CD4 Promoter Research Articles

Rs2564978(T) allele associated with severe influenza a disrupts binding site for myeloid differentiation factor PU.1 and reduces <i>CD55/DAF</i> gene promoter activity in macrophages

An inhibitor of the complement system CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity during influenza A infection, as well as with vascular complications in pathologies involving excessive activation of the complement system. Using a luciferase reporter system, we performed functional analysis of the single nucleotide polymorphism rs2564978 located in the promoter of the CD55 gene in human pro-monocytic cell line U937. We have shown a decreased activity in activated U937 cells of the CD55 gene promoter carrying minor rs2564978(T) allele associated with the severe course of influenza A(H1N1)pdm09. Using bioinformatic resources, we determined that transcription factor PU.1 can potentially bind to the CD55 promoter region containing rs2564978 in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was determined by genetic knockdown of PU.1 using small interfering RNAs under specific monocyte activation conditions.

A novel peptide encoded by circ-SLC9A6 promotes lipid dyshomeostasis through the regulation of H4K16ac-mediated CD36 transcription in NAFLD.

As the leading cause of end-stage liver disease, nonalcoholic fatty liver disease (NAFLD) is mainly induced by lipid dyshomeostasis. The translation of endogenous circular RNAs (circRNAs) is closely related to the progression of various diseases, but the involvement of circRNAs in NAFLD has not been determined. Combined high-throughput circRNA profiles were used to identify circRNAs with translational potential. The underlying molecular mechanisms were investigated by RNA sequencing, pull-down/MS and site-specific mutagenesis. In this study, we focused on circ-SLC9A6, an abnormally highly expressed circRNA in human and mouse liver tissue during NAFLD development that exacerbates metabolic dyshomeostasis in hepatocytes by encoding a novel peptide called SLC9A6-126aa in vivo and in vitro. YTHDF2-mediated degradation of m6A-modified circ-SLC9A6 was found to be essential for the regulation of SLC9A6-126aa expression. We further found that the phosphorylation of SLC9A6-126aa by AKT was crucial for its cytoplasmic localization and the maintenance of physiological homeostasis, whereas high-fat stress induced substantial translocation of unphosphorylated SLC9A6-126aa to the nucleus, resulting in a vicious cycle of lipid metabolic dysfunction. Nuclear SLC9A6-126aa promotes transcriptional activation of the target gene CD36 and enhances its occupancy of the CD36 promoter locus by regulating MOF-mediated histone H4K16 acetylation. Hepatic CD36 depletion significantly ameliorated hyperactivated MAPK signalling and lipid disturbance in SLC9A6-126aa transgenic mice. Clinically, increasing levels of SLC9A6-126aa were observed during NAFLD progression and were found to be positively correlated with the CD36 and MAPK cascades. This study revealed the role of circ-SLC9A6-derived SLC9A6-126aa in the epigenetic modification-mediated regulation of lipid metabolism. Our findings may provide promising therapeutic targets for NAFLD and new insights into the pathological mechanisms of metabolic diseases. Under normal circumstances, driven by m6A modification, YTHDF2 directly recognizes and degrades circ-SLC9A6, thereby inhibiting the translation of SLC9A6-126aa. Additionally, AKT1 phosphorylates and inhibits the nuclear translocation of SLC9A6-126aa. In NAFLD, lipid overload leads to YTHDF2 and AKT1 deficiency, ultimately increasing the expression and nuclear import of SLC9A6-126aa. Nuclear SLC9A6-126aa binds directly to the CD36 promoter and initiates CD36 transcription, which induces lipid dyshomeostasis.

MAPK4 facilitates angiogenesis by inhibiting the ERK pathway in non-small cell lung cancer.

