Musashi-2 (MSI2) promotes neuroblastoma tumorigenesis through targeting MYC-mediated glucose-6-phosphate dehydrogenase (G6PD) transcriptional activation.

Neuroblastoma (NB) is the deadliest pediatric solid tumor due to its rapid proliferation. Aberrant expression of MYCN is deemed as the most remarkable feature for the predictive hallmark of NB progression and recurrence. However, the phenomenon that only detection of MYCN in the nearly 20% of NB patients hints that there should be other vital oncogenes in the progression of NB. Here, we firstly show that MSI2 mRNA is augmented by analyzing public GEO datasets in the malignant stage according to International Neuroblastoma Staging System (INSS) stages. Although accumulating evidences uncover the emerging roles of MSI2 in several cancers, the regulatory functions and underlying mechanisms of MSI2 in NB remain under-investigated. Herein, we identified that high-expressed MSI2 and low-expressed n-Myc group account for 43.1% of total NB clinical samples (n = 65). Meanwhile, MSI2 expression is profoundly upregulated along with NB malignancy and negatively associated with the survival outcome of NB patients in the NB tissue microarray (NB: n = 65; Ganglioneuroblastoma: n = 31; Ganglioneuroma: n = 27). In vitro, our results revealed that MSI2 promoted migration, invasion, and proliferation of NB cells via enhancing pentose phosphate pathway. Mechanistically, MSI2 upregulated the key enzyme glucose-6-phosphate dehydrogenase (G6PD) via directly binding to 3'-untranslated regions of c-Myc mRNA to facilitate its stability, resulting in enhancing pentose phosphate pathway. Our findings reveal that MSI2 promotes pentose phosphate pathway via activating c-Myc-G6PD signaling, suggesting that MSI2 exhibits a novel and powerful target for the diagnosis and treatment of NB.

Performance of two tools for pulmonary vein occlusion assessment with a novel navigation system in cryoballoon ablation procedure.

INTRODUCTION: Optimal occlusion of pulmonary vein (PV) is essential for atrial fibrillation (AF) cryoballoon ablation (CBA). The aim of the study was to investigate the performance of two different tools for the assessment of PV occlusion with a novel navigation system in CBA procedure. METHODS: In consecutive patients with paroxysmal AF who underwent CBA procedure with the guidance of the novel 3-dimentional mapping system, the baseline tool, injection tool and pulmonary venography were all employed to assess the degree of PV occlusion, and the corresponding cryoablation parameters were recorded. RESULTS: In 23 patients (mean age 60.0 ± 13.9 years, 56.5% male), a total of 149 attempts of occlusion and 122 cryoablations in 92 PVs were performed. Using pulmonary venography as the gold standard, the overall sensitivity, specificity of the baseline tool was 96.7% (95% confidence interval [CI] 90.0%-99.1%), and 40.5% (95% CI 26.0%-56.7%), respectively, while the corresponding value of the injection tool was 69.6% (95% CI 59.7%-78.1%), and 100.0% (95% CI 90.6%-100.0%), respectively. Cryoablation with optimal occlusion showed lower nadir temperature (baseline tool: -44.3 ± 8.4°C vs. -35.1 ± 6.5°C, p < .001; injection tool: -46.7 ± 6.4°C vs. -38.3 ± 9.2°C, p < .001) and longer total thaw time (baseline tool: 53.3 ± 17.0 s vs. 38.2 ± 14.9 s, p = .003; injection tool: 58.5 ± 15.5 s vs. 41.7 ± 15.2 s, p < .001) compared with those without. CONCLUSIONS: Both tools were able to accurately assess the degree of PV occlusion and predict the acute cryoablation effect, with the baseline tool being more sensitive and the injection tool more specific.

Long-Term prognosis of radiofrequency catheter ablation for atrial fibrillation with different subtypes of heart failure in the era of ablation index guidance.

