Comparison of genomic alterations in Epstein-Barr virus-positive and Epstein-Barr virus-negative diffuse large B-cell lymphoma.

BACKGROUND: Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV-posDLBCL) is an aggressive B-cell lymphoma that often presents similar morphological and immune phenotype features to that of EBV-negative DLBCL (EBV-negDLBCL). AIMS AND METHODS: To better understand their difference in genomic landscape, we performed whole-exome sequencing (WES) of EBV-posDLBCL and EBV-negDLBCL. RESULTS: This analysis revealed a new mutational signature 17 (unknown) and signature 29 (smoking) in EBV-posDLBCL as well as a specific mutational signature 24 (associated with aflatoxin) in EBV-negDLBCL. Compared with EBV-negDLBCL, more somatic copy number alterations (CNAs) and deletions were detected in EBV-posDLBCL (p = 0.01). The most frequent CNAs specifically detected in EBV-posDLBCL were gains at 9p24.1 (PDL1 and JAK2), 8q22.2-q24.23 (DEPTOR and MYC), and 7q31.31-q32.2 (MET), which were validated in additional EBV-posDLBCL cases. Overall, 53.7% (22/41) and 62.9% (22/35) of the cases expressed PD-L1 and c-MET, respectively, in neoplastic cells, whereas only 15.4% (4/26) expressed c-MYC. Neoplastic c-MET expression was positively correlated with PD-L1 (p < 0.001) and MYC expression (p = 0.016). However, EBV-posDLBCL cases did not show any differences in overall survival between PD-L1-, c-MET-, or c-MYC-positive and -negative cases or between age-related groups. Analysis of the association between somatic mutation load and EBV status showed no difference in the distribution of tumor mutant burden between the two lymphomas (p = 0.41). Recurrent mutations in EBV-posDLBCL implicated several genes, including DCAF8L1, KLF2, and NOL9, while in EBV-negDLBCL, ANK2, BPTF, and CNIH3 were more frequently mutated. Additionally, PIM1 is the most altered gene in all the WES-detected cases. CONCLUSIONS: Our results confirm that genomic alteration differs significantly between EBV-posDLBCL and EBV-negDLBCL, and reveal new genetic alterations in EBV-posDLBCL. The positive correlation of c-MET and PD-L1/c-Myc expression may be involved in the pathogenesis of EBV-posDLBCL, which is should be explored prospectively in trials involving MET-directed therapies.

Inhibition of proteasomal deubiquitinases USP14 and UCHL5 overcomes tyrosine kinase inhibitor resistance in chronic myeloid leukaemia.

BACKGROUND: Chronic myeloid leukaemia (CML) is a haematological cancer featured by the presence of BCR-ABL fusion protein with abnormal tyrosine kinase activation. Classical tyrosine kinase inhibitor (TKI)-based therapies are available to patients with CML. However, acquired resistance to TKI has been a challenging obstacle, especially stubborn T315I mutation is the most common cause. Therefore, it is especially urgent to find more effective targets to overcome TKI resistance induced by BCR-ABLT315I . Proteasomal deubiquitinases (USP14 and UCHL5) have fundamental roles in the ubiquitin-proteasome system and possess multiple functions during cancer progression. METHODS: The human peripheral blood mononuclear cells were collected to measure the mRNA expression of USP14 and UCHL5, as well as to detect the toxicity effect of b-AP15. We explored the effect of b-AP15 on the activity of proteasomal deubiquitinases. We detected the effects of b-AP15 on BCR-ABLWT and BCR-ABLT315I CML cells in vitro and in the subcutaneous tumour model. We knocked down USP14 and/or UCHL5 by shRNA to explore whether these proteasomal deubiquitinases are required for cell proliferation of CML. RESULTS: In this study, we found that increased expression of the proteasomal deubiquitinase USP14 and UCHL5 in primary cancer cells from CML patients compared to healthy donors. b-AP15, an inhibitor of USP14 and UCHL5, exhibited potent tumour-killing activity in BCR-ABLWT and BCR-ABLT315I CML cell lines, as well as in CML xenografts and primary CML cells. Mechanically, pharmacological or genetic inhibition of USP14 and UCHL5 induced cell apoptosis and decreased the protein level of BCR-ABL in CML cells expressing BCR-ABLWT and BCR-ABLT315I . Moreover, b-AP15 synergistically enhanced the cytotoxic effect caused by TKI imatinib in BCR-ABLWT and BCR-ABLT315I CML cells. CONCLUSION: Collectively, our results demonstrate targeting USP14 and UCHL5 as a potential strategy for combating TKI resistance in CML.

