Short Hairpin RNA Interference Research Articles

CD155 on Multiple Myeloma Cells Is Epigenetically Regulated, Reduce Patients Survival and Compromises the Action of Novel Immunotherapies

Epigenetics, especially DNA methylation, is a key molecular regulator of gene expression. This regulation is important for cytotoxic cell activation (i.e., T-cells and NK cells) and is crucial for the clearance of malignant cells. Immune checkpoint (IC) events mediate cytotoxicity, one IC receptor involved in the process is CD155. Such receptor interacts primarily with TIGIT on cytotoxic cells and induces the inhibition of cytotoxicity. Multiple myeloma (MM) represents the 10% of all hematological malignancies and it has been described that MM cells hinder cytotoxic cell activation by inhibition and exhaustion. Furthermore, novel immunotherapeutic strategies such as Bispecific T-cell Engagers (BiTE) and Chimeric Antigen Receptor (CAR) therapies are being evaluated in MM treatment, with promising results. Nevertheless, they will confront a hostile environment. This is a very active field with frequent descriptions of new targets and therapeutic approaches being studied each day. We investigated whether CD155 is epigenetically regulated in MM and its importance in inducing inhibition of anti-tumor response in the context of novel immunotherapies through the CD155/TIGIT axis. To achieve our objectives, we focused on the promoter region of CD155 (the region up to 1500 base pairs upstream of the TSS) and characterized it at DNA, RNA and protein levels through bisulfite sequencing, quantitative PCR, and flow cytometry. We confirmed CD155 as an epigenetically regulated gene, thus demonstrating an existing correlation between CD155 promoter methylation and gene expression. In vitro treatment with azacytidine was performed to assess the active repression of CD155 expression by DNA methylation. Six different MM cell lines were characterized (two not expressing: AMO-1, KMS-12-BM, four expressing: RPMI-8226, MM.1S, EJM and JJN-3). From there, we created CD155 depletion models on expressing cell lines by short hairpin RNA interference and developed an in vitro coculture system with T-cells (from healthy donors). We observed a loss of expression between the 60% to 90% (when compared with controls). Depletion of CD155 did not significantly impact cell growth, apoptosis, or cell cycle. On the coculture with healthy T-cells, more cytotoxicity was detected against CD155-depleted cells (in RPMI-8226 Mann-Whitney P=0.020; JJN-3 and EJM P<0.001). To incorporate novel immunotherapeutic strategies, we carried out the same cocultures with 100pg/mL of anti-BCMA/CD3 BiTE, against anti-BCMA CAR-T or anti-BCMA CAR-NK cells (for RPMI-8226 and JJN-3 Mann-Whitney P=0.008). Coculture combinations were also performed in the presence of 10μg/ml of neutralizing αTIGIT (RPMI-8226, Mann-Whitney P =0.005; JJN-3 P=0.004) and/or αPD1 antibody. When novel immunotherapy approaches were included in the system, we observed how the depletion of CD155 was related to a cytotoxicity increase in all cases. In the presence of neutralizing αTIGIT, cytotoxicity was increased against CD155-expressing cells, while we observed no difference against CD155-depleted cells. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and the antibody combination showed a synergic effect in CD155 expressing cells. Finally, we analyzed the existence of a correlation between CD155 gene expression and survival in newly diagnosed MM samples from CoMMpass project public data (N= 793). These data supported our results as the newly diagnosed MM patients with higher expression of CD155 had shorter overall survival than lower expressing ones (Logrank test P<0.001). In summary, in the context of MM, the expression of CD155 is regulated by the methylation status of its promoter region. Expression of CD155 effectively reduces cytotoxic cell response, which is mediated by the interaction of CD155 and TIGIT. The addition of αTIGIT and αPD1 validated that the CD155 cytotoxic cell inhibition is mediated in part by TIGIT interaction, independently of PD1/PD-L1 axis. This interaction seems to impact patients treated with current therapies, since their outcome correlates with CD155 expression. Based on our results, novel immunotherapies focused on enhancing immune cytotoxicity will benefit from CD155/TIGIT axis blockade. These results warrant further investigation on CD155 as a biomarker and target for novel immunotherapies for MM and other malignancies.

