Cbl-b inhibition promotes less differentiated phenotypes of T cells with enhanced cytokine production.

For adoptive therapy with T cell receptor engineered T (TCR-T) cells, the quantity and quality of the final cell product directly affect their anti-tumor efficacy. The post-transfer efficacy window of TCR-T cells is keen to optimizing attempts during the manufacturing process. Cbl-b is a E3 ubiquitin ligase previously shown with critical negative impact in T cell functions. This study investigated whether strategic inclusion of a commercially available small inhibitor targeting Cbl-b (Cbl-b-IN-1) prior to T cell activation could enhance the quality of the final TCR-T cell product. Examination with both PBMCs and TCR-T cells revealed that Cbl-b-IN-1 treatment promoted TCR expression efficiency, T cell proliferation potential and, specifically, cell survival capability post antigenic stimulation. Cbl-b-IN-1 exposure facilitated T cells in maintaining less differentiated states with enhanced cytokine production. Further, we found that Cbl-b-IN-1 effectively augmented the activation of TCR signaling, shown by increased phosphorylation levels of Zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase c-γ1 (PLCγ1). In conclusion, our results evidence that the inclusion of Cbl-b inhibitor immediately prior to TCR-T cell activation may enhance their proliferation, survival, and function potentials, presenting an applicable optimization strategy for immunotherapy with adoptive cell transfer.

Exploring the role of CBLB in acute myocardial infarction: transcriptomic, microbiomic, and metabolomic analyses.

BACKGROUND: Specific alterations in gut microbiota and metabolites have been linked to AMI, with CBLB potentially playing an essential role. However, the precise interactions remain understudied, creating a significant gap in our understanding. This study aims to address this by exploring these interactions in CBLB-intervened AMI mice using transcriptome sequencing, 16 S rDNA, and non-targeted metabolite analysis. METHODS: To probe the therapeutic potential and mechanistic underpinnings of CBLB overexpression in AMI, we utilized an integrative multi-omics strategy encompassing transcriptomics, metabolomics, and 16s rDNA sequencing. We selected these particular methods as they facilitate a holistic comprehension of the intricate interplay between the host and its microbiota, and the potential effects on the host's metabolic and gene expression profiles. The uniqueness of our investigation stems from utilizing a multi-omics approach to illuminate the role of CBLB in AMI, an approach yet unreported to the best of our knowledge. Our experimental protocol encompassed transfection of CBLB lentivirus-packaged vectors into 293T cells, followed by subsequent intervention in AMI mice. Subsequently, we conducted pathological staining, fecal 16s rDNA sequencing, and serum non-targeted metabolome sequencing. We applied differential expression analysis to discern differentially expressed genes (DEGs), differential metabolites, and differential microbiota. We performed protein-protein interaction analysis to identify core genes, and conducted correlation studies to clarify the relationships amongst these core genes, paramount metabolites, and key microbiota. RESULTS: Following the intervention of CBLB in AMI, we observed a significant decrease in inflammatory cell infiltration and collagen fiber formation in the infarcted region of mice hearts. We identified key changes in microbiota, metabolites, and DEGs that were associated with this intervention. The findings revealed that CBLB has a significant correlation with DEGs, differential metabolites and microbiota, respectively. This suggests it could play a pivotal role in the regulation of AMI. CONCLUSION: This study confirmed the potential of differentially expressed genes, metabolites, and microbiota in AMI regulation post-CBLB intervention. Our findings lay groundwork for future exploration of CBLB's role in AMI, suggesting potential therapeutic applications and novel research directions in AMI treatment strategies.

Toll-like Receptor Agonist CBLB502 Protects Against Radiation-induced Intestinal Injury in Mice.

BACKGROUND/AIM: The small intestine is one of the organs most vulnerable to ionizing radiation (IR) damage. However, methods to protect against IR-induced intestinal injury are limited. CBLB502, a Toll-like receptor 5 (TLR5) agonist from Salmonella flagellin, exerts radioprotective effects on various tissues and organs. However, the molecular mechanisms by which CBLB502 protects against IR-induced intestinal injury remain unclear. Thus, this study aimed to elucidate the mechanisms underlying IR-induced intestinal injury and the protective effects of CBLB502 against this condition in mice. MATERIALS AND METHODS: Mice were administered 0.2 mg/kg CBLB502 before IR at different doses for different time points, and then the survival rate, body weight, hemogram, and histopathology of the mice were analyzed. RESULTS: CBLB502 reduced IR-induced intestinal injury. RNA-seq analysis revealed that different doses and durations of IR induced different regulatory patterns. CBLB502 protected against intestinal injury mainly after IR by reversing the expression of IR-induced genes and regulating immune processes and metabolic pathways. CONCLUSION: This study preliminarily describes the regulatory mechanism of IR-induced intestinal injury and the potential molecular protective mechanism of CBLB502, providing a basis for identifying the functional genes and molecular mechanisms that mediate protection against IR-induced injury.

