CTLA-4 tail fusion enhances CAR-T antitumor immunity.

Chimeric antigen receptor (CAR)-T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail, we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex or triplex CTLA-4 cytoplasmic tails (CCTs) fused to the C terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of proinflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior antitumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

Applications of CRISPR technology in cellular immunotherapy.

CRISPR technology has transformed multiple fields, including cancer and immunology. CRISPR-based gene editing and screening empowers direct genomic manipulation of immune cells, opening doors to unbiased functional genetic screens. These screens aid in the discovery of novel factors that regulate and reprogram immune responses, offering novel drug targets. The engineering of immune cells using CRISPR has sparked a transformation in the cellular immunotherapy field, resulting in a multitude of ongoing clinical trials. In this review, we discuss the development and applications of CRISPR and related gene editing technologies in immune cells, focusing on functional genomics screening, gene editing-based cell therapies, as well as future directions in this rapidly advancing field.

Reduction in MicroRNA-4488 Expression Induces NFκB Translocation in Venous Endothelial Cells Under Arterial Flow.

PURPOSE: Little is known about the molecular interactions among inflammatory responses that damage venous endothelial cells (vECs) during venous-to-arterial flow transition in vein graft diseases. Because arterial flow triggers excessive autophagy and inflammation in vECs, this study aimed to investigate the mediator of inflammation and methods to prevent vEC damage. METHODS: Arterial laminar shear stress (ALSS; 12 dynes/cm2) was applied to vECs via in vitro and ex vivo perfusion systems. Inflammation in vECs was measured using inflammatory protein markers, NFκB translocation, cyclooxygenase-2 (COX-2) and COX-2 and NFκB promoter assays. The involvement of microRNA-4488 (miR-4488) was measured and confirmed by altering the specific miR using a miR-4488 mimic or inhibitor. The potential anti-inflammatory drugs and/or nitric oxide (NO) donor L-arginine (L-Arg) to prevent damage to vECs under ALSS was investigated. RESULTS: ALSS triggered reactive oxygen species production, excessive autophagy, COX-2 protein expression, and NFκB translocation during vEC inflammation. Reduction in miR-4488 expression was detected in inflamed vECs treated with LPS, lipopolysaccharide (LPS) TNFα, and ALSS. Transfection of miR-4488 mimic (50 nM) prior to ALSS application inhibited the accumulation of inflammatory proteins as well as the translocation of NFκB. Combined treatment of vECs with COX-2-specific inhibitor (SC-236) and L-Arg alleviated the ALSS-induced inflammatory responses. Protective effects of the combined treatment on vECs against ALSS-induced damage were abolished by the application of miR-4488 inhibitor. CONCLUSION: We showed that ALSS triggered the COX-2/NFκB pathway to induce vEC inflammation with a reduction in miR-4488. Combination of SC-236 and L-Arg prevented ALSS-induced vEC damage, thus, shows high potential for preventing vein graft diseases.

Synthesis and biological evaluation of 7-methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazoles as new colchicine site inhibitors.

The colchicine site inhibitors (CSIs) displayed both antimitotic and vascular disrupting activities, therefore are promising potential antitumor agents. In this study, a series 1-phenyl-4,5-dihydro-2H-benzo[e]indazoles were found as new CSIs of which the bioactive configuration was locked. Among them, compounds C1 and C2 displayed the best activity, with tubulin polymerization IC50 of 3.4 and 1.5 µM, and growth IC50 of low nanomolar concentrations against human colon cancer cell lines. In addition, compound C1 showed excellent broad-spectrum antitumor activity in the NCI-60 Human Tumor Cell Lines Screen, encouraging further study of this antitumor compound.

Design, synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target inhibitors for the treatment of multiple myeloma.

Multiple myeloma (MM) is the second most common haematological malignancy. Almost all patients with MM eventually relapse, and most recommended treatment protocols for the patients with relapsed refractory MM comprise a combination of drugs with different mechanisms of action. Therefore novel drugs are in urgent need in clinic. Bcl-2 inhibitors and HDAC inhibitors were proved their anti-MM effect in clinic or under clinical trials, and they were further discovered to have synergistic interactions. In this study, a series of Bcl-2/HDAC dual-target inhibitors were designed and synthesized. Among them, compounds 7e-7g showed good inhibitory activities against HDAC6 and high binding affinities to Bcl-2 protein simultaneously. They also displayed good growth inhibitory activities against human MM cell line RPMI-8226, which proved their potential value for the treatment of multiple myeloma.