Angiogenesis plays an important role in the occurrence and development of non-small cell lung cancer (NSCLC). The atypical mitogen-activated protein kinase 4 (MAPK4) has been shown to be involved in the pathogenesis of various diseases. However, the potential role of MAPK4 in the tumor angiogenesis of NSCLC remains unclear. Adult male C57BL/6 wild-type mice were randomly divided into the control group and p-siMAPK4 intervention group, respectively. The cell proliferation was analyzed withflow cytometry and immunofluorescence staining. The vascular density in tumor mass was analyzed by immunofluorescence staining. The expressions of MAPK4 and related signaling molecules were detected by western blot analysis and immunofluorescence staining, and so on. We found that the expression of MAPK4, which was dominantly expressed in local endothelial cells (ECs), was correlated with tumor angiogenesis of NSCLC. Furthermore, MAPK4 silencing inhibited the proliferation and migration abilities of human umbilical vein ECs (HUVECs). Global gene analysis showed that MAPK4 silencing altered the expression of multiple genes related to cell cycle and angiogenesis pathways, and that MAPK4 silencing increased transduction of the extracellular regulated protein kinases 1/2 (ERK1/2) pathwaybut not Akt and c-Jun n-terminal kinase pathways. Further analysis showed that MAPK4 silencing inhibited the proliferation and migration abilities of HUVECs cultured in tumor cell supernatant, which was accompanied with increased transduction of the ERK1/2 pathway. Clinical data analysis suggested that the higher expression of MAPK4 and CD34 were associated with poor prognosis ofpatients with NSCLC. Targeted silencing of MAPK4 in ECs using small interfering RNA driven by the CD34 promoter effectively inhibited tumor angiogenesis and growth of NSCLC in vivo. Our results reveal that MAPK4 plays an important role in the angiogenesis and development of NSCLC. MAPK4 may thus represent a new target for NSCLC.

The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

Poorly Differentiated Hepatocellular Carcinoma Cells Avoid Apoptosis by Interacting with T Cells via CD40–CD40 Ligand Linkage

Hepatocellular carcinoma (HCC) is associated with elevated soluble CD40 levels in our previous study. Here we aimed to investigate CD40's role in liver tumor progression. We examined CD40 levels in HCC patient tissues and various HCC cell lines, studying their interaction with CD4+T cells. RNA sequencing analysis was performed to explore the mechanisms of CD40 induction. Poorly differentiated HCC tumour tissues exhibited high membrane-bound CD40 (mCD40) expression, in contrast to non-tumour areas. Poorly differentiated HCC cell lines showed high expression of mCD40 with low CD40 promoter methylation which were opposite of well-differentiated ones. Solely modulating CD40 expression in HCC cells exerted no direct consequences on cell growth or appearance. Interestingly, HLFs co-cultured with activated (CD40L+) CD4+ T cells elevated CD40 levels and showed a modest 3.2% of dead cells, then increased to 10.9% underwent pre-neutralizing CD40 condition, while pre-blocking both CD40 and Integrin α5β1 concomitantly caused only 1.9% of cell death. RNA sequencing of co-cultured HLFs with activated CD4+ T cells revealed the upregulation of interferon (IFN) and immune response pathways. Elevated IFN-γ levels in the activated T cell media stimulated the JAK1/STAT3 pathway, resulting in increased CD40 expression in HLF. Collectively, CD40 expression in poorly differentiated HCC cells prevents cell death by interacting with CD40L in activated T cells. Targeting CD40 may represent a promising anticancer therapy.

Abstract 1223: KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma

Abstract Daratumumab (Dara), a first-in-class humanized monoclonal antibody targeting CD38, demonstrates notable efficacy in both newly diagnosed and relapsed/refractory multiple myeloma (MM) when administrated alone or in combination with other agents. However, the development of Dara resistance and subsequent relapse is common.To identify the tumor-intrinsic genes involved in resistance to NK-mediated cytotoxicity in the presence of Dara in MM cells, we conducted an in vitro genome-wide CRISPR-Cas9 knockout (KO) screen and found 433 genes exhibiting a positive correlation with MM cytotoxicity elicited by Dara and primary NK cells. Remarkably, 16 of these genes demonstrated a consistent correlation with cytolytic activity across the majority of the 36 cancer types in the TCGA database.The efficacy of Dara is closely linked to the expression of CD38, with nonresponders exhibiting low CD38 expression. Therefore, we conducted another CRISPR screen to identify the genes whose KO resulted in diminished CD38 expression. The genes that overlapped in these two screens are implicated in regulating both Dara-mediated ADCC and CD38 expression. Our focus turned to KDM6A among the overlapping genes. We validated that KDM6A KO significantly decreased CD38 expression and that reintroducing KDM6A into KDM6A KO cells restored the expression of CD38 and Dara-induced cytotoxicity.KDM6A demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. Our ChIP-seq data unveiled that KDM6A-KO cells exhibited an increased H3K27me3 level at the CD38 promoter area than control cells. Remarkably, this pattern was consistent with Dara-resistant cell lines, which displayed reduced CD38 expression alongside elevated levels of H3K27me3 at the CD38 promoter. Interestingly, we found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment, implying the existence of an additional mechanism. We performed RNA-seq in KDM6A KO cells and found CD48, an NK-activating ligand, was significantly repressed. Overexpression of CD48 in KDM6A KO cells resulted in a substantial increase in the secretion of Granzyme B and Perforin by NK cells and restored the activity of NK cells and re-sensitized KDM6A KO cells to Dara treatment. These data suggest that KDM6A mediates Dara-mediated ADCC sensitivity through the dual regulation of CD38 and CD48. KDM6A acts to oppose the EZH2/PRC2 complex by demethylating H3K27me3, and we found that Tazemetostat (Taze), an FDA-approved EZH2 inhibitor, increased the expression of CD38 and CD48, especially in KDM6A-KO cells. Importantly, Taze also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. In summary, our findings illuminate the dual role of KDM6A in enhancing the efficacy of Dara in MM. Imitating KDM6A function using an FDA-approved inhibitor holds promise in overcoming Dara resistance and improving patient outcomes in MM. Citation Format: Jiye Liu, Lijie Xing, Jiang Li, Kenneth Wen, Ning Liu, Yuntong Liu, Annamaria Gulla, Nikhil Munshi, Teru Hideshima, Kenneth Carl Anderson. KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1223.

