A new immunotherapy strategy targeted CD30 in peripheral T-cell lymphomas: CAR-modified T-cell therapy based on CD30 mAb.

Chimeric antigen receptor T-cell immunotherapy (CAR-T) has shown remarkable efficacy in treating tumors of lymphopoietic origin. Herein, we demonstrate an effective CAR-T cell treatment for recurrent and malignant CD30-positive peripheral T-cell lymphomas (PTCL) has been demonstrated. The extracellular fragment gene sequences of CD30 were obtained from tumor tissues of PTCL patients and cloned into a plasmid vector to express the CD30 antigen. The CD30 targeting single-chain antibody fragment (scFv) was obtained from CD30-positive monoclonal hybridoma cells, which were obtained from CD30 antigen immunized mice. After a second-generation of CAR lentiviral construction, CD30 CAR T cells were produced and used to determine the cytotoxicity of this construct toward Karpas 299 cells. The results of CD30 CAR T-mediated cell lysis show that 9C11-2 CAR T cells could significantly promote the lysis of CD30-positive Karpas 299 cells in both LDH and real-time cell electronic sensing (RTCA) assays. In vivo data show that 9C11-2 CAR T cells effectively suppress the tumor growth in a Karpas 299 cell xenograft NCG mouse model. The CD30 CAR T cells exhibited an efficient cytotoxic effect after being co-cultured with the target cells and they also exhibited a significant tumor-inhibiting ability after being intravenously injected into PTCL xenograft tumors; these observations suggest that the new CD30 CAR-T cell may be a promising therapeutic candidate for cancer therapy.

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis.

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.

Bendamustine treatment of Chinese patients with relapsed indolent non-Hodgkin lymphoma: a multicenter, open-label, single-arm, phase 3 study.

BACKGROUND: Bendamustine was approved in China on May 26th, 2019 by the National Medical Product Administration for the treatment of indolent B-cell non-Hodgkin lymphoma (NHL). The current study was the registration trial and the first reported evaluation of the efficacy, safety, and pharmacokinetics of bendamustine in Chinese adult patients with indolent B-cell NHL following relapse after chemotherapy and rituximab treatment. METHODS: This was a prospective, multicenter, open-label, single-arm, phase 3 study (NCT01596621; C18083/3076) with a 2-year follow-up period. Eligible patients received bendamustine hydrochloride 120 mg/m2 infused intravenously on days 1 and 2 of each 21-day treatment cycle for at least six planned cycles (and up to eight cycles). The primary endpoint was the overall response rate (ORR); and secondary endpoints were duration of response (DoR), progression-free survival (PFS), safety, and pharmacokinetics. Patients were classified according to their best overall response after initiation of therapy. Proportions of patients in each response category (complete response [CR], partial response [PR], stable disease, or progressive disease) were summarized along with a two-sided binomial exact 95% confidence intervals (CIs) for the ORR. RESULTS: A total of 102 patients were enrolled from 20 centers between August 6th, 2012, and June 18th, 2015. At the time of the primary analysis, the ORR was 73% (95% CI: 63%-81%) per Independent Review Committee (IRC) including 19% CR and 54% PR. With the follow-up period, the median DoR was 16.2 months by IRC and 13.4 months by investigator assessment; the median PFS was 18.6 months and 15.3 months, respectively. The most common non-hematologic adverse events (AEs) were gastrointestinal toxicity, pyrexia, and rash. Grade 3/4 neutropenia was reported in 76% of patients. Serious AEs were reported in 29 patients and five patients died during the study. Pharmacokinetic analysis indicated that the characteristics of bendamustine and its metabolites M3 and M4 were generally consistent with those reported for other ethnicities. CONCLUSION: Bendamustine is an active and effective therapy in Chinese patients with relapsed, indolent B-cell NHL, with a comparable risk/benefit relationship to that reported in North American patients. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, No. NCT01596621; https://clinicaltrials.gov/ct2/show/NCT01596621.