Effects of Dispatcher-Assisted Public-Access Defibrillation Programs on the Outcomes of Out-of-Hospital Cardiac Arrest: A Before-and-After Study.

BACKGROUND: Public access defibrillation (PAD) programs have been implemented globally over the past decade. Although PAD can substantially increase the survival of cardiac arrest, PAD use remains low. This study aimed to evaluate whether drawing upon the successful experiences of dispatcher-assisted cardiopulmonary resuscitation programs would increase the use of PAD in dispatcher-assisted PAD programs. METHODS AND RESULTS: This study using a before-and-after design was conducted in Taoyuan City using a local out-of-hospital cardiac arrest registry system and data of dispatcher performance derived from audio recordings. The primary outcomes were the rate of bystander PAD use, sustained return of spontaneous circulation, survival to discharge, and favorable neurological outcomes. The secondary outcomes were the performance of dispatchers in terms of PAD instruction and dispatcher-assisted cardiopulmonary resuscitation administration, the time interval indicators of dispatcher-assisted cardiopulmonary resuscitation. A total of 1159 patients were included and divided into 2 groups: the before-run-in group (502 patients) and the after-run-in group (657 patients). No significant difference was observed between the 2 groups in terms of baseline characteristics. The rate of PAD use in the after-run-in group significantly increased from 5.0% to 8.7% (P=0.015). The rate of favorable neurological outcomes increased from 4.4% to 5.9%, which was not a statistically significant difference. Compared with the before-run-in group, the rate of successful automated external defibrillator acquisition was 13.5% in the after-run-in group (P<0.001). CONCLUSIONS: Implementing a dispatcher-assisted PAD protocol in a municipality setting significantly increased bystander PAD use without affecting dispatcher performance in out-of-hospital cardiac arrest recognition, cardiopulmonary resuscitation instruction, or dispatcher-assisted cardiopulmonary resuscitation time indicators.

CTLA-4 tail fusion enhances CAR-T anti-tumor immunity.

Chimeric antigen receptor (CAR) T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here, utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CT), we reprogram CAR function and substantially enhance CAR-T efficacy in vivo . CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen, and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior anti-tumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

Cas12a/Cpf1 knock-in mice enable efficient multiplexed immune cell engineering.

Cas9 transgenic animals have drastically accelerated the discovery of novel immune modulators. But due to its inability to process its own CRISPR RNAs (crRNAs), simultaneous multiplexed gene perturbations using Cas9 remains limited, especially by pseudoviral vectors. Cas12a/Cpf1, however, can process concatenated crRNA arrays for this purpose. Here, we created conditional and constitutive LbCas12a knock-in transgenic mice. With these mice, we demonstrated efficient multiplexed gene editing and surface protein knockdown within individual primary immune cells. We showed genome editing across multiple types of primary immune cells including CD4 and CD8 T cells, B cells, and bone-marrow derived dendritic cells. These transgenic animals, along with the accompanying viral vectors, together provide a versatile toolkit for a broad range of ex vivo and in vivo gene editing applications, including fundamental immunological discovery and immune gene engineering.

Skp2-mediated Zeb1 expression facilitates cancer migration by a ubiquitination-independent pathway.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) constitutes one of the most dismal malignancies worldwide. Despite multidisciplinary involvement in interventions involving surgery, radiotherapy, and chemotherapy, most pancreatic cancer patients eventually develop distant metastasis. S-phase kinase-associated protein 2 (Skp2) plays an important role in cell-cycle regulation in pancreatic cancer. However, the role of Skp2 in individualized PDAC treatment is largely unknown. MAIN METHODS: Immunoblotting, quantitative reverse transcription polymerase chain reaction, cell viability test, chromatin immunoprecipitation assay, and xenograft in vivo assay were performed in parental and Skp2-depleted cells. The immunohistochemistry of Skp2 was analyzed on the tissue microarrays of 45 PDAC cases and mice tissues. KEY FINDINGS: In this study, we observed that Skp2 is a marker for poor prognosis in PDAC patients. Upregulation of the inhibitor of κB (IκB)-inducing kinase-nuclear factor kappa B (NF-κB) signal cascade mediated Skp2 expression thereby promoting epithelial-mesenchymal transition (EMT). Depletion of NF-κB-associated signaling effectively prevented Skp2-mediated pancreatic cancer cell migration. As a functional consequence, Skp2 orchestrated with Myc to induce zinc finger E-box binding homeobox 1 (Zeb1) transcription by recruiting p300 to the Zeb1 promoter independent of Skp2 E3-ligase activity. Therefore, blockade of Skp2 could significantly reduce the expression of Zeb1 and inhibit cancer cell migration. In conclusion, Skp2 regulated Zeb1 activity to control the migration and invasion abilities of pancreatic cancer cells. Skp2 expression in PDAC may affect cell vulnerability to standard chemotherapy regimens. SIGNIFICANCE: Therefore, in patients with PDAC, modulation of Skp2 expression could be a novel strategy for preventing cancer cell metastasis.