Ribonucleicacid interference or small molecule inhibition of Runx1 in the border zone prevents cardiac contractile dysfunction following myocardial infarction.

Myocardial infarction (MI) is a major cause of death worldwide. Effective treatments are required to improve recovery of cardiac function following MI, with the aim of improving patient outcomes and preventing progression to heart failure. The perfused but hypocontractile region bordering an infarct is functionally distinct from the remote surviving myocardium and is a determinant of adverse remodelling and cardiac contractility. Expression of the transcription factor RUNX1 is increased in the border zone 1-day after MI, suggesting potential for targeted therapeutic intervention. This study sought to investigate whether an increase in RUNX1 in the border zone can be therapeutically targeted to preserve contractility following MI. In this work we demonstrate that Runx1 drives reductions in cardiomyocyte contractility, calcium handling, mitochondrial density, and expression of genes important for oxidative phosphorylation. Both tamoxifen-inducible Runx1-deficient and essential co-factor common β subunit (Cbfβ)-deficient cardiomyocyte-specific mouse models demonstrated that antagonizing RUNX1 function preserves the expression of genes important for oxidative phosphorylation following MI. Antagonizing RUNX1 expression via short-hairpin RNA interference preserved contractile function following MI. Equivalent effects were obtained with a small molecule inhibitor (Ro5-3335) that reduces RUNX1 function by blocking its interaction with CBFβ. Our results confirm the translational potential of RUNX1 as a novel therapeutic target in MI, with wider opportunities for use across a range of cardiac diseases where RUNX1 drives adverse cardiac remodelling.

Regulation of neural stem cell self-renewal, proliferation and differentiation by the RhoA guanine nucleotide exchange factor Arhgef 1

The role of the Arhgef1 as a RhoA-specific guanine nucleotide exchange factor has been widely investigated in the immune system. Our previous findings reveal that Arhgef 1 is highly expressed in neural stem cells (NSCs) and controls the process of neurite formation. However, the functional role of Arhgef 1 in NSCs remains poorly understood. In order to investigate the role of Arhgef 1 in NSCs, Arhgef 1 expression in NSCs was reduced by using lentivirus-mediated short hairpin RNA interference. Our results indicate that down-regulated expression of Arhgef 1 reduced the self-renewal, proliferation capacity of NSCs and affect cell fate determination. In addition, the comparative transcriptome analysis from RNA-seq data determines the mechanisms of deficits in Arhgef 1 knockdown NSCs. Altogether, our present studies show that Arhgef 1 down-regulation leads to interruption of the cell cycle procession. The importance of Arhgef 1 for regulating self-renewal, proliferation and differentiation in NSCs is reported for the first time.

Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice.

The mitochondrial permeability transition pore is a nonspecific transmembrane channel. Inhibition of mitochondrial permeability transition pore opening has been shown to alleviate mitochondrial swelling, calcium overload, and axonal degeneration. Cyclophilin D is an important component of the mitochondrial permeability transition pore. Whether cyclophilin D participates in mitochondrial impairment and axonal injury after intracerebral hemorrhage is not clear. In this study, we established mouse models of intracerebral hemorrhage in vivo by injection of autologous blood and oxyhemoglobin into the striatum in Thy1-YFP mice, in which pyramidal neurons and axons express yellow fluorescent protein. We also simulated intracerebral hemorrhage in vitro in PC12 cells using oxyhemoglobin. We found that axonal degeneration in the early stage of intracerebral hemorrhage depended on mitochondrial swelling induced by cyclophilin D activation and mitochondrial permeability transition pore opening. We further investigated the mechanism underlying the role of cyclophilin D in mouse models and PC12 cell models of intracerebral hemorrhage. We found that both cyclosporin A inhibition and short hairpin RNA interference of cyclophilin D reduced mitochondrial permeability transition pore opening and mitochondrial injury. In addition, inhibition of cyclophilin D and mitochondrial permeability transition pore opening protected corticospinal tract integrity and alleviated motor dysfunction caused by intracerebral hemorrhage. Our findings suggest that cyclophilin D is used as a key mediator of axonal degeneration after intracerebral hemorrhage; inhibition of cyclophilin D expression can protect mitochondrial structure and function and further alleviate corticospinal tract injury and motor dysfunction after intracerebral hemorrhage. Our findings provide a therapeutic target for preventing axonal degeneration of white matter injury and subsequent functional impairment in central nervous diseases.