Roles of Mso1 and the SM protein Sec1 in efficient vesicle fusion during fission yeast cytokinesis.

Membrane trafficking during cytokinesis is essential for the delivery of membrane lipids and cargoes to the division site. However, the molecular mechanisms are still incompletely understood. In this study, we demonstrate the importance of uncharacterized fission yeast proteins Mso1 and Sec1 in membrane trafficking during cytokinesis. Fission yeast Mso1 shares homology with budding yeast Mso1 and human Mint1, proteins that interact with Sec1/Munc18 family proteins during vesicle fusion. Sec1/Munc18 proteins and their interactors are important regulators of SNARE complex formation during vesicle fusion. The roles of these proteins in vesicle trafficking during cytokinesis have been barely studied. Here, we show that fission yeast Mso1 is also a Sec1-binding protein and Mso1 and Sec1 localize to the division site interdependently during cytokinesis. The loss of Sec1 localization in mso1Δ cells results in a decrease in vesicle fusion and cytokinesis defects such as slow ring constriction, defective ring disassembly, and delayed plasma membrane closure. We also find that Mso1 and Sec1 may have functions independent of the exocyst tethering complex on the plasma membrane at the division site. Together, Mso1 and Sec1 play essential roles in regulating vesicle fusion and cargo delivery at the division site during cytokinesis.

An Assay to Study Intra-Chromosomal Deletions in Yeast.

An accurate DNA damage response pathway is critical for the repair of DNA double-strand breaks. Repair may occur by homologous recombination, of which many different sub-pathways have been identified. Some recombination pathways are conservative, meaning that the chromosome sequences are preserved, and others are non-conservative, leading to some alteration of the DNA sequence. We describe an in vivo genetic assay to study non-conservative intra-chromosomal deletions at regions of non-tandem direct repeats in Schizosaccharomyces pombe. This assay can be used to study both spontaneous breaks arising during DNA replication and induced double-strand breaks created with the S. cerevisiae homothallic endonuclease (HO). The preliminary genetic validation of this assay shows that spontaneous breaks require rad52+ but not rad51+, while induced breaks require both genes, in agreement with previous studies. This assay will be useful in the field of DNA damage repair for studying mechanisms of intra-chromosomal deletions.

Distinct Roles of Myosin-II Isoforms in Cytokinesis under Normal and Stressed Conditions.

To address the question of why more than one myosin-II isoform is expressed in a single cell to drive cytokinesis, we analyzed the roles of the myosin-II isoforms, Myo2 and Myp2, of the fission yeast Schizosaccharomyces pombe, in cytokinesis under normal and stressed conditions. We found that Myp2 controls the disassembly, stability, and constriction initiation of the Myo2 ring in response to high-salt stress. A C-terminal coiled-coil domain of Myp2 is required for its immobility and contractility during cytokinesis, and when fused to the tail of the dynamic Myo2, renders the chimera the low-turnover property. We also found, by following distinct processes in real time at the single-cell level, that Myo2 and Myp2 are differentially required but collectively essential for guiding extracellular matrix remodeling during cytokinesis. These results suggest that the dynamic and immobile myosin-II isoforms are evolved to carry out cytokinesis with robustness under different growth conditions.

The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis.

F-BAR proteins bind the plasma membrane (PM) to scaffold and organize the actin cytoskeleton. To understand how F-BAR proteins achieve their PM association, we studied the localization of a Schizosaccharomyces pombe F-BAR protein Rga7, which requires the coiled-coil protein Rng10 for targeting to the division site during cytokinesis. We find that the Rga7 F-BAR domain directly binds a motif in Rng10 simultaneously with the PM, and that an adjacent Rng10 motif independently binds the PM. Together, these multivalent interactions significantly enhance Rga7 F-BAR avidity for membranes at physiological protein concentrations, ensuring the division site localization of Rga7. Moreover, the requirement for the F-BAR domain in Rga7 localization and function in cytokinesis is bypassed by tethering an Rga7 construct lacking its F-BAR to Rng10, indicating that at least some F-BAR domains are necessary but not sufficient for PM targeting and are stably localized to specific cortical positions through adaptor proteins.