Differential Nonenrollment of Taiwanese Children in School-Based Substance Use Prevention Studies: A Multilevel Analysis.

OBJECTIVE: The present study investigated the extent to which individual and school characteristics may differentially affect parental consent and child assent in the enrollment of a school-based substance use prevention study in Taiwan. METHOD: This study linked field notes on response and consent status during enrollment of the school-based prevention study with administrative survey data reported by the targeted students when they were in fourth grade (age 10-11) (N = 2,560; 53% male, 97.8% matched). The outcome variables, defined by the combined status of parental consent/child assent, were nonresponse and negative, discordant, and positive consent. Individual characteristics included family (parental education, employment) and child (psychological/behavioral, substance use) factors. Aggregate school-level substance use and percentage of aboriginal students and nonnative parents served as school-level factors. Multilevel multinomial regression analyses were performed. RESULTS: Successful consent was obtained from only 820 students (32%). Male gender and feeling neglected by families were associated with failing to respond (adjusted odds ratio = 1.78 and 1.71, respectively). Higher parental educational attainment reduced the odds of negative consent by 30%, whereas having unemployed parents increased the odds of discordant consent by 326%. Children attending schools with a higher percentage of indigenous students were two times more likely to have nonresponse, negative consent, and discordant consent. CONCLUSIONS: Nonresponse to the consent request or negative consent appeared to be associated with disadvantaged background and unfavorable parent-child interaction. This suggests complex pathways underlying ascertainment and a need to modify the consent practices in school-based prevention studies involving minors, especially in schools with higher ethnic minority composition.

The discovery of novel small molecule allosteric activators of aldehyde dehydrogenase 2.

Aldehyde dehydrogenase 2 (ALDH2) plays important role in ethanol metabolism, and also serves as an important shield from the damage occurring under oxidative stress. A special inactive variant was found carried by 35-45% of East Asians. The variant carriers have recently been found at the higher risk for the diseases related to the damage occurring under oxidative stress, such as cardiovascular and cerebrovascular diseases. As a result, ALDH2 activators may potentially serve as a new class of therapeutics. Herein, N-benzylanilines were found as novel allosteric activators of ALDH2 by computational virtual screening using ligand-based and structure-based screening parallel screening strategy. Then a structural optimization was performed and has led to the discovery of the compound C6. It has good activity in vitro and in vivo, which could reduce infarct size by ∼70% in ischemic stroke rat models. This study provided good lead compounds for the further development of ALDH2 activators.

Curcumol Suppresses Triple-negative Breast Cancer Metastasis by Attenuating Anoikis Resistance via Inhibition of Skp2-mediated Transcriptional Addiction.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is a unique subtype that lacks expression of several conventional biomarkers and has a higher incidence of lymph node invasion and distal metastasis among all breast cancers. Anoikis resistance is the fundamental reason behind tumor cells' survival without their attachment to the extracellular matrix and metastasis to distal organs. Therefore, finding novel anti-cancer drugs that can suppress anoikis resistance in cancer cells is critical for patients with TNBC. MATERIALS AND METHODS: Curcumol, a natural compound, was used to assess whether it can inhibit the anoikis resistance and affects cell mortality and motility of IV2-1 TNBC cells. RESULTS: Curcumol suppressed anoikis resistance and inhibited TNBC cell survival in suspension. Additionally, these anti-cancer effects induced by curcumol could be related to the YAP1/Skp2 molecular pathway. CONCLUSION: Curcumol is an effective Skp2-targeted therapy that attenuates anoikis resistance and metastasis in TNBC cells.

Traditional versus blended CPR training program: A randomized controlled non-inferiority study.

