, our results reveal a critical metabolism-centric role of RALY in tumour progression, which may lead to cancer therapeutics targeting RALY for treating CRC.

Raloxifene alleviates amyloid-ß-induced cytotoxicity in HT22 neuronal cells via inhibiting oligomeric and fibrillar species formation.

Raloxifene, a selective estrogen receptor modulator, displays benefits for Alzheimer's disease (AD) prevention in postmenopausal women as hormonal changes during menopause have the potential to influence AD pathogenesis, but the underlying mechanism of its neuroprotection is not entirely clear. In this study, the effects of raloxifene on amyloid-ß (Aß) amyloidogenesis were evaluated. The results demonstrated that raloxifene inhibits Aß42 aggregation and destabilizes preformed Aß42 fibrils through directly interacting with the N-terminus and middle domains of Aß42 peptides. Consequently, raloxifene not only reduces direct toxicity of Aß42 in HT22 neuronal cells, but also suppresses expressions of tumor necrosis factor-α and transforming growth factor-ß induced by Aß42 peptides, and then alleviates microglia-mediated indirect toxicity of Aß42 to HT22 neuronal cells. Our results suggested an alternative possible explanation for the neuroprotective activity of raloxifene in AD prevention.

(+)-Isobicyclogermacrenal and spathulenol from Aristolochia yunnanensis alleviate cardiac fibrosis by inhibiting transforming growth factor ß/small mother against decapentaplegic signaling pathway.

Cardiac fibrosis contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. Antifibrotic therapies are likely to be a crucial strategy in curbing many fibrosis-related cardiac diseases. In our previous study, an ethyl acetate extract of a traditional Chinese medicine Aristolochia yunnanensis Franch. was found to have a therapeutic effect on myocardial fibrosis in vitro and in vivo. However, the exact chemicals and their mechanisms responsible for the activity of the crude extract have not been illustrated yet. In the current study, 10 sesquiterpenoids (1-10) were isolated from the active extract, and their antifibrotic effects were systematically evaluated in transforming growth factor ß 1 (TGFß1)-stimulated cardiac fibroblasts and NIH3T3 fibrosis models. (+)-Isobicyclogermacrenal (1) and spathulenol (2) were identified as the main active components, being more potent than the well-known natural antifibrotic agent oxymatrine. Compounds 1 and 2 could inhibit the TGFß1-induced cardiac fibroblasts proliferation and suppress the expression of the fibrosis biomarkers fibronectin and α-smooth muscle actin via down-regulation of their mRNA levels. The mechanism study revealed that 1 and 2 could inhibit the phosphorylation of TGFß type I receptor, leading to the decrease of the phosphorylation levels of downstream Smad2/3, then consequently blocking the nuclear translocation of Smad2/3 in the TGFß/Smad signaling pathway. These findings suggest that 1 and 2 may serve as promising natural leads for the development of anticardiac fibrosis drugs.

Inhibitory effect of flavonoids on human glutaminyl cyclase.

Glutaminyl cyclase (QC) plays an important role in the pathogenesis of Alzheimer's disease (AD) and can be a potential target for the development of novel anti-AD agents. However, the study of QC inhibitors are still less. Here, phenol-4' (R1-), C5-OH (R2-) and C7-OH (R3-) modified apigenin derivatives were synthesized as a new class of human QC (hQC) inhibitors. The efficacy investigation of these compounds was performed by spectrophotometric assessment and the structure-activity relationship (SAR) was evaluated. Molecular docking was also carried out to analyze the binding mode of the synthesized flavonoid to the active site of hQC.

Nodal promotes the generation of M2-like macrophages and downregulates the expression of IL-12.

Nodal, a member of the TGF-ß superfamily, is an embryonic morphogen that is upregulated in different types of tumors. Nodal increases the tumorigenesis by inducing angiogenesis and promoting metastasis. Importantly, Nodal inhibition suppresses the growth and invasion of tumor. Since tumor-associated macrophages (TAMs) are the major infiltrating leukocytes in most cancers, we investigated whether Nodal is involved in the differentiation of TAMs. Our results revealed that Nodal inhibition in tumor microenvironment upregulated the production of IL-12 in macrophages and reversed TAMs to classically activated macrophage phenotype. In contrast, treatment with recombinant Nodal (rNodal) decreased the expression of IL-12 in murine macrophages. Furthermore, rNodal promoted macrophage polarization to an alternatively activated macrophage-like/TAM phenotype and modulated its function. These results suggest that Nodal may play an important role in macrophage polarization and downregulation of IL-12. The rescued antitumor function of TAMs via the inhibition of Nodal expression could be a new therapeutic strategy for cancer treatment.

Isolation and cytotoxicity evaluation of taxanes from the barks of Taxus wallichiana var. mairei.