Fc receptor-like 1 (FCRL1) is a novel biomarker for prognosis and a possible therapeutic target in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma, which can involve various types of mature B-cells. Considering that the incidence of DLBCL has increased, additional research is required to identify novel and effective prognostic and therapeutic molecules. Fc receptor-like 1 (FCRL1) acts as an activation co-receptor of human B-cells. Aberrant expression of this molecule has been reported in a number of B-cell-related disorders. Moreover, the clinical significance and prognosis value of FCRL1 in DLBCL are not completely identified. In this study, the expression levels of FCRL1 were determined in thirty patients with DLBCL and 15 healthy controls (HCs). In addition, the correlation between FCRL1 expressions with clinicopathological variables of DLBCL patients were examined. Then, the potential roles of FCRL1 in proliferation, apoptosis, and cell cycle distribution of B-cells from DLBCL patients were determined using flow cytometry analysis, after knockdown of this marker using retroviral short hairpin RNA interference. Quantitative real time-PCR, western blotting, and enzyme-linked immunosorbent assay were also used to identify the possible effects of FCRL1 knockdown on the expression levels of BCL-2, BID, BAX, intracellular signaling pathway PI3K/p-Akt, and p65 nuclear factor-kappa B (NF-κB) in the B-cells of DLBCL. Statistical analysis revealed higher levels of FCRL1 expression in the B-cells of DLBCL patients compared to HCs at both protein and mRNA levels. A positive correlation was observed between the FCRL1 expression and some clinicopathological parameters of DLBCL patients. In addition, FCRL1 knockdown significantly decreased cell proliferation and stimulated apoptosis as well as G1 cell cycle arrest in the B-cells of DLBCL patients. The levels of p65 NF-κB and PI3K/p-Akt expressions were markedly reduced after knockdown of FCRL1 expression. These results suggested that FCRL1 could be a potential novel biomarker for prognosis and/or a possible effective therapeutic target for treatment of patients with DLBCL.

Therapeutic Potential of Combinative shRNA-Encoded Lentivirus-Mediated Gene Silencing to Accelerate Somatosensory Recovery After Spinal Cord Trauma.

Neuropathic pain following spinal cord injury (SCI) remains a difficult problem that affects more than 80% of SCI patients. Growing evidence indicates that neuroinflammatory responses play a key role in neuropathic pain after SCI. Short hairpin RNA (shRNA) interference is an efficient tool for the knockdown of disease-related specific gene expression after SCI, yet insufficient data is available to establish guidelines. In this study, we have constructed the transient receptor potential ankyrin 1 (TRPA1) shRNA encoded-lentiviral vector (LV-shTRPA1) and P38 MAPK shRNA encoded-lentiviral vector (LV-shP38) to investigate the silencing effects of shRNAs and their ability to reprogram the neuroinflammatory responses, thereby enhancing somatosensory recovery after SCI. Our in vitro data employing HEK293-FT and activated macrophages demonstrated that delivered LV-shRNAs showed high transduction efficacy with no cytotoxicity. Furthermore, a combination of LV-shP38 and LV-shTRPA1 was found to be most effective at suppressing target genes, cutting the expression of pro-inflammatory and pro-nociceptive factors in the dorsal horn of the spinal cord and dorsal root ganglia, thus contributing to the alleviation of neuronal hypersensitivities after SCI. Overall, our data demonstrated that the combination LV-shP38/shTRPA1 produced a synergistic effect for immunomodulation and reduced neuropathic pain with a favorable risk-to-benefit ratio. Collectively, our LV-mediated shRNA delivery will provide an efficient tool for gene silencing therapeutic approaches to treat various incurable disorders.

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18β-glycyrrhetinic acid (18β-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18β-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18β-GA in Treg of ITP patients. Furthermore, we found that 18β-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18β-GA treatment. In addition, 18β-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18β-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A.