T cell specific deletion of Casitas B lineage lymphoma-b reduces atherosclerosis, but increases plaque T cell infiltration and systemic T cell activation.

Introduction: Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. Methods: To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Results: Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. Conclusion: In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.

Casitas b cell lymphoma­B (Cbl-b): A new therapeutic avenue for small-molecule immunotherapy.

Immunotherapy has revolutionized the area of cancer treatment. Although most immunotherapies now are antibodies targeting membrane checkpoint molecules, there is an increasing demand for small-molecule drugs that address intracellular pathways. The E3 ubiquitin ligase Casitas B cell lymphoma­b (Cbl-b) has been regarded as a promising intracellular immunotherapy target. Cbl-b regulates the downstream proteins of multiple membrane receptors and co-receptors, restricting the activation of the innate and adaptive immune system. Recently, Cbl-b inhibitors have been reported with promising effects on immune surveillance activation and anti-tumor efficacy. Several molecules have entered phase Ⅰ clinical trials. In this review, the biological rationale of Cbl-b as a promising target for cancer immunotherapy and the latest research progress of Cbl-b are summarized, with special emphasis on the allosteric small-molecule inhibitors of Cbl-b.

E3 ubiquitin ligase casitas B-lineage lymphoma-b modulates T-cell anergic resistance via phosphoinositide 3-kinase signaling in patients with immune thrombocytopenia.

BACKGROUND: The E3 ubiquitin ligase casitas B-lineage lymphoma-b (CBLB) is a newly identified component of the ubiquitin-dependent protein degradation system and is considered an important negative regulator of immune cells. CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms. However, the involvement of CBLB in the pathogenesis of immune thrombocytopenia (ITP) is unknown. OBJECTIVES: We aimed to investigate the expression and role of CBLB in CD4+ T cells obtained from patients with ITP through quantitative proteomics analyses. METHODS: CD4+ T cells were transfected with adenoviral vectors overexpressing CBLB to clarify the effect of CBLB on anergic induction of T cells in patients with ITP. DNA methylation levels of the CBLB promoter and 5' untranslated region (UTR) in patient-derived CD4+ T cells were detected via MassARRAY EpiTYPER assay (Agena Bioscience). RESULTS: CD4+ T cells from patients with ITP showed resistance to anergic induction, highly activated phosphoinositide 3-kinase-protein kinase B (AKT) signaling, decreased CBLB expression, and 5' UTR hypermethylation of CBLB. CBLB overexpression in T cells effectively attenuated the elevated phosphorylated protein kinase B level and resistance to anergy. Low-dose decitabine treatment led to significantly elevated levels of CBLB expression in CD4+ T cells from 7 patients showing a partial or complete response. CONCLUSION: These results indicate that the 5' UTR hypermethylation of CBLB in CD4+ T cells induces resistance to T-cell anergy in ITP. Thus, the upregulation of CBLB expression by low-dose decitabine treatment may represent a potential therapeutic approach to ITP.

Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity.

IMPORTANCE: This is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

The inducible secreting TLR5 agonist, CBLB502, enhances the anti-tumor activity of CAR133-NK92 cells in colorectal cancer.

OBJECTIVE: CAR-T/NK cells have had limited success in the treatment of solid tumors, such as colorectal cancer (CRC), in part because of the heterogeneous nature of tumor-associated antigens that lead to antigen-negative relapse after the initial response. This barrier might be overcome by enhancing the recruitment and durability of endogenous immune cells. METHODS: Immunohistochemistry and flow cytometry were used to assess the expression of CD133 antigen in tissue microarrays and cell lines, respectively. Retroviral vector transduction was used to generate CBLB502-secreting CAR133-NK92 cells (CAR133-i502-NK92). The tumor killing capacity of CAR133-NK92 cells in vitro and in vivo were quantified via LDH release, the RTCA assay, and the degranulation test, as well as measuring tumor bioluminescence signal intensity in mice xenografts. RESULTS: We engineered CAR133-i502-NK92 cells and demonstrated that those cells displayed enhanced proliferation (9.0 × 104 cells vs. 7.0 × 104 cells) and specific anti-tumor activities in vitro and in a xenogeneic mouse model, and were well-tolerated. Notably, CBLB502 secreted by CAR133-i502-NK92 cells effectively activated endogenous immune cells. Furthermore, in hCD133+/hCD133- mixed cancer xenograft models, CAR133-i502-NK92 cells suppressed cancer growth better than the counterparts (n = 5, P = 0.0297). Greater T-cell infiltration was associated with greater anti-tumor potency (P < 0.0001). CONCLUSIONS: Armed with a CBLB502 TLR5 agonist, CAR133-NK92 cells were shown to be capable of specifically eliminating CD133-positive colon cancer cells in a CAR133-dependent manner and indirectly eradicating CD133-negative colon cancer cells in a CBLB502-specific endogenous immune response manner. This study describes a novel technique for optimizing CAR-T/NK cells for the treatment of antigenically-diverse solid tumors.

Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells.

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the hepatocyte growth factor (HGF)-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE) and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (double KO [DKO] cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude, and duration of c-Met receptor signaling via immunoblot and receptor trafficking by immunofluorescence. Single KO cells displayed a decrease in receptor ubiquitylation and an increase in phosphorylation compared to control. DKO cells had no detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live-cell time-lapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Toll-like Receptor Agonist CBLB502 Protects Against Cisplatin-induced Liver and Kidney Damage in Mice.

BACKGROUND/AIM: CBLB502, a Toll-like receptor-5 agonist derived from Salmonella flagellin, exerts protective roles against irradiation and chemical drugs in mammalian tissues and stimulates tissue regeneration. This study aimed to investigate whether CBLB502 can protect against liver and kidney damage induced by the chemotherapeutic drug cisplatin (CDDP) and the underlying mechanism of the protective effect. MATERIALS AND METHODS: Mice were pretreated with CBLB502 [0.2 mg/kg, intraperitoneal (i.p.) injection] 0.5 h prior to administration of CDDP (20 mg/kg, i.p. injection), and analyses of the liver and kidney indices, blood biochemistry, and histopathology were performed. RESULTS: Pretreatment with CBLB502 alleviated CDDP-induced liver and kidney damage. RNA sequencing and bioinformatic analysis indicated that CDDP induced a similar damage-promoting gene regulation pattern in the liver and kidney. CBLB502 protected against liver and kidney damage only after CDDP treatment primarily via different pathways. However, some CBLB502-regulated genes were common between the liver and kidney, including those involved in blood coagulation, fibrinolysis, hemostasis, apoptotic regulation, NF-kappaB signaling, and response to lipopolysaccharide, suggesting a general protective effect by CBLB502. CONCLUSION: Our data provide insights into the protective mechanism of CBLB502 against CDDP-induced tissue damage in the liver and kidney and might provide a basis for future studies on functional genes and regulatory mechanisms that mediate protection against chemoradiotherapy-induced damage.

Postoperative circulating tumor DNA detection and CBLB mutations are prognostic biomarkers for gastric cancer.

BACKGROUND: Several studies have demonstrated that circulating tumor DNA (ctDNA) can be used to predict the postoperative recurrence of several cancers. However, there are few studies on the use of ctDNA as a prognosis tool for gastric cancer (GC) patients. OBJECTIVE: This study aims to determine whether ctDNA could be used as a prognostic biomarker in GC patients through multigene-panel sequencing. METHODS: Using next-generation sequencing (NGS) Multigene Panels, the mutational signatures associated with the prognosis of GC patients were identified. We calculated the survival probability with Kaplan-Meier and used the Log-rank test to compare survival curves between ctDNA-positive and ctDNA-negative groups. Potential application of radiology combined with tumor plasma biomarker analysis of ctDNA in GC patients was carried out. RESULTS: Disease progression is more likely in ctDNA-positive patients as characterized clinically by a generally higher T stage and a poorer therapeutic response (P < 0.05). ctDNA-positive patients also had worse overall-survival (OS: P = 0.203) and progression-free survival (PFS: P = 0.037). The combined analysis of ctDNA, radiological, and serum biomarkers in four patients indicated that ctDNA monitoring can be a good complement to radiological and plasma tumor markers for GC patients. Kaplan-Meier analysis using a cohort of GC patients in the TCGA database showed that patients with CBLB mutations had shorter OS and PFS than wild-type patients (OS: P = 0.0036; PFS: P = 0.0027). CONCLUSIONS: This study confirmed the utility and feasibility of ctDNA in the prognosis monitoring of gastric cancer.

Cbl-b inhibited CD4+ T cell activation by regulating the expression of miR-99a/miR-125b.