Fifteen taxanes (1-15) including a new taxane glucoside, 7ß,9α,10ß-triacetoxy-13α-hydroxy-5α-O-(ß-d-glucopyranosyl)taxa-4(20),11-diene (1), were isolated from the barks of Taxus wallichiana var. mairei. Compounds 1-15 representing three sub-types of 6/8/6-taxane were evaluated in vitro for anti-proliferative activity against a panel of parental and drug-resistant cancer cells. Potent compounds were found while several exhibited selective cytotoxicity. Especially, 3, 8, and 10 showed selective inhibition to breast carcinoma cell line MCF-7, while 13 selectively inhibited taxol resistant human ovarian carcinoma cell line A2780/TAX (IC50=0.19µM), being more potent than the clinical drugs taxol (IC50=4.4µM) and docetaxol (IC50=0.42µM), and less cytotoxic to mouse embryonic fibroblast cell line NIH-3T3, a cell line close to normal cell line. The possible P-glycoprotein evasion mechanism of 13 against A2780/TAX and the preliminary structure-activity relationships (SARs) of this group of compounds were also discussed.

Formyl peptide enhances cancer immunotherapy by activating antitumoral neutrophils, and T cells.

Neutrophils are heterogeneous and plastic, with the ability to polarize from antitumour to protumour phenotype and modulate tumour microenvironment components. While some advances have been made, the neutrophil-targeting therapy remains underexplored. Activation of formyl peptide receptors (FPRs) by formylated peptides is needed for local control of infection through the recruitment of activated neutrophils while the potential contribution of antitumour activity remains underexplored. Here, we demonstrate that neutrophils can be harnessed to suppress tumour growth through the action of the formyl peptide (FP) on the formyl peptide receptor (FPR). Mechanistically, FP efficiently recruits neutrophils to produce reactive oxygen species production (ROS), resulting in the direct killing of tumours. Antitumour functions disappeared when neutrophils were depleted by anti-Ly6G antibodies. Interestingly, extensive T-cell activation was observed in mouse tumours treated with FP, showing the potential to alter the immune suppressed tumour microenvironment (TME) and further sensitize mice to anti-PD1 therapy. Transcriptomic and flow cytometry analyses revealed the mechanisms of FP-sensitized anti-PD1 therapy, mainly including stimulated neutrophils and an altered immune-suppressed tumour microenvironment. Collectively, these data establish FP as an effective combination partner for sensitizing anti-PD1 therapy by stimulating tumour-infiltrated neutrophils.

Nilotinib in combination with sunitinib renders MCL-1 for degradation and activates autophagy that overcomes sunitinib resistance in renal cell carcinoma.

PURPOSE: Sunitinib is a recommended drug for metastatic renal cell carcinoma (RCC). However, the therapeutic potential of sunitinib is impaired by toxicity and resistance. Therefore, we seek to explore a combinatorial strategy to improve sunitinib efficacy of low-toxicity dose for better clinical application. METHODS: We screen synergistic reagents of sunitinib from a compound library containing 1374 FDA-approved drugs by in vitro cell viability evaluation. The synergistically antiproliferative and proapoptotic effects were demonstrated on in vitro and in vivo models. The molecular mechanism was investigated by phosphoproteomics, co-immunoprecipitation, immunofluorescence and western-blot assays, etc. RESULTS: From the four-step screening, nilotinib stood out as a potential synergistic killer combined with sunitinib. Subsequent functional evaluation demonstrated that nilotinib and sunitinib synergistically inhibit RCC cell proliferation and promote apoptosis in vitro and in vivo. Mechanistically, nilotinib activates E3-ligase HUWE1 and in combination with sunitinib renders MCL-1 for degradation via proteasome pathway, resulting in the release of Beclin-1 from MCL-1/Beclin-1 complex. Subsequently, Beclin-1 induces complete autophagy flux to promote antitumor effect. CONCLUSION: Our findings revealed that a novel mechanism that nilotinib in combination with sunitinib overcomes sunitinib resistance in RCC. Therefore, this novel rational combination regimen provides a promising therapeutic avenue for metastatic RCC and rationale for evaluating this combination clinically.

A multidimensional platform of patient-derived tumors identifies drug susceptibilities for clinical lenvatinib resistance.

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.

Curcumin analogue T83 exhibits potent antitumor activity and induces radiosensitivity through inactivation of Jab1 in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy that is most common in East Asia, Africa, and Alaska. Radiotherapy is the main treatment option; unfortunately, disease response to concurrent radiotherapy and chemotherapy varies among patients with NPC, and in many cases, NPC becomes resistant to radiotherapy. Our previous studies indicated that Jab1/CSN5 was overexpressed and plays a role in the pathogenesis and radiotherapy resistance in NPC. Therefore, it is important to seek for innovative therapeutics targeting Jab1/CSN5 for NPC. In this study, we explored the antitumor effect of a curcumin analogue T83 in NPC, and found T83 exhibits antitumor activity and induces radiosensitivity through inactivation of Jab1 in NPC. METHODS: NPC cell viability and proliferation were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. Cell cycle distribution was detected with use of flow cytometry. Apoptosis was examined by using the Annexin V/propidium iodide staining assay and cleavage poly(ADP-ribose polymerase (PARP) and cleavage caspase-3 expression. Jab1 expression was examined by Western blotting. RESULTS: A growth inhibitory effect was observed with T83 treatment in a dose- and time-dependent manner. T83 significantly induced G2/M arrest and apoptosis in NPC. In addition, T83 inhibited Jab1 expression and sensitized NPC cells to radiotherapy. CONCLUSION: Our data indicate that T83 exhibits potent inhibitory activity in NPC cells and induces radiotherapy sensitivity. Thus, T83 has translational potential as a chemopreventive or therapeutic agent for NPC.