Background: The long-term outcomes of ablation index (AI)-guided radiofrequency catheter ablation (RFCA) on atrial fibrillation (AF) and different subtypes of heart failure (HF) remain unknown. The aim of the study was to evaluate the long-term prognosis of AI-guided RFCA procedures in patients with AF and concomitant HF. Methods: We retrospectively included consecutive patients with AF and HF who underwent the initial RFCA procedure with AI guidance from March 2018 to June 2021 in our institution. The patients were categorized into two groups: HF with preserved ejection fraction (HFpEF) group and HF with mid-range ejection fraction (HFmrEF) +HF with reduced ejection fraction (HFrEF) group. Results: A total of 101 patients were included. HFpEF and HFmrEF + HFrEF groups consisted of 71 (70.3%) and 30 patients (29.7%), respectively. During a median follow-up of 32.0 (18.2, 37.6) months, no significant difference was detected in AF recurrence between groups (21.1 vs. 33.3%) after multiple procedures, whereas the incidence of the composite endpoint of all-cause death, thromboembolic events, and HF hospitalization was significantly lower in HFpEF group (9.9 vs. 25.0%, Log-rank p = 0.018). In multivariable analysis, a history of hypertension [hazard ratio (HR) 4.667, 95% confidence interval (CI) 1.433-15.203, p = 0.011], left ventricular ejection fraction (LVEF) < 50% (HR 5.390, 95% CI 1.911-15.203, p = 0.001) and recurrent AF after multiple procedures (HR 7.542, 95% CI 2.355-24.148, p = 0.001) were independently associated with the incidence of the composite endpoint. Conclusion: Long-term success could be achieved in 75% of patients with AF and concomitant HF after AI-guided RFCA procedures, irrespective of different HF subtypes. Preserved LVEF was associated with a reduction in the composite endpoint compared with impaired LVEF. Patients with recurrent AF tend to have a poorer prognosis.

Development of Microfluidic Chip-Based Loop-Mediated Isothermal Amplification (LAMP) Method for Detection of Carbapenemase Producing Bacteria.

The rapid and accurate diagnostic methods to identify carbapenemase-producing organisms (CPO) is of great importance for controlling the CPO infection. Herein, we have developed a microfluidic chip-based technique to detect CPO and assessed its clinical value in detecting CPO directly from blood cultures (BCs). The detection performance of the microfluidic chip-based LAMP amplification method was analyzed retrospectively on a collection of 192 isolates including molecularly characterized 108 CPO and 84 non-CPO and prospectively on a collection of 133 positive BCs with or without CPO suspicion, respectively. In the retrospective study, the microfluidic chip-based LAMP amplification method exhibited 87.5% accuracy (95% CI [82.0-91.5]), 97.7% sensitivity (95% CI [91.2-99.6]), 78.8% specificity (95% CI [69.5-86.0]), 79.6% positive predictive value (PPV) (95% CI [70.6-86.5]) and 97.6% negative predictive value (NPV) (95% CI [90.9-99.6]). Among the 192 isolates, 22 (11.5%) false-positives (FP) and 2 (1.0%) false negatives (FN) were observed. In the prospective study, the 133 routine isolates of positive BCs including 18 meropenem-resistant CPO and 115 non-CPO were assessed, and 4 FP were observed in non-CPO and CPO, respectively. The current method showed a total detection performance of 94.0% accuracy (95% CI [88.4-97.1]), 100.0% sensitivity (95% CI [73.2-100.0]), 93.2% specificity (95% CI [86.7-96.8]), 63.6% PPV (95% CI [40.8-82.0]) and 100.0% NPV (95% CI [95.8-100.0]). In summary, the microfluidic chip-based LAMP amplification method is reliable for the rapid screening and detection of CPO with high accuracy, sensitivity, and specificity, and could easily be implemented in clinical microbiology laboratories. IMPORTANCE Rapid and accurate identification of CPO may reduce the genetic exchanges among bacteria and prevent further dissemination of carbapenemases to non-CPO. The current method had designed microfluidic chip-based LAMP amplification method for multiplex detection of carbapenemase genes and evaluated the detection performance of the newly method. The current method can rapidly screen and detect CPO with high accuracy, sensitivity, and specificity, and could easily be implemented in clinical microbiology laboratories, as this will reduce the carbapenem resistance issues worldwide.

Microgel Single-Cell Culture Arrays on a Microfluidic Chip for Selective Expansion and Recovery of Colorectal Cancer Stem Cells.