The molecular regulation of T cell activation has always been a hot topic in immunology. It has been reported that Cbl-b inhibits T cell activation, but the specific molecular mechanism especially for transcriptional regulation has not been very clear so far. Our present study showed that ablation of Cbl-b resulted in the increased expression of miR-99a and miR-125b, and the antagonism of miR-99a or miR-125b could inhibit the Cbl-b-/- T cell over-activation partly. Further study demonstrated that Cbl-b could bind and ubiquitinate SHP-2 in the activated T cells. The activation of SHP-2 deficient T cells was significantly inhibited. Western blot showed that SHP-2 could dephosphorylate HOXA10, and HOXA10 could enter the nucleus under the stimulation of anti-CD3 antibody alone in Cbl-b deficient T cells. Luciferase reporter assay and CUT&Tag qPCR showed that HOXA10 could regulate the expression of miR-99a/miR-125b. Real-time PCR and western blot further indicated that miR-99a/miR-125b functioned on PI3K/AKT pathway to regulate T cell activation. In conclusion, our study demonstrated that Cbl-b ubiquitinated SHP-2 to arrest HOXA10-mediated CD4+ T cell activation by regulating the expression of miR-99a/miR-125b and their function on PI3K/AKT pathway, which might providing a new explanation for the regulation of T cell activation and potential new idea for autoimmune diseases and tumor immunotherapies.

Involvement of the E3 ubiquitin ligase Cblb in host defense and evaluation of transcriptome during Trueperella pyogenes infection.

Trueperella pyogenes (T. pyogenes) is a versatile and ingenious bacterium that causes severe suppurative injuries in lots of economically important ruminants. The underlying pathogenesis of T. pyogenes infection remains poorly understood. In the current study, we performed transcriptome sequencing of mouse blood tissue infected with T. pyogenes. A total of 36.73 G clean data were collected, and 136 differentially expressed genes were obtained in the infection group compared to the control group. In addition, we found that the E3 ubiquitin ligase Cblb exhibited significant upregulation in the infection groups compared to the control group. Mechanistically, T. pyogenes infection markedly enhanced the expression of Cblb and regulated the host defense response. Inhibiting Cblb expression with Cblb siRNA impaired the inflammatory response and reduced the effect of phagocytosis in RAW264.7 murine macrophages. Intriguingly, overexpression of Cblb induced a strong inflammatory response and enhanced phagocytosis against T. pyogenes infection in macrophages. More importantly, the overexpression of Cblb significantly reduced the bacterial load and protected mice from the T. pyogenes infections. Therefore, our findings reveal that Cblb is a novel and potential regulator in response to T. pyogenes infection and shed new light on the development of promising treatments against T. pyogenes-related diseases.

Preclinical Evaluation of CRISPR-Edited CAR-NK-92 Cells for Off-the-Shelf Treatment of AML and B-ALL.

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines' cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.

Targeting the Cbl-b-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cell responses.

A critical feature of cancer is the ability to induce immunosuppression and evade immune responses. Tumor-induced immunosuppression diminishes the effectiveness of endogenous immune responses and decreases the efficacy of cancer immunotherapy. In this study, we describe a new immunosuppressive pathway in which adenosine promotes Casitas B-lineage lymphoma b (Cbl-b)-mediated Notch1 degradation, causing suppression of CD8+ T-cells effector functions. Genetic knockout and pharmacological inhibition of Cbl-b prevents Notch1 degradation in response to adenosine and reactivates its signaling. Reactivation of Notch1 results in enhanced CD8+ T-cell effector functions, anti-cancer response and resistance to immunosuppression. Our work provides evidence that targeting the Cbl-b-Notch1 axis is a novel promising strategy for cancer immunotherapy.

Role of Long Noncoding RNA Regulator of Reprogramming in Colon Cancer Progression via Epidermal Growth Factor Receptor Signaling.

Background: Long intergenic noncoding RNA regulator of reprogramming (linc-ROR) is a novel long noncoding RNA that exhibits significant effects on cancer progression. This research presented that linc-ROR had a crucial part in promoting biological characteristics associated with worse prognosis in colon cancer. Method: Bioinformatics analysis was performed to predict signaling pathways related to linc-ROR. In addition, western blot, quantitative reverse transcription-polymerase chain reaction, RNA-pulldown, cell proliferation assays, colony formation assays, wound healing assays, and transwell assays were applied to detect the role and regulation of particular molecules. Results: Our results showed that the knockdown of linc-ROR reduced cell invasion, proliferative ability, and migration in colon cancer. Further evaluation verified that downregulating linc-ROR inhibited the activation of epidermal growth factor receptor (EGFR) signaling. In addition, cbl-b, a kind of E3 ubiquitin ligase that increases the degradation of EGFR, was found to be a potential linc-ROR target. Conclusions: Based on our findings, it was presented that linc-ROR served a role as a tumor-promoting factor via repressing the ubiquitination and degradation of EGFR signaling, which indicated that it could be a possible prognostic marker and therapeutic target for colon cancer.