The BCMA-Targeted Fourth-Generation CAR-T Cells Secreting IL-7 and CCL19 for Therapy of Refractory/Recurrent Multiple Myeloma.

Chimeric antigen receptor (CAR) technology has revolutionized cancer treatment, particularly in malignant hematological tumors. Currently, the BCMA-targeted second-generation CAR-T cells have showed impressive efficacy in the treatment of refractory/relapsed multiple myeloma (R/R MM), but up to 50% relapse remains to be addressed urgently. Here we constructed the BCMA-targeted fourth-generation CAR-T cells expressing IL-7 and CCL19 (i.e., BCMA-7 × 19 CAR-T cells), and demonstrated that BCMA-7 × 19 CAR-T cells exhibited superior expansion, differentiation, migration and cytotoxicity. Furthermore, we have been carrying out the first-in-human clinical trial for therapy of R/R MM by use of BCMA-7 × 19 CAR-T cells (ClinicalTrials.gov Identifier: NCT03778346), which preliminarily showed promising safety and efficacy in first two enrolled patients. The two patients achieved a CR and VGPR with Grade 1 cytokine release syndrome only 1 month after one dose of CAR-T cell infusion, and the responses lasted more than 12-month. Taken together, BCMA-7 × 19 CAR-T cells were safe and effective against refractory/relapsed multiple myeloma and thus warranted further clinical study.

Pseudolaric acid B induces mitotic arrest and apoptosis in both imatinib-sensitive and -resistant chronic myeloid leukaemia cells.

The selective BCR-ABL tyrosine kinase inhibitor imatinib is one of the first-line therapies in the management of chronic myeloid leukaemia (CML). However, acquired resistance to this inhibitor, which is especially conferred by the T315I point mutation in BCR-ABL, impedes the efficacy of imatinib therapy. Therefore, the discovery and development of novel agents to overcome imatinib resistance is urgently needed. Pseudolaric acid B (PAB), a small molecule isolated from the traditional Chinese medicine Cortex pseudolaricis, has been reported to be a potential candidate for immune disorders and cancer treatment. However, its effects on CML and the involved molecular mechanism have not been reported. In the current study, by performing both in vitro and in vivo experiments in CML cells, we showed that PAB blocked the cell cycle at G2/M phase and subsequently activated the caspase pathway, cleaved the BCR-ABL protein and inhibited the BCR-ABL downstream pathways, ultimately leading to cell proliferation inhibition, cytotoxicity and apoptosis. These events were observed in both imatinib-sensitive and imatinib-insensitive CML cell lines. Moreover, PAB decreased the viability of primary blood mononuclear cells from CML patients and induced apoptosis in these cells. Our findings suggest that PAB could be used as a novel agent to sensitize imatinib-resistant CML.

Proteasomal cysteine deubiquitinase inhibitor b-AP15 suppresses migration and induces apoptosis in diffuse large B cell lymphoma.

BACKGROUND: The first line therapy for patients with diffuse large B cell (DLBCL) is R-CHOP. About half of DLBCL patients are either refractory to, or will relapse, after the treatment. Therefore, identifying novel drug targets and effective therapeutic agents is urgently needed for improving DLBCL patient survival. b-AP15, a selective small molecule inhibitor of proteasomal USP14 and UCHL5 deubiquitinases (DUBs), has shown selectivity and efficacy in several other types of cancer cells. This is the first study to report the effect of b-AP15 in DLBCL. METHODS: Cell lines of two DLBCL subtypes, Germinal Center B Cell/ GCB (SU-DHL-4, OCI-LY-1, OCI-LY-19) and Activated B Cell/ABC (SU-DHL-2), were used in the current study. Cell viability was measured by MTS assay, proliferation by trypan blue exclusion staining assay, cellular apoptosis by Annexin V-FITC/PI staining and mitochondrial outer membrane permeability assays, the activities of 20S proteasome peptidases by cleavage of specific fluorogenic substrates, and cell migration was detected by transwell assay in these GCB- and ABC-DLBCL cell lines. Mouse xenograft models of SU-DHL-4 and SU-DHL-2 cells were used to determine in vivo effects of b-AP15 in DLBCL tumors. RESULTS: b-AP15 inhibited proteasome DUB activities and activated cell death pathway, as evident by caspase activation and mitochondria apoptosis in GCB- and ABC- DLBCL cell lines. b-AP15 treatment suppressed migration of GCB- and ABC-DLBCL cells via inhibiting Wnt/ß-catenin and TGFß/Smad pathways. Additionally, b-AP15 significantly inhibited the growth of GCB- and ABC DLBCL in xenograft models. CONCLUSIONS: These results indicate that b-AP15 inhibits cell migration and induces apoptosis in GCB- and ABC-DLBCL cells, and suggest that inhibition of 19S proteasomal DUB should be a novel strategy for DLBCL treatment.