Cardiopulmonary resuscitation (CPR) training and its quality are critical in improving the survival rate of cardiac arrest. This randomized controlled study investigated the efficacy of a newly developed CPR training program for the public in a Taiwanese setting. A total of 832 adults were randomized to either a traditional or blended (18-minute e-learning plus 30-minute hands-on) compression-only CPR training program. The primary outcome was compression depth. Secondary outcomes included CPR knowledge test, practical test, quality of CPR performance, and skill retention. The mean compression depth was 5.21 cm and 5.24 cm in the blended and traditional groups, respectively. The mean difference in compression depth between groups was -0.04 (95% confidence interval -0.13 to infinity), demonstrating that the blended CPR training program was non-inferior to the traditional CPR training program in compression depth after initial training. Secondary outcome results were comparable between groups. Although the mean compression depth and rate were guideline-compliant, only half of the compressions were delivered with adequate depth and rate in both groups. CPR knowledge and skill retained similarly in both groups at 6 and 12 months after training. The blended CPR training program was non-inferior to the traditional CPR training program. However, there is still room for improvement in optimizing initial skill performance as well as skill retention. Clinical Trial Registration: NCT03586752; www.clinicaltrial.gov.

1-Phenyl-dihydrobenzoindazoles as novel colchicine site inhibitors: Structural basis and antitumor efficacy.

The colchicine site inhibitors (CSIs) showed promising prospects as antitumor agents due to their vascular disrupting activities besides antimitotic activities. 1-Phenyl-dihydrobenzoindazole was found as a novel scaffold of CSI without the cis-trans isomerization problem. The X-ray co-crystal structure of the lead compound with tubulin was determined, which revealed the binding mode including special water-bridged hydrogen bonds. The structure also provided guidance for the structural optimization of this type of CSI, which led to the discovery of the most potent inhibitor A3, with growth IC50 lower than 1 nM against human colon cancer cell lines and tubulin polymerization IC50 of 1.6 µM. In addition, its water-soluble prodrug B1 showed good in vivo antitumor activity on two human colon cancer xenograft nude mice models, encouraging further study of this type of antitumor compound.

Impact of BMI1 expression on the apoptotic effect of paclitaxel in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Despite advances in treatment, no treatment modality specific for the different CRC phenotypes has been developed. BMI1 has been previously reported to play an important role in the regulation of cancer stem cells and cell cycle in CRC. However, the role of BMI1 in individualized treatment for CRC is largely unknown. In this study, we found that the apoptotic effect of paclitaxel is more profound in BMI1-depleted cells. The apoptotic effect is exerted by promoting caspase-8-independent apoptotic pathways after combination with paclitaxel in BMI1 knockdown cells. This effect could be totally recovered by pretreatment with caspase inhibitor compared with caspase-8 inhibitor alone. It has been reported that the levels of MCL-1 play a role in regulating cell resistance to paclitaxel treatment. Our data indicated that the downregulation of MCL-1 through the activation of GSK3beta and JNK is driven by BMI1 depletion. Consistent with in vitro data, a synergic anti-growth effect of BMI1 depletion with paclitaxel treatment was shown in vivo. In conclusion, paclitaxel has a stronger suppressive effect on tumor growth and proliferation in CRC with low BMI1 expression. Thus, in CRC patients, paclitaxel could be specifically indicated for patients with low BMI1 expression.

Identification of CHD4-ß1 integrin axis as a prognostic marker in triple-negative breast cancer using next-generation sequencing and bioinformatics.

AIMS: Triple-negative breast cancer (TNBC) is a special subtype of breast cancer that lacks receptor expression and is difficult to cure. Epigenetic regulators have been suggested as targets for cancer therapy in recent years. Our previous study indicated that the chromodomain-helicase-DNA-binding protein 4 (CHD4) is a prognostic biomarker of TNBC and therapeutic target in patients with TNBC. However, the exact mechanisms regulated by CHD4 are still unclear. METHODS: In this study, we compared differences in gene expression in parental and CHD4-deficient cells by next-generation sequencing and Ingenuity Pathway Analysis. KEY FINDINGS: We found that ß1 integrin is a downstream target gene of CHD4, which could be transcriptionally regulated by CHD4 in TNBC cells. Consistent with in vitro data, immunohistochemistry revealed that co-expression of ß1 integrin and CHD4 was significantly associated with metastatic state, recurrence, and survival status in TNBC patients. It also showed a positive correlation between ß1 integrin and CHD4 in vivo. SIGNIFICANCE: This is the first study to suggest that CHD4 regulates ß1 integrin in TNBC. Overall, CHD4-ß1 integrin axis could potentially be a predictive marker in patients with TNBC and the use of ß1 integrin inhibitors may be a therapeutic option for TNBC patients with high CHD4 expression.