BACKGROUNDColorectal cancer (CRC) is one of the most common malignant tumors worldwide. The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative. Immunohistochemical analysis has revealed high expression of centromere protein K (CENPK) in CRC. However, the role of CENPK in the progression of CRC is not well characterized.AIMTo evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A (CUL4A) in RKO and HCT116 cells.METHODSHuman colon cancer samples were collected and tested using a human gene expression chip. We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis. In vitro experiments verified the function of this gene. We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction (qPCR), western blot, and flow cytometry. The effect of short hairpin RNA (shRNA) virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging. To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells, we performed a series of in vitro experiments, using qPCR, western blot, MTT assay, and flow cytometry.RESULTSWe demonstrated overexpression of CENPK in human colon cancer samples. CENPK was an independent risk factor in patients with CRC. The downstream genes FBX32, CUL4A, and Yes-associated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells. Significantly delayed xenograft tumor emergence, slower growth rate, and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group. The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo. The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells, with overexpression of the CUL4A.CONCLUSIONWe indicated a potential role of CENPK in promoting tumor proliferation, and it may be a novel diagnostic and prognostic biomarker for CRC.

Mesenchymal stem cells regulate M1 polarization of peritoneal macrophages through the CARD9-NF-κB signaling pathway in severe acute pancreatitis.

Macrophages release large numbers of proinflammatory cytokines that trigger inflammatory cascade reactions, which promote the rapid development of severe acute pancreatitis (SAP) from local to systemic inflammation. The ability of mesenchymal stem cells (MSCs) to suppress inflammation is related to inhibition of M1 polarization of macrophages. Our previous studies revealed that caspase recruitment domain protein 9 (CARD9) was involved in SAP inflammation and activation of the CARD9-NF-κB signaling pathway plays an important proinflammatory role in SAP. At present, there is no effective treatment to control the inflammatory response in SAP. Therefore, the aim of the present study was to determine whether MSCs regulate the polarization of macrophages through the CARD9-NF-κB signaling pathway in SAP. Short hairpin RNA interference technology and coculture in vitro were used to assess the activation status of the CARD9-NF-κB signal pathway in macrophages. Furthermore, flow cytometry was used to determine the polarization state of macrophages. The results showed MSCs inhibited CARD9 expression in vivo and in vitro (P < .05), alleviated inflammation induced by proinflammatory cytokines, and inhibited the phosphorylation of NF-κB in macrophages both in vivo and in vitro. Meanwhile, MSCs downregulated the CARD9-NF-κB signal pathway and inhibited M1 polarization of macrophages. In conclusion, MSCs regulate M1 polarization of peritoneal macrophages through the CARD9-NF-κB signaling pathway in SAP and transplantation of MSCs presents an effective treatment option for SAP.

Impact of SLC43A3/ENBT1 Expression and Function on 6-Mercaptopurine Transport and Cytotoxicity in Human Acute Lymphoblastic Leukemia Cells.

6-Mercaptopurine (6-MP) is used extensively in the treatment of acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases. Our laboratory determined previously, using a recombinant HEK293 cell model, that the SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) transports 6-MP into cells and significantly impacts the cytotoxicity of 6-MP in that model. To further investigate the clinical relevance of this finding, we now extend this work to an analysis of the impact of SLC43A3/ENBT1 expression and function on 6-MP uptake and cytotoxicity in leukemic lymphoblasts, the therapeutic target of 6-MP in ALL. A panel of ALL cell lines was assessed for SLC43A3/ENBT1 expression, ENBT1 function, and sensitivity to 6-MP. There was a significant difference in SLC43A3 expression among the cell lines that positively correlated with the rate of ENBT1-mediated 6-MP uptake. Cells with the lowest expression of SLC43A3 (SUP-B15: Vmax = 22± 5 pmol/µl per second) were also significantly less sensitive to 6-MP-induced cytotoxicity than were the highest expressing cells (ALL-1: Vmax = 69 ± 10 pmol/µl per second). Furthermore, knockdown of ENBT1 using short hairpin RNA interference (shRNAi) in RS4;11 cells caused a significant decrease in ENBT1-mediated 6-MP uptake (Vmax: RS4;11 = 40 ± 4 pmol/µl per second; RS4;11 shRNAi = 26 ± 3 pmol/µl per Second) and 6-MP cytotoxicity (EC50: RS4;11 = 0.58 ± 0.05 µM; RS4;11 shRNAi =1.44 ± 0.59 µM). This study showed that ENBT1 is a major contributor to 6-MP uptake in leukemia cell lines and may prove to be a biomarker for the therapeutic efficacy of 6-MP in patients with ALL. SIGNIFICANCE STATEMENT: This study shows that SLC43A3-encoded equilibrative nucleobase transporter 1 is responsible for the transport of 6-mercaptopurine (6-MP) into leukemia cells and that its level of expression can impact the cytotoxicity of 6-MP. Further studies are warranted to investigate the therapeutic implications in patient populations.