Molecular mechanisms of contractile-ring constriction and membrane trafficking in cytokinesis.

In this review, we discuss the molecular mechanisms of cytokinesis from plants to humans, with a focus on contribution of membrane trafficking to cytokinesis. Selection of the division site in fungi, metazoans, and plants is reviewed, as well as the assembly and constriction of a contractile ring in fungi and metazoans. We also provide an introduction to exocytosis and endocytosis, and discuss how they contribute to successful cytokinesis in eukaryotic cells. The conservation in the coordination of membrane deposition and cytoskeleton during cytokinesis in fungi, metazoans, and plants is highlighted.

Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein.

The ubiquitin-like protein Atg8, in its lipidated form, plays central roles in autophagy. Yet, remarkably, Atg8 also carries out lipidation-independent functions in non-autophagic processes. How Atg8 performs its moonlighting roles is unclear. Here we report that in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, the lipidation-independent roles of Atg8 in maintaining normal morphology and functions of the vacuole require its interaction with a vacuole membrane protein Hfl1 (homolog of human TMEM184 proteins). Crystal structures revealed that the Atg8-Hfl1 interaction is not mediated by the typical Atg8-family-interacting motif (AIM) that forms an intermolecular ß-sheet with Atg8. Instead, the Atg8-binding regions in Hfl1 proteins adopt a helical conformation, thus representing a new type of AIMs (termed helical AIMs here). These results deepen our understanding of both the functional versatility of Atg8 and the mechanistic diversity of Atg8 binding.

Three-year Follow-up on the Safety and Effectiveness of Rituximab Plus Chemotherapy as First-Line Treatment of Diffuse Large B-Cell Lymphoma and Follicular Lymphoma in Real-World Clinical Settings in China: A Prospective, Multicenter, Noninterventional Study.

BACKGROUND: Prospective real-life data on the safety and effectiveness of rituximab in Chinese patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL) are limited. This real-world study aimed to evaluate long-term safety and effectiveness outcomes of rituximab plus chemotherapy (R-chemo) as first-line treatment in Chinese patients with DLBCL or FL. Hepatitis B virus (HBV) reactivation management was also investigated. METHODS: A prospective, multicenter, single-arm, noninterventional study of previously untreated CD20-positive DLBCL or FL patients receiving first-line R-chemo treatment at 24 centers in China was conducted between January 17, 2011 and October 31, 2016. Enrolled patients underwent safety and effectiveness assessments after the last rituximab dose and were followed up for 3 years. Effectiveness endpoints included progression-free survival (PFS) and overall survival (OS). Safety endpoints were adverse events (AEs), serious AEs, drug-related AEs, and AEs of special interest. We also reported data on the incidence of HBV reactivation. RESULTS: In total, 283 previously untreated CD20-positive DLBCL and 31 FL patients from 24 centers were enrolled. Three-year PFS was 59% (95% confidence interval [CI]: 50-67%) for DLBCL patients and 46% (95% CI: 20-69%) for FL patients. For DLBCL patients, multivariate analyses showed that PFS was not associated with international prognostic index, tumor maximum diameter, HBV infection status, or number of rituximab treatment cycles, and OS was only associated with age >60 years (P < 0.05). R-chemo was well tolerated. The incidence of HBV reactivation in hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative/hepatitis B core antibody-positive patients was 13% (3/24) and 4% (3/69), respectively. CONCLUSIONS: R-chemo is effective and safe in real-world clinical practice as first-line treatment for DLBCL and FL in China, and that HBV reactivation during R-chemo is manageable with preventive measures and treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01340443; https://clinicaltrials.gov/ct2/show/NCT01340443.

Roles of the fission yeast UNC-13/Munc13 protein Ync13 in late stages of cytokinesis.