Cancer stem cells (CSCs) are rare and lack definite biomarkers, necessitating new methods for a robust expansion. Here, we developed a microfluidic single-cell culture (SCC) approach for expanding and recovering colorectal CSCs from both cell lines and tumor tissues. By incorporating alginate hydrogels with droplet microfluidics, a high-density microgel array can be formed on a microfluidic chip that allows for single-cell encapsulation and nonadhesive culture. The SCC approach takes advantage of the self-renewal property of stem cells, as only the CSCs can survive in the SCC and form tumorspheres. Consecutive imaging confirmed the formation of single-cell-derived tumorspheres, mainly from a population of small-sized cells. Through on-chip decapsulation of the alginate microgel, ∼6000 live cells can be recovered in a single run, which is sufficient for most biological assays. The recovered cells were verified to have the genetic and phenotypic characteristics of CSCs. Furthermore, multiple CSC-specific targets were identified by comparing the transcriptomics of the CSCs with the primary cancer cells. To summarize, the microgel SCC array offers a label-free approach to obtain sufficient quantities of CSCs and thus is potentially useful for understanding cancer biology and developing personalized CSC-targeting therapies.

The Transferrin Receptor-Directed CAR for the Therapy of Hematologic Malignancies.

As many patients ultimately relapse after chimeric antigen receptor (CAR) T-cell therapy, identification of alternative targets is currently being evaluated. Substantial research efforts are underway to develop new targets. The transferrin receptor (TfR) is prevalently expressed on rapidly proliferating tumor cells and holds the potential to be the alternative target. In order to investigate the efficacy and challenges of TfR-targeting on the CAR-based therapy strategy, we generated a TfR-specific CAR and established the TfR-CAR-modified T cells. To take the advantage of TfR being widely shared by multiple tumors, TfR-CAR T cells were assessed against several TfR+ hematological malignant cell lines. Data showed that TfR-CAR T cells were powerfully potent in killing all these types of cells in vitro and in killing T-ALL cells in vivo. These findings suggest that TfR could be a universal target to broaden and improve the therapeutic efficacy of CAR T cells and warrant further efforts to use these cells as an alternative CAR T cell product for the therapy of hematological malignancies.

Establishment of a hTfR mAb-functionalized HPPS theranostic nanoplatform.

Rational: Many efforts have been made to develop ligand-directed nanotheranostics in cancer management which could afford both therapeutic and diagnostic functions as well as tumor-tailored targeting. Theranostic nanoplatform targeting transferrin receptor (TfR) is an effective system for favorable delivery of diagnostic and therapeutic agents to malignancy site. Methods: To enable amalgamation of therapy and diagnosis to many TfR+ tumor, hTfR (human TfR) monoclonal antibody (mAb)-functionalized HPPS nanoparticle (HPPS-mAb) was prepared with hTfR mAb on the shell and with fluorophore DiR-BOA in the core. The targeting specificity was investigated in vitro by immunostaining and in vivo using a double-tumor-engrafted mouse model. HPPS-mAb/siRNA effect on HepG2 cells was determined by RT-PCR and western blot. Results: HPPS-mAb could specifically target cancer cells through TfR and achieve tumor accumulation at an early valuable time node, thus efficiently delivering therapeutic survivin siRNA into TfR+ HepG2 cells and mediating cell apoptosis. DiR-BOA can act as an imaging tool to diagnose cancer. Conclusions: Our studies provide a promising TfR mAb-directed theranostic nanoplatform candidate in tumor molecular imaging and in TfR targeted tumor therapy.

Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats.

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR+ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.

LINC01149 variant modulates MICA expression that facilitates hepatitis B virus spontaneous recovery but increases hepatocellular carcinoma risk.