Identification of sodium homeostasis genes in Camelus bactrianus by whole transcriptome sequencing.

Salt dietary intake is tightly coupled to human health, and excessive sodium can cause strokes and cardiovascular diseases. Research into the renal medulla of camels exhibiting high salt resistance may aid identification of the mechanisms governing resistance to high salinity. In this study, we used RNA sequencing (RNA‐seq) to show that in the renal medulla of camels under salt stress, 22 mRNAs, 2 long noncoding RNAs (lncRNAs), and 31 microRNAs (miRNAs) exhibited differential expression compared with the free salt‐intake diet group. Using fluorescence in situ hybridization and dual‐luciferase reporter assays, we demonstrated that the lncRNA LNC003834 can bind miRNA‐34a and thereby relieve suppression of the salt‐absorption‐inhibiting SLC14A1 mRNA from miRNA‐34a, suggesting that the above lncRNA‐miRNA‐mRNA act as competing endogenous RNAs (ceRNAs). We subsequently performed short hairpin RNA and small RNA interference and reactive oxygen species (ROS) detection assays to show that SLC6A1, PCBP2, and PEX5L can improve the antioxidation capacity of renal medulla cells of camel by decreasing ROS levels. Our data suggest that camels achieve sodium homeostasis through regulating the expression of salt‐reabsorption‐related genes in the renal medulla, and this involves ceRNAs (SLC14A1 mRNA, LNC003834, and miRNA‐34a) and antioxidant genes (SLC6A1, PCBP2, and PEX5L). These data may assist in the development of treatments for diseases induced by high salt diets.

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway.

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.

HER4 Promotes Osteosarcoma Progression and Predicts Poor Prognosis through the PTEN-PI3K/AKT Pathway.

Studies have reported a relationship between human epidermal growth factor receptor 4 (HER4), a ubiquitously expressed and unique member of the ErbB family, and clinicopathological features of osteosarcoma. However, further investigation is warranted. HER4 expression was analyzed by quantitative reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry. The relationship between HER4 expression and the prognosis of patients with osteosarcoma was determined by constructing a Kaplan-Meier curve. Cell viability and proliferation were investigated by MTT and colony formation assays. The mechanism underlying HER4-modulated proliferation and invasion/migration of osteosarcoma cells was determined by short hairpin RNA (shRNA) interference, colony formation, migration, invasion, and western blotting experiments. Spheroid formation assay and CD133+ cell populations were used to examine HER4-induced stem-like traits.The present findings revealed that HER4 was overexpressed in both osteosarcoma cells and tissues. Moreover, this overexpression was associated with high Enneking stage, metastasis, and recurrence. Sh-HER4 showed obviously suppressed cell viability, colony formation, and invasion/migration. In addition, knockdown of HER4 markedly attenuated the spheroid size and proportion of CD133-positive cells, as well as the expression of stemness markers. Sh-HER4 also reduced the tumor size, downregulated the expression of phosphorylated-PI3K (p-PI3K) and p-AKT, and increased that of p-phosphatase and tensin homolog (p-PTEN) in mouse tissue. From a mechanistic perspective, HER4 knockdown activated p-PTEN and suppressed p-PI3K and p-AKT expression. HER4 promoted osteosarcoma progression through inactivation of the PTEN-PI3K/AKT pathway.Taken together, the results indicate that HER4 represents a novel target in osteosarcoma progression and stemness modulation, and may be of value for the development of treatments against osteosarcoma.