The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

Injectable Supramolecular Hydrogels for In Situ Programming of Car-T Cells toward Solid Tumor Immunotherapy.

Chimeric antigen receptor (CAR)-T cell immunotherapy is approved in the treatment of hematological malignancies, but remains far from satisfactoryin solid tumor treatment due to inadequate intra-tumor CAR-T cell infiltration. Herein, an injectable supramolecular hydrogel system, based on self-assembly between cationic polymer mPEG-PCL-PEI (PPP) conjugated with T cell targeting anti-CD3e f(ab')2 fragment and α-cyclodextrin (α-CD), is designed to load plasmid CAR (pCAR) with a T cell specific CD2 promoter, which successfully achieves in situ fabrication and effective accumulation of CAR-T cells at the tumor site in humanized mice models. More importantly, due to this tumor microenvironment reprogramming, secretion of cellular inflammatory cytokines (interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) or tumor killer protein granzyme B is significantly promoted, which reverses the immunosuppressive microenvironment and significantly enhances the intra-tumor CAR-T cells and cytotoxic T cells infiltration. To the best of the current knowledge, this is a pioneer report of using injectable supramolecular hydrogel for in situ reprogramming CAR-T cells, which might be beneficial for solid tumor CAR-T immunotherapy.

Fatty acid translocase (FAT/CD36) in silver pomfret (Pampus argenteus): Molecular cloning and functional characterization

Understanding the mechanisms of lipid transport and metabolism in fish is crucial to enhance dietary lipid utilization. Here, fatty acid translocase (CD36) gene was characterized in silver pomfret (Pampus argenteus). The open reading frame of silver pomfret cd36 gene was 1395 bp, encoding 464 amino acids. The silver pomfret CD36 protein contained typical transmembrane regions and N-glycosylation modification sites, and was localized to the cytomembrane. The cd36 gene was ubiquitously expressed in all tested tissues, with the highest expression observed in brain tissue. In vivo, both fasting and short-term high-fat feeding could increase cd36 expression in intestinal tissue. In vitro, cd36 expression was induced by palmitic acid, oleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid treatment in intestinal tissue. Furthermore, dual-luciferase reporter assay results indicated that peroxisome proliferator-activated receptor gamma (PPARγ) could enhance cd36 promoter activity, and the co-expression of cd36 and pparγ was observed in EPA-incubated intestine, suggesting that EPA may regulate the expression of cd36 via PPARγ to maintain the homeostasis of intestinal lipid metabolism in silver pomfret. These results highlighted the crucial role of CD36 in silver pomfret, and suggested that the cd36 expression may be regulated by PPARγ. This study could contribute to a greater understanding of lipid metabolism and the development of effective strategies for nutrient requirements in fish.

Dual action of macrophage miR-204 confines cyclosporine A-induced atherosclerosis.