Interpreting disease-causing variants, especially in noncoding regions by genome-wide association studies (GWAS), has become one of the most challenging and demanding tasks. We hypothesized that functional lncRNAs variants in GWAS-identified loci might alter expression level of genes associated with persistent HBV infection and hepatocellular carcinoma (HCC). Integrated bioinformatics approaches were used to prioritize potentially functional variants and a two-stage case-control study (2473 HBV positive HCC patients, 2248 persistent HBV carriers and 2294 spontaneously recovered subjects) was performed to assess the roles of these variants. The rs2844512 G > C variant in LINC01149 was identified to facilitate HBV spontaneous recovery (OR = 0.84, 95% CI = 0.77-0.92) but increase the risk of HCC (OR = 1.21, 95% CI = 1.11-1.32) in combined samples. Subsequent biological assays indicated this variant created a binding site for miR-128-3p and upregulated MICA expression by serving as a miRNA sponge, which might recruit NK-cells to lyse infected cells, but release highly soluble MICA by shedding to induce NK-cells exhaustion and tumor immune evasion. These findings highlight a regulatory circuit between LINC01149 and MICA, mediating by miR-128-3p, and the important role of upregulated MICA in conferring susceptibility to persistent HBV infection and HCC.

Therapeutic Bispecific T-Cell Engager Antibody Targeting the Transferrin Receptor.

Bispecific T-cell engager antibodies (BiTE) have been explored as a means to recruit cytolytic T cells to kill tumor cells. The transferrin receptor (TfR) is highly expressed on the surface of rapidly proliferating tumor cells. Therefore, it holds great potential in T cell redirecting therapies. In this research, we developed a BiTE targeting TfR and CD3 (TfR-BiTE) and studied its therapeutic impact on TfR-positive cancer. TfR-BiTE had the ability to induce the selective lysis of various TfR-positive cancer cells through the activation of T cells, the release of cytokines, and then the coming proliferation of T cells, whereas TfR-negative cells were not affected. In a subcutaneous HepG2 xenograft model, low concentrations of TfR-BiTE inhibited tumor growth. Overall, these results reveal that TfR-BiTE can selectively deplete TfR-positive HepG2 cells; hence, it represents a novel immunotherapeutic approach for the treatment of hepatocellular carcinoma.

Systematic Functional Interrogation of Genes in GWAS Loci Identified ATF1 as a Key Driver in Colorectal Cancer Modulated by a Promoter-Enhancer Interaction.

Genome-wide association studies (GWASs) have identified approximately 100 colorectal cancer (CRC) risk loci. However, the causal genes in these loci have not been systematically interrogated. We conducted a high-throughput RNA-interference functional screen to identify the genes essential for proliferation in the CRC risk loci of Asian populations. We found that ATF1, located in the 12q13.12 region, functions as an oncogene that facilitates cell proliferation; ATF1 has the most significant effect of the identified genes and promotes CRC xenograft growth by affecting cell apoptosis. Next, by integrating a fine-mapping analysis, a two-stage affected-control study consisting of 6,213 affected individuals and 10,388 controls, and multipronged experiments, we elucidated that two risk variants, dbSNP: rs61926301 and dbSNP: rs7959129, that located in the ATF1 promoter and first intron, respectively, facilitate a promoter-enhancer interaction, mediated by the synergy of SP1 and GATA3, to upregulate ATF1 expression, thus synergistically predisposing to CRC risk (OR = 1.77, 95% CI = 1.42-2.21, p = 3.16 × 10-7; Pmultiplicative-interaction = 1.20 × 10-22; Padditive-interaction = 6.50 × 10-3). Finally, we performed RNA-seq and ChIP-seq assays in CRC cells treated with ATF1 overexpression in order to dissect the target programs of ATF1. Results showed that ATF1 activates a subset of genes, including BRAF, NRAS, MYC, BIRC2, DAAM1, MAML2, STAT1, ID1, and NKD2, related to apoptosis, Wnt, TGF-ß, and MAPK pathways, and these effects could cooperatively increase the risk of CRC. These findings reveal the clinical potential of ATF1 in CRC development and illuminate a promoter-enhancer interaction module between the ATF1 regulatory elements dbSNP: rs61926301 and dbSNP: rs7959129, and they bring us closer to understanding the molecular drivers of cancer.

Quinoline and thiazolopyridine allosteric inhibitors of MALT1.