Cytokinesis is a complicated yet conserved step of the cell-division cycle that requires the coordination of multiple proteins and cellular processes. Here we describe a previously uncharacterized protein, Ync13, and its roles during fission yeast cytokinesis. Ync13 is a member of the UNC-13/Munc13 protein family, whose animal homologues are essential priming factors for soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly during exocytosis in various cell types, but no roles in cytokinesis have been reported. We find that Ync13 binds to lipids in vitro and dynamically localizes to the plasma membrane at cell tips during interphase and at the division site during cytokinesis. Deletion of Ync13 leads to defective septation and exocytosis, uneven distribution of cell-wall enzymes and components of cell-wall integrity pathway along the division site and massive cell lysis during cell separation. Interestingly, loss of Ync13 compromises endocytic site selection at the division plane. Collectively, we find that Ync13 has a novel function as an UNC-13/Munc13 protein in coordinating exocytosis, endocytosis, and cell-wall integrity during fission yeast cytokinesis.

Loss of ARID1A expression is associated with poor prognosis in patients with gastric cancer.

Deletion of the frequently mutated AT-rich interacting domain-containing protein 1A (ARID1A), an SWI/SNF subunit, is associated with poor prognosis in various tumors. This study observed and analyzed ARID1A expression and its correlation with prognosis in gastric carcinoma. Postoperative sections of 98 patients with primary gastric cancer and 40 patients with gastric benign lesions were examined by immunohistochemistry. ARID1A deficiency was observed in 19.39% of gastric cancer tissues, 4.08% of matched paracancerous tissues, and 2.5% of normal gastric mucosa tissues. ARID1A expression was significantly down-regulated in gastric cancer tissues compared with paracancerous tissues (P = .001) and normal gastric mucosa tissues (P = .011). ARID1A deletion significantly correlated with tumor size (P = .022), lymph node metastasis (P = .030), and tumor differentiation (P = .009). In the 90 gastric cancer tissues with tumor stages II and III, the clinical outcome of the ARID1A-negative patients was significantly poorer than that of the ARID1A-positive patients (P = .005). Univariate analysis revealed that tumor invasion depth (P = .025), stage (P = .032), poor differentiation (P = .046), lymph node metastasis (P = .038), and ARID1A expression (P = .023) were significantly related to the overall survival of gastric cancer patients. Multivariate analysis demonstrated that tumor invasion depth (P = .029) and ARID1A expression (P = .031) were independent factors that indicate poor prognosis. In conclusion, the loss of ARID1A expression in gastric cancer patients significantly correlated with poor survival.

Sbg1 Is a Novel Regulator for the Localization of the ß-Glucan Synthase Bgs1 in Fission Yeast.

Glucan synthases synthesize glucans, complex polysaccharides that are the major components in fungal cell walls and division septa. Studying regulation of glucan synthases is important as they are essential for fungal cell survival and thus popular targets for anti-fungal drugs. Linear 1,3-ß-glucan is the main component of primary septum and is synthesized by the conserved ß-glucan synthase Bgs1 in fission yeast cytokinesis. It is known that Rho1 GTPase regulates Bgs1 catalytic activity and the F-BAR protein Cdc15 plays a role in Bgs1 delivery to the plasma membrane. Here we characterize a novel protein Sbg1 that is present in a complex with Bgs1 and regulates its protein levels and localization. Sbg1 is essential for contractile-ring constriction and septum formation during cytokinesis. Sbg1 and Bgs1 physically interact and are interdependent for localization to the plasma membrane. Bgs1 is less stable and/or mis-targeted to vacuoles in sbg1 mutants. Moreover, Sbg1 plays an earlier and more important role in Bgs1 trafficking and localization than Cdc15. Together, our data reveal a new mode of regulation for the essential ß-glucan synthase Bgs1 by the novel protein Sbg1.

Cytokinesis: Going Super-Resolution in Live Cells.

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying molecular organization, but mostly in fixed cells. New work using high-speed fluorescence photoactivation localization microscopy now reveals the organization of cytokinesis nodes and contractile rings in live fission yeast cells.

Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis.

Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast.

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis.

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.

Real-Time Visualization and Quantification of Contractile Ring Proteins in Single Living Cells.

Single-cell microscopy provides a powerful tool to visualize cellular and subcellular processes in wild-type and mutant cells by observing fluorescently tagged proteins. Here, we describe three simple methods to visualize fission yeast cells: gelatin slides, coverslip-bottom dishes, and tetrad fluorescence microscopy. These imaging methods and data analysis using free software make it possible to quantify protein localization, dynamics, and concentration with high spatial and temporal resolution. In fission yeast, the actomyosin contractile ring is essential for cytokinesis. We use the visualization and quantification of contractile ring proteins as an example to demonstrate how to use these methods.

Mechanistic insights into the anchorage of the contractile ring by anillin and Mid1.

Anillins and Mid1 are scaffold proteins that play key roles in anchorage of the contractile ring at the cell equator during cytokinesis in animals and fungi, respectively. Here, we report crystal structures and functional analysis of human anillin and S. pombe Mid1. The combined data show anillin contains a cryptic C2 domain and a Rho-binding domain. Together with the tethering PH domain, three membrane-associating elements synergistically bind to RhoA and phospholipids to anchor anillin at the cleavage furrow. Surprisingly, Mid1 also binds to the membrane through a cryptic C2 domain. Dimerization of Mid1 leads to high affinity and preference for PI(4,5)P2, which stably anchors Mid1 at the division plane, bypassing the requirement for Rho GTPase. These findings uncover the unexpected general machinery and the divergent regulatory logics for the anchorage of the contractile ring through the anillin/Mid1 family proteins from yeast to humans.

MicroRNA-377 predicts poor clinical outcome of gastric cancer and induces tumorigenesis by targeting multiple tumor-suppressor genes.

Gastric cancer (GC) is a major cause of cancer mortality worldwide. MicroRNAs are evolutionally conserved small non-coding RNAs that are critical for the regulation of gene expression. The aberrant expression of microRNA (miRNA) is involved in tumorigenesis and prognosis. In the present study, the clinical significance of miR-377 was assessed using RT-qPCR and MTT assay. The results showed that the expression of miR-377 was upregulated in GC compared with normal gastric tissues, and its expression level was increased in GC cell lines compared with normal gastric cells. In addition, there was a significant association between miR-377 expression and clinicopathological characteristics, in particular distant metastasis, TNM stage and early recurrence. GC patients with a higher miR-377 expression showed significantly poorer overall survival (OR) and shorter time to recurrence than those with a lower miR-377 expression. The Cox regression analysis identified miR-377 overexpression as an independent prognostic factor for GC. Overexpression of miR-377 in MKN-45 GC cells significantly promoted cell proliferation, whereas the suppression of miR-377 inhibited these effects. Furthermore, miR-377 downregulated p53, PTEN and TIMP1 expression by directly targeting the 3'-untranslated region of these target genes. Collectively, miR-377 potentially served as a new molecular predictive biomarker of GC tumorigenesis and prognosis, which may be useful in targeted therapy and the prognosis of GC patients.

SOAX: a software for quantification of 3D biopolymer networks.

Filamentous biopolymer networks in cells and tissues are routinely imaged by confocal microscopy. Image analysis methods enable quantitative study of the properties of these curvilinear networks. However, software tools to quantify the geometry and topology of these often dense 3D networks and to localize network junctions are scarce. To fill this gap, we developed a new software tool called "SOAX", which can accurately extract the centerlines of 3D biopolymer networks and identify network junctions using Stretching Open Active Contours (SOACs). It provides an open-source, user-friendly platform for network centerline extraction, 2D/3D visualization, manual editing and quantitative analysis. We propose a method to quantify the performance of SOAX, which helps determine the optimal extraction parameter values. We quantify several different types of biopolymer networks to demonstrate SOAX's potential to help answer key questions in cell biology and biophysics from a quantitative viewpoint.