Atherosclerosis induced by cyclosporine A (CsA), an inhibitor of the calcineurin/nuclear factor of activated T cells (NFAT) pathway, is a major concern after organ transplantation. However, the atherosclerotic mechanisms of CsA remain obscure. We previously demonstrated that calcineurin/NFAT signalling inhibition contributes to atherogenesis via suppressing microRNA-204 (miR-204) transcription. We therefore hypothesised that miR-204 is involved in the development of CsA-induced atherosclerosis. ApoE-/- mice with macrophage-miR-204 overexpression were generated to determine the effects of miR-204 on CsA-induced atherosclerosis. Luciferase reporter assays and chromatin immunoprecipitation sequencing were performed to explore the targets mediating miR-204 effects. CsA alone did not significantly affect atherosclerotic lesions or serum lipid levels. However, it exacerbated high-fat diet-induced atherosclerosis and hyperlipidemia in C57BL/6J and ApoE-/- mice, respectively. miR-204 levels decreased in circulating monocytes and plaque lesions during CsA-induced atherosclerosis. The upregulation of miR-204 in macrophages inhibited CsA-induced atherosclerotic plaque formation but did not affect serum lipid levels. miR-204 limited the CsA-induced foam cell formation by reducing the expression of the scavenger receptors SR-BII and CD36. SR-BII was post-transcriptionally regulated by mature miR-204-5p via 3'-UTR targeting. Additionally, nuclear-localised miR-204-3p prevented the CsA-induced binding of Ago2 to the CD36 promoter, suppressing CD36 transcription. SR-BII or CD36 expression restoration dampened the beneficial effects of miR-204 on CsA-induced atherosclerosis. Macrophage miR-204 ameliorates CsA-induced atherosclerosis, suggesting that miR-204 may be a potential target for the prevention and treatment of CsA-related atherosclerotic side effects.

Role of SH3GLB1 in the regulation of CD133 expression in GBM cells

BackgroundGlioblastoma (GBM), a malignant brain tumor, has poor survival outcomes due to recurrence or drug resistance. We found that SH3GLB1 is a crucial factor for cells to evade temozolomide (TMZ) cytotoxicity through autophagy-mediated oxidative phosphorylation, which is associated with CD133 levels. Therefore, we propose that SH3GLB1 participate in the impact on tumor-initiating cells (TICs).MethodsThe parental, the derived resistant cell lines and their CD133+ cells were used, and the levels of the proteins were compared by western blotting. Then RNA interference was applied to observe the effects of the target protein on TIC-related features. Finally, in vitro transcription assays were used to validate the association between SH3GLB1 and CD133.ResultsThe CD133+ cells from resistant cells with enhanced SH3GLB1 levels more easily survived cytotoxic treatment than those from the parental cells. Inhibition of SH3GLB1 attenuated frequency and size of spheroid formation, and the levels of CD133 and histone 4 lysine 5 (H4K5) acetylation can be simultaneously regulated by SH3GLB1 modification. The H4K5 acetylation of the CD133 promoter was later suggested to be the mediating mechanism of SH3GLB1.ConclusionsThese data indicate that SH3GLB1 can regulate CD133 expression, suggesting that the protein plays a crucial role in TICs. Our findings on the effects of SH3GLB1 on the cells will help explain tumor resistance formation.

CD38-Specific CAR Integrated into CD38 Locus Driven by Different Promoters Causes Distinct Antitumor Activities of T and NK Cells.

The robust and stable expression of CD38 in T-cell acute lymphoblastic leukemia (T-ALL) blasts makes CD38 chimeric antigen receptor (CAR)-T/natural killer (NK) a potential therapy for T-ALL. However, CD38 expression in normal T/NK cells causes fratricide of CD38 CAR-T/NK cells. Here a "2-in-1" gene editing strategy is developed to generate fratricide-resistant locus-specific CAR-T/NK cells. CD38-specific CAR is integrated into the disrupted CD38 locus by CRISPR/Cas9, and CAR is placed under the control of either endogenous CD38 promoter (CD38KO/KI ) or exogenous EF1α promoter (CD38KO/KI EF1α). CD38 knockout reduces fratricide and allows the expansion of CAR-T cells. Meanwhile, CD38KO/KI EF1α results in higher CAR expression than CD38KO/KI in both CAR-T and CAR-NK cells. In a mouse T-ALL model, CD38KO/KI EF1α CAR-T cells eradicate tumors better than CD38KO/KI CAR-T cells. Surprisingly, CD38KO/KI CAR-NK cells show superior tumor control than CD38KO/KI EF1α CAR-NK cells. Further investigation reveals that endogenous regulatory elements in NK cells lead to higher expression of CD38 CAR than in T cells, and the expression levels of CAR affect the therapeutic outcome of CAR-T and CAR-NK cells differently. Therefore, these results support the efficacy of CD38 CAR-T/NK against T-ALL and demonstrate that the "2-in-1" strategy can resolve fratricide and enhance tumor eradication, paving the way for clinical translation.