Dual Cross-Link Networks To Preserve Physical Interactions Induced by Soaking Methods: Developing a Strong and Biocompatible Protein-Based Hydrogel.

The engineering of natural protein-based hydrogels with outstanding mechanics and functionalities is an eternal pursuit in biomaterials science. Soaking methods have recently been used to simply and efficiently tune the macroscopic mechanical properties of hydrogels consisting of natural polymers (proteins or polysaccharides). However, the high concentration of salts or organic solvent within the soaked hydrogels is harmful to cells. Once the salts were eliminated, however, these hydrogels experience mechanics attenuation and swelling, limiting their applications as biomaterials. Here we utilize dual cross-link networks to address these problems by preserving the physical interactions formed during the soaking treatment. As an example, a dual cross-link gelatin-chitosan hydrogel is prepared by a click reaction and hydrophobic interactions, which achieve an unusual combination of abundant water content (>87%), strong mechanics (ultimate strength ∼1.8 MPa), antiswelling properties (swelling ratio of 11.25% in PBS for 7 days), thermal stability, and biocompatibility. We expect that this strategy can be generalized for the development of tough, biocompatible, and environmentally stable protein-based hydrogels.

Fabrication of gelatin-TiO2 nanocomposite film and its structural, antibacterial and physical properties.

Biodegradable fish skin gelatin-titanium dioxide (TiO2) nanocomposite films were fabricated and characterized as a function of incorporating amount of TiO2 nanoparticles (gelatin/TiO2 ratio of 30:1, 20:1 and 10:1). A uniform distribution of TiO2 nanoparticles into gelatin matrix was observed using atomic force microscopy (AFM) micrographs. The data of intrinsic fluorescence spectra, Fourier transform infrared spectra (FTIR) and X-ray diffraction confirmed the interaction between protein and nanoparticles through hydrogen bonding. The TiO2-incorporated gelatin nanocomposite films exhibited more effective antibacterial activity for Escherichia coli after irradiating 120 min by UV light (365 nm), which were 54.38% for E. coli and 44.89% for Staphylococcus aureus, respectively. The analysis of physical properties revealed that addition of TiO2 nanoparticles to gelatin films significantly increased the tensile strength and elongation at break, while decreased its water vapor permeability. The light barrier measurements indicated that these films were highly transparent, and they had excellent barrier properties against UVC light at the same time. The results demonstrated the feasibility of incorporating nanoparticles to improve the properties of gelatin films, and it is of significance in utilizing the gelatin and titanium dioxide to produce biodegradable nanocomposite film as packaging material in food industry.

[The Transmission of KPC-2 Gene in Klebsiella Species].

OBJECTIVES: To explore the mechanisms of imipenem resistance inKlebsiella spp. and the transmission of Klebsiella pneumoniae Carbapenemase-2(KPC-2) gene in Klebsiella species. METHODS: The imipenem resistant Klebsiella pneumoniae and Klebsiella oxytoca were isolated in the West China Hospital of Sichuan University in 2009/2010 and 2012/2013. Their minimal inhibitory concentration (MIC) was determined by agar dilution method. CARB ChromID plate and improved Hodge test were undertaken to detect carbapenemases resistant phenotype. PCR method was used for detecting KPC-2 gene. Plasmid transmission was detected by plasmid conjugation tests. The homology of the plasmids and the strains was analyzed using random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus PCR(ERIC-PCR) methods. RESULTS: Three strains of Klebsiella oxytoca collected in 2009/2010 and 7 Klebsiella pneumoniae collected in 2012/2013 developed carbapenemases resistance, all of which carried KPC-2 gene. The 3 KPC-2 positive plasmids isolated from Klebsiella oxytoca transited to recipient organisms and showed homology with the 7 KPC-2 gene positive plasmids isolated from Klebsiella pneumoniae. The ERIC-PCR showed homology of the 7 KPC positive Klebsiella pneumoniae. CONCLUSIONS: Carbapenemases inKlebsiella spp with expressed KPC-2 gene contribute to the development of resistance in this hospital. The transmission of KPC-2 plasmid in Klebsiella oxytoca may cause imipenem resistance in Klebsiella pneumonia. The horizontal transmission may be the main mechanism in the spread of imipenem resistance inKlebsiella spp.