Synthesis, antioxidant, and antimicrobial evaluation of some 2-arylbenzimidazole derivatives.

A series of 2-arylbenzimidazole derivatives (3a-3p and 4a-4i) were synthesized and evaluated as potential antioxidant and antimicrobial agents. Their antioxidant properties were evaluated by various in vitro assays including hydroxyl radical (HO) scavenging, superoxide radical anion (O2(-)) scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, and ferric reducing antioxidant power. Results demonstrated that compounds with hydroxyl group at the 5-position of benzimidazole ring had a comparable or better antioxidant activity in comparison to standard antioxidant tert-butylhydroquinone (TBHQ). Markedly, compound 4h that showed the highest HO scavenging activity (EC50=46µM) in vitro had a significant reduction of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced intracellular oxidative stress and H2O2-induced cell death. In addition, these compounds showed moderate to good inhibitory activity against Staphylococcus aureus selectively at noncytotoxic concentrations.

Development of cyclopeptide inhibitors of cGAS targeting protein-DNA interaction and phase separation.

Cyclic GMP-AMP synthase (cGAS) is an essential sensor of aberrant cytosolic DNA for initiating innate immunity upon invading pathogens and cellular stress, which is considered as a potential drug target for autoimmune and autoinflammatory diseases. Here, we report the discovery of a class of cyclopeptide inhibitors of cGAS identified by an in vitro screening assay from a focused library of cyclic peptides. These cyclopeptides specifically bind to the DNA binding site of cGAS and block the binding of dsDNA with cGAS, subsequently inhibit dsDNA-induced liquid phase condensation and activation of cGAS. The specificity and potency of one optimal lead XQ2B were characterized in cellular assays. Concordantly, XQ2B inhibited herpes simplex virus-1 (HSV-1)-induced antiviral immune responses and enhanced HSV-1 infection in vitro and in vivo. Furthermore, XQ2B significantly suppressed the elevated levels of type I interferon and proinflammatory cytokines in primary macrophages from Trex1-/- mice and systemic inflammation in Trex1-/- mice. XQ2B represents the specific cGAS inhibitor targeting protein-DNA interaction and phase separation and serves as a scaffold for the development of therapies in the treatment of cGAS-dependent inflammatory diseases.

Disulfiram enhances cisplatin cytotoxicity by forming a novel platinum chelate Pt(DDTC)3.

Despite the use of targeted therapy in non-small cell lung cancer (NSCLC) patients, cisplatin (DDP)-based chemotherapy is still the main option. However, DDP resistance is the major factor contributing to the failure of chemotherapy. In this study, we tried to screen DDP sensitizers from an FDA-approved drug library containing 1374 small-molecule drugs to overcome DDP resistance in NSCLC. As a result, disulfiram (DSF) was identified as a DDP sensitizer: DSF and DDP had synergistic anti-NSCLC effects, which are mainly reflected in inhibiting tumor cell proliferation, plate colony formation and 3D spheroidogenesis and inducing apoptosis in vitro, as well as the growth of NSCLC xenografts in mice. Although DSF has recently been reported to promote the antitumor effect of DDP by inhibiting ALDH activity or modulating some important factors or pathways, unexpectedly, we found that DSF reacted with DDP to form a new platinum chelate, Pt(DDTC)3+, which might be one of the important mechanisms for their synergistic effect. Moreover, Pt(DDTC)3+ has a stronger anti-NSCLC effect than DDP, and its antitumor activity is broad-spectrum. These findings reveal a novel mechanism underlying the synergistic antitumor effect of DDP and DSF, and provide a drug candidate or a lead compound for the development of a new antitumor drug.

Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1-BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy.

Radiotherapy is widely used in the management of advanced colorectal cancer (CRC). However, the clinical efficacy is limited by the safe irradiated dose. Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation. The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination (HR) DSB repair, and its functions may be affected by HERC2 or BAP1. Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes; however, the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn't been defined. Through activity-based profiling, we identified PT33 as an active entity for HR repair suppression. Subsequently, we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen. Mechanistically, pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction, interrupting HR repair. Consequently, PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo. Overall, these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.

Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction.

BACKGROUND: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction. RESULTS: We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A), lysine (K) or aspartic acid (D), respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously. CONCLUSIONS: We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.