Early-life stress perturbs the epigenetics of Cd36 concurrent with adult onset of NAFLD in mice

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the U.S. and worldwide. The roles of early postnatal life stress (EPLS) and the fatty acid translocase (CD36) on the pathogenesis of adult-onset NAFLD remain unknown. We hypothesized that EPLS, in the form of neonatal maternal separation (NMS), would predispose mice towards developing adult NAFLD, increase hepatic CD36 expression, and differentially methylate Cd36 promoter concurrently.MethodsNMS was performed on mice from postnatal day 1 to 21 and a high-fat/high-sucrose (HFS) diet was started at 4 weeks of age to generate four experimental groups: Naive-control diet (CD), Naive-HFS, NMS-CD, and NMS-HFS.ResultsNMS alone caused NAFLD in adult male mice at 25 weeks of age. The effects of NMS and HFS were generally additive in terms of NAFLD, hepatic Cd36 mRNA levels, and hepatic Cd36 promoter DNA hypomethylation. Cd36 promoter methylation negatively correlated with Cd36 mRNA levels. Two differentially methylated regions (DMRs) within Cd36 promoter regions appeared to be vulnerable to NMS in the mouse.ConclusionsOur findings suggest that NMS increases the risk of an individual, particularly male, towards NAFLD when faced with a HFS diet later in life.ImpactThe key message of this article is that neonatal maternal separation and a postweaning high-fat/high-sucrose diet increased the risk of an individual, particularly male, towards NAFLD in adult life.What this study adds to the existing literature includes the identification of two vulnerable differentially methylated regions in hepatic Cd36 promoters whose methylation levels very strongly negatively correlated with Cd36 mRNA.The impact of this article is that it provides an early-life environment-responsive gene/promoter methylation model and an animal model for furthering the mechanistic study on how the insults in early-life environment are “transmitted” into adulthood and caused NAFLD.

Reverse effect of Semaphorin-3F on rituximab resistance in diffuse large B-cell lymphoma via the Hippo pathway.

Exploring the underlying mechanism of rituximab resistance is critical to improve the outcomes of patients with diffuse large B-cell lymphoma (DLBCL). Here, we tried to identify the effects of the axon guidance factor semaphorin-3F (SEMA3F) on rituximab resistance as well as its therapeutic value in DLBCL. The effects of SEMA3F on the treatment response to rituximab were investigated by gain- or loss-of-function experiments. The role of the Hippo pathway in SEMA3F-mediated activity was explored. A xenograft mouse model generated by SEMA3F knockdown in cells was used to evaluate rituximab sensitivity and combined therapeutic effects. The prognostic value of SEMA3F and TAZ (WW domain-containing transcription regulator protein 1) was examined in the Gene Expression Omnibus (GEO) database and human DLBCL specimens. We found that loss of SEMA3F was related to a poor prognosis in patients who received rituximab-based immunochemotherapy instead of chemotherapy regimen. Knockdown of SEMA3F significantly repressed the expression of CD20 and reduced the proapoptotic activity and complement-dependent cytotoxicity (CDC) activity induced by rituximab. We further demonstrated that the Hippo pathway was involved in the SEMA3F-mediated regulation of CD20. Knockdown of SEMA3F expression induced the nuclear accumulation of TAZ and inhibited CD20 transcriptional levels via direct binding of the transcription factor TEAD2 and the CD20 promoter. Moreover, in patients with DLBCL, SEMA3F expression was negatively correlated with TAZ, and patients with SEMA3F low TAZ high had a limited benefit from a rituximab-based strategy. Specifically, treatment of DLBCL cells with rituximab and a YAP/TAZ inhibitor showed promising therapeutic effects in vitro and in vivo . Our study thus defined a previously unknown mechanism of SEMA3F-mediated rituximab resistance through TAZ activation in DLBCL and identified potential therapeutic targets in patients.