Early cardiomyopathy without severe metabolic dysregulation in a patient with cblB-type methylmalonic acidemia.

BACKGROUND: Cardiomyopathy is a known complication of organic acidemias but generally thought to be secondary to poor metabolic control. METHODS: Our patient was found through biochemical testing and Sanger sequencing to harbor an Icelandic founder mutation: NM_052845.4(MMAB):c.571C > T(p.Arg191Trp), leading to an early presentation (4 h after birth) of cblB-type methylmalonic acidemia (MMA). Biochemical testing of this patient suggested B-12-responsiveness and thus the patient was treated with cyanocobalamin throughout life. Informed parental consent was obtained for this report. RESULTS: Our patient had three metabolic decompensations in her life (at birth, at 1 month, and at 5 months). The first decompensation was probably linked to stress of delivery, second to rhinovirus infection, and third by co-infection of norovirus and enterovirus. At 3 months, the patient was noted to be tachypneic, although this was attributed to her underlying metabolic acidosis. At 5 months and 10 days, the patient was admitted with minor flu-like symptoms but developed severe diarrhea in hospital and upon rehydration had cardiac decompensation and was found to have undiagnosed dilated cardiomyopathy. Although, patient was treated aggressively with dextrose, hemodialysis, levocarnitine, and vasoactive agents, there was limited response to medications to treat cardiac failure, and eventually the patient passed away before turning 6 months old. CONCLUSIONS: Other than these three mild decompensations, patient had very good metabolic control, thus demonstrating that even without frequent metabolic decompensation, cardiomyopathy can be an observed phenotype in cblB-type MMA even very early in life, suggesting that this phenotype may be independent of metabolic control.

Codelivery of SARS-CoV-2 Prefusion-Spike Protein with CBLB502 by a Dual-Chambered Ferritin Nanocarrier Potentiates Systemic and Mucosal Immunity.

Thousands of breakthrough infections are confirmed after intramuscular (i.m.) injection of the approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two major factors might contribute to breakthrough infections. One is the emergence of mutant variants of SARS-CoV-2, and the other is that i.m. injection has an inefficient ability to activate mucosal immunity in the upper respiratory tract. Here, we devised a dual-chambered nanocarrier that can codeliver the adjuvant CBLB502 with prefusion-spike (pre-S) onto a ferritin nanoparticle. This vaccine enabled enhanced systemic and local mucosal immunity in the upper and lower respiratory tract. Further, codelivery of CBLB502 with pre-S induced a Th1/Th2-balanced immunoglobulin G response. Moreover, the codelivery nanoparticle showed a Th1-biased cellular immune response as the release of splenic INF-γ was significantly heightened while the level of IL-4 was elevated to a moderate extent. In general, the developed dual-chambered nanoparticle can trigger multifaceted immune responses and shows great potential for mucosal vaccine development.

Hepatocyte-like cells differentiated from methylmalonic aciduria cblB type induced pluripotent stem cells: A platform for the evaluation of pharmacochaperoning.

Methylmalonic aciduria cblB type (MMA cblB type, MMAB OMIM #251110), caused by a deficiency in the enzyme ATP:cob(I)alamin adenosyltransferase (ATR, E.C_2. 5.1.17), is a severe metabolic disorder with a poor prognosis despite treatment. We recently described the potential therapeutic use of pharmacological chaperones (PCs) after increasing the residual activity of ATR in patient-derived fibroblasts. The present work reports the successful generation of hepatocyte-like cells (HLCs) differentiated from two healthy and two MMAB induced pluripotent stem cell (iPSC) lines, and the use of this platform for testing the effects of PCs. The MMAB cells produced little ATR, showed reduced residual ATR activity, and had higher concentrations of methylmalonic acid compared to healthy HLCs. Differential proteome analysis revealed the two MMAB HCLs to show reproducible differentiation, but this was not so for the healthy HLCs. Interestingly, PC treatment in combination with vitamin B12 increased the amount of ATR available, and subsequently ATR activity, in both MMAB HLCs. More importantly, the treatment significantly reduced the methylmalonic acid content of both. In summary, the HLC model would appear to be an excellent candidate for the pharmacological testing of the described PCs, for analyzing the effects of new drugs, and investigating the repurposing of older drugs, before testing in animal models.