Quinolines and thiazolopyridines were developed as allosteric inhibitors of MALT1, with good cellular potency and exquisite selectivity. Mouse pharmacokinetic (PK) profiling showed these to have low in vivo clearance, and moderate oral exposure. The thiazolopyridines were less lipophilic than the quinolines, and one thiazolopyridine example was active in our hIL10 mouse pharmacodynamic (PD) model upon oral dosing.

IL-23 enhances the malignant properties of hepatoma cells by attenuation of HNF4α.

Chronic infection with hepatitis B virus (HBV) is one of the major risk factors for hepatocellular carcinoma. HBV infection can induce the expression of IL-23. However, the effects of IL-23 on carcinogenesis are rare and contradictory. To investigate the potential role of IL-23 on malignant properties of hepatoma cells, in the present study, first, we confirmed that HBV drove infected hepatoma cells to produce more IL-23. And then we found that at low concentration, human recombinant IL-23 (hrIL-23) enhanced malignant properties of hepatoma cells through increasing the proportion of stem/progenitor cells, promoting proliferation and colony formation, reducing apoptosis and inducing motility and invasivity of them. Hepatocyte nuclear factor 4 alpha (HNF4α), which is essential for liver development and hepatocyte function, was found to be downregulated in HBV integrated or transiently transfected hepatoma cells. Its expression was also decreased in cells treated by hrIL-23 or by HepG2.215 culture supernatant and this decrease could be abolished by supplementation of anti-IL-23p19 antibody. Hence, it is speculated that HBV related IL-23 can enhance malignant properties of hepatoma cells through attenuation of HNF4α. The findings identified a potential target of interventional strategies for treating hepatitis B patients through manipulation of the IL-23.

GRP78 Impairs Production of Lipopolysaccharide-Induced Cytokines by Interaction with CD14.

The 78-kDa glucose-regulated protein (GRP78) is a stress-inducible chaperone that resides primarily in the endoplasmic reticulum. GRP78 has been described to be released at times of cellular stress and as having extracellular properties that are anti-inflammatory or favor the resolution of inflammation. In the current study, we confirmed that GRP78 impaired the production of lipopolysaccharide-induced pro-inflammatory cytokines in GRP78-treated bone-marrow-derived dendritic cells (DCs). To explore the underlying mechanism, first of all, GRP78 was checked to be bound to the plasma membrane. Interestingly, such binding promoted endocytosis of toll-like receptor (TLR) 4 and reduction in TLR4 on the plasma surface had a key role in desensitization of GRP78-treated DCs to lipopolysaccharide. Given that cluster of differentiation (CD)14 is a crucial regulator of TLR4 endocytosis, interaction of GRP78 with CD14 was investigated next. Data showed that GRP78 co-localized with CD14 on the plasma membrane and glutathione-S-transferase-GRP78 precipitated CD14. In CD14 knockout mice, down-regulation of tumor necrosis factor-α and reduction in TLR4 on the plasma surface were abrogated in GRP78-treated DCs. Overall, these data suggested that GRP78 mediates endocytosis of TLR4 by targeting CD14 to favor the resolution of inflammation.

Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress.

Chinese hamster ovary (CHO) cells are regarded as one of the most commonly used mammalian hosts, which decreases the productivity due to loss in culture viability. Overexpressing antiapoptosis genes in CHO cells was developed as a means of limiting cell death upon exposure to environmental insults. Glucose-regulated protein 78 (GRP78) is traditionally regarded as a major ER chaperone that participates in protein folding and other cell processes. It is also a potent antiapoptotic protein and plays a critical role in cell survival, proliferation, and metastasis. In this study, the impact of GRP78 on CHO cells in response to environmental insults such as serum deprivation and oxidative stress was investigated. First, it was confirmed that CHO cells were very sensitive to environmental insults. Then, GRP78 overexpressing CHO cell line was established and exposed to serum deprivation and H2O2. Results showed that GRP78 engineering increased the viability and decreased the apoptosis of CHO cells. The survival advantage due to GRP78 engineering could be mediated by suppression of caspase-3 involved in cell death pathways in stressed cells. Besides, GRP78 engineering also enhanced yields of antibody against transferrin receptor in CHO cells. GRP78 should be a potential application in the biopharmaceutical industries.