Single cell analysis of the localization of the hematopoietic stem cells within the bone marrow architecture identifies niche-specific proliferation dynamics.

Hematopoietic stem cells (HSC) reside in the bone marrow (BM) in specialized niches which provide support for their self-replication and differentiation into the blood cells. Recently, numerous studies using sophisticated molecular and microscopic technology have provided snap-shots information on the identity of the BM niches in mice. In adults, HSC are localized around arterioles and sinusoids/venules whereas in juvenile mice they are in close to the osteoblasts. However, although it is well recognized that in mice the nature of the hematopoietic niche change with age or after exposure to inflammatory insults, much work remains to be done to identify changes occurring under these conditions. The dynamic changes occurring in niche/HSC interactions as HSC enter into cycle are also poorly defined. We exploit mice harboring the hCD34tTA/Tet-O-H2BGFP transgene to establish the feasibility to assess interactions of the HSC with their niche as they cycle. In this model, H2BGFP expression is driven by the TET trans-activator under the control of the human CD34 promoter which in mice is active only in the HSC. Since Doxycycline inhibits TET, HSC exposed to this drug no longer express H2BGFP and loose half of their label every division allowing establishing the dynamics of their first 1-3 divisions. To this aim, we first validated user-friendly confocal microscopy methods to determine HSC divisions by hemi-decrement changes in levels of GFP expression. We then tracked the interaction occurring in old mice between the HSC and their niche during the first HSC divisions. We determined that in old mice, most of the HSC are located around vessels, both arterioles which sustain quiescence and self-replication, and venules/sinusoids, which sustain differentiation. After just 1 week of exposure to Doxycycline, great numbers of the HSC around the venules lost most of their GFP label, indicating that they had cycled. By contrast, the few HSC surrounding the arterioles retained maximal levels of GFP expression, indicating that they are either dormant or cycle at very low rates. These results reveal that in old mice, HSC cycle very dynamically and are biased toward interactions with the niche that instructs them to differentiate.

All-trans retinoic acid improves NSD2-mediated RARα phase separation and efficacy of anti-CD38 CAR T-cell therapy in multiple myeloma

BackgroundImmunotherapies targeting CD38 have demonstrated salient efficacy in relapsed/refractory multiple myeloma (MM). However, loss of CD38 antigen and outgrowth of CD38 negative plasma cells have emerged as a major obstacle...

The genetic and epigenetic regulation of CD55 and its pathway analysis in colon cancer.

CD55 plays an important role in the development of colon cancer. This study aims to evaluate the expression of CD55 in colon cancer and discover how it is regulated by transcriptional factors and miRNA. The expression of CD55 was explored by TIMER2.0, UALCAN, and Human Protein Atlas (HPA) databases. TRANSFAC and Contra v3 were used to predict the potential binding sites of transcription factors in the CD55 promoter. TargetScan and starBase v2.0 were used to predict the potential binding ability of miRNAs to the 3' untranslated region (3'UTR) of CD55. SurvivalMeth was used to explore the differentially methylated sites in the CD55 promoter. Western blotting was used to detect the expression of TFCP2 and CD55. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were performed to determine the targeting relationship of TFCP2, NF-κB, or miR-27a-3p with CD55. CD55-related genes were explored by constructing a protein-protein interaction (PPI) network and performing pathway analysis by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). CD55 was highly expressed in colon cancer tissues. The mRNA and protein expression levels of TFCP2 were reduced by si-TFCP2. NF-κB mRNA was obviously reduced by NF-κB inhibitor and increased by NF-κB activator. CD55 protein was also inhibited by miR-27a-3p. Dual-luciferase reporter assays showed that after knocking down TFCP2 or inhibiting NF-κB, the promoter activity of CD55 was decreased by 21% and 70%, respectively; after activating NF-κB, the promoter activity of CD55 increased by 2.3 times. As TFCP2 or NF-κB binding site was mutated, the transcriptional activity of CD55 was significantly decreased. ChIP assay showed that TFCP2 and NF-κB combined to the promoter of CD55. The luciferase activity of CD55 3'UTR decreased after being co-transfected with miR-27a-3p mimics and increased by miR-27a-3p antagomir. As the miR-27a-3p binding site was mutated, we did not find any significant effect of miR-27a-3p on reporter activity. PPI network assay revealed a set of CD55-related genes, which included CFP, CFB, C4A, and C4B. GO and KEGG analyses revealed that the target genes occur more frequently in immune-related pathways. Our results indicated that CD55 is regulated by TFCP2, NF-κB, miR-27a-3p, and several immune-related genes, which in turn affects colon cancer.

A Novel in Duck Myoblasts: The Transcription Factor Retinoid X Receptor Alpha (RXRA) Inhibits Lipid Accumulation by Promoting CD36 Expression.

Retinoid X receptor alpha (RXRA) is a well-characterized factor that regulates lipid metabolism; however, the regulatory mechanism in muscle cells of poultry is still unknown. The overexpression and the knockdown of RXRA in myoblasts (CS2 cells), RT-PCR, and western blotting were used to detect the expression levels of genes and proteins related to PPAR-signaling pathways. Intracellular triglycerides (TGs), cholesterol (CHOL), and nonesterified free fatty acids (NEFAs) were detected by the Elisa kit. Fat droplets were stained with Oil Red O. The double-fluorescein reporter gene and chromatin immunoprecipitation (CHIP) were used to verify the relationship between RXRA and candidate target genes. The RXRA gene was highly expressed in duck breast muscle, and its mRNA and its protein were reduced during the differentiation of CS2 cells. The CS2 cells, with the overexpression of RXRA, showed reduced content in TGs, CHOL, NEFAs, and lipid droplets and upregulated the mRNA expression of CD36, ACSL1, and PPARG genes and the protein expression of CD36 and PPARG. The knockdown of RXRA expression in CS2 cells enhanced the content of TGs, CHOL, NEFAs, and lipid droplets and downregulated the mRNA and protein expression of CD36, ACLS1, ELOVL6, and PPARG. The overexpression of the RXRA gene, the activity of the double-luciferase reporter gene of the wild-type CD36 promoter was higher than that of the mutant type. RXRA bound to -860/-852 nt, -688/-680 nt, and -165/-157 nt at the promoter region of CD36. Moreover, the overexpression of CD36 in CS2 cells could suppress the content of TGs, CHOL, NEFAs, and lipid droplets, while the knockdown expression of CD36 increased the content of TGs, CHOL, NEFAs, and lipid droplets. In this study, the transcription factor, RXRA, inhibited the accumulation of TGs, CHOL, NEFAs, and fat droplets in CS2 cells by promoting CD36 expression.

Hypoxia-Inducible Factor 2α Attenuates Renal Ischemia-Reperfusion Injury by Suppressing CD36-Mediated Lipid Accumulation in Dendritic Cells in a Mouse Model.

Hypoxia and hypoxia-inducible factors (HIFs) play essential and multiple roles in renal ischemia-reperfusion injury (IRI). Dendritic cells (DCs) comprise a major subpopulation of the immunocytes in the kidney and are key initiators and effectors of the innate immune responses after IRI. The role of HIF-2α in DCs remains unclear in the context of renal IRI. To investigate the importance of HIF-2α in DCs upon renal IRI, we examined the effects of DC-specific HIF-2α ablation in a murine model. Bone marrow-derived DCs (BMDCs) from DC-specific HIF-2α-ablated mice and wild-type mice were used for functional studies and transcriptional profiling. DC-specific ablation of HIF-2α led to hyperactivation of natural killer T (NKT) cells, ultimately exacerbating murine renal IRI. HIF-2α deficiency in DCs triggered IFN-γ and IL-4 production in NKT cells, along with upregulation of type I IFN and chemokine responses that were critical for NKT cell activation. Mechanistically, loss of HIF-2α in DCs promoted their expression of CD36, a scavenger receptor for lipid uptake, increasing cellular lipid accumulation. Furthermore, HIF-2α bound directly to a reverse hypoxia-responsive element (rHRE) in the CD36 promoter. Importantly, CD36 blockade by sulfo-N-succinimidyl oleate (SSO) reduced NKT cell activation and abolished the exacerbation of renal IRI elicited by HIF-2α knockout. Our study reveals a previously unrecognized role of the HIF-2α/CD36 regulatory axis in rewiring DC lipid metabolism under IRI-associated hypoxia. These findings suggest a potential therapeutic target to resolve long-standing obstacles in treatment of this severe complication.

EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies. Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019). Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance. We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression. Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38neg and CD38low cell lines compared to CD38highcells. In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3). Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro. Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.