Super Enhancer-Mediated Upregulation of HJURP Promotes Growth and Survival of t(4;14)-Positive Multiple Myeloma.

Multiple myeloma is an incurable malignancy with marked clinical and genetic heterogeneity. The cytogenetic abnormality t(4;14) (p16.3;q32.3) confers aggressive behavior in multiple myeloma. Recently, essential oncogenic drivers in a wide range of cancers have been shown to be controlled by super-enhancers (SE). We used chromatin immunoprecipitation sequencing of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs in t(4;14)-translocated multiple myeloma. The histone chaperone HJURP was aberrantly overexpressed in t(4;14)-positive multiple myeloma due to transcriptional activation by a distal SE induced by the histone lysine methyltransferase NSD2. Silencing of HJURP with short hairpin RNA or CRISPR interference of SE function impaired cell viability and led to apoptosis. Conversely, HJURP overexpression promoted cell proliferation and abrogated apoptosis. Mechanistically, the NSD2/BRD4 complex positively coregulated HJURP transcription by binding the promoter and active elements of its SE. In summary, this study introduces SE profiling as an efficient approach to identify new targets and understand molecular pathogenesis in specific subtypes of cancer. Moreover, HJURP could be a valuable therapeutic target in patients with t(4;14)-positive myeloma. SIGNIFICANCE: A super-enhancer screen in t(4;14) multiple myeloma serves to identify genes that promote growth and survival of myeloma cells, which may be evaluated in future studies as therapeutic targets.

Targeted CHO cell engineering approaches can reduce HCP-related enzymatic degradation and improve mAb product quality.

Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable short hairpin RNA interference or monoallelic deletion of the target gene using CRISPR-Cas9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification.

Effect of fibulin-5 on aldosterone-induced apoptosis in human ascending aortic smooth muscle cells.

The aim of the current study was to investigate the effect of aldosterone on apoptosis in human aortic smooth muscle cells (HA-VSMC) and to determine the role of fibulin-5 in the aldosterone-induced apoptosis of HA-VSMC cells. Through the construction of a fibulin-5 eukaryotic overexpression vector and a short hairpin RNA interference plasmid, fibulin-5 was overexpressed and silenced, respectively. The role of fibulin-5 in the aldosterone-induced apoptosis of HA-VSMC was subsequently determined. The overexpression of fibulin-5 inhibited the apoptosis of cells, particularly at low concentrations of aldosterone; a smaller effect on apoptosis was induced by high concentrations of aldosterone. fibulin-5 knockdown promoted the apoptosis of cells induced by high concentrations of aldosterone but had a smaller effect on the apoptosis of cells induced by low concentrations of aldosterone. Therefore, the results of the current study indicate that fibulin-5 inhibits the aldosterone-induced apoptosis of HA-VSMC cells and that this effect may be altered by changing the aldosterone concentration.

MACC1 Is Associated With Epithelial-Mesenchymal Transition and Can Predict Poor Prognosis in Nasopharyngeal Carcinoma.

BackgroundGiven the reported correlation between the oncogene metastasis-associated in colon cancer 1 (MACC1) and nasopharyngeal carcinoma (NPC), as well as between MACC1 and epithelial–mesenchymal transition (EMT), we speculated that EMT is a likely causative link between MACC1 expression and poor NPC prognosis. Thus, we aim to clarify the relationship between MACC1 and EMT in NPC prognosis.Material and MethodsWe performed immunohistochemical examination of tissue sections from 128 NPC patients that were divided into six groups corresponding to high and low protein expression of MACC1 and two EMT-related proteins, vimentin and E-cadherin, and Kaplan–Meier (KM) survival analyses were performed.ResultsKM survival analysis showed that upregulation of MACC1 and vimentin and downregulation of E-cadherin were significantly associated with reduced survival in NPC. Short hairpin RNA (shRNA) interference and immunoblotting in the NPC cell line HNE-1 led to increased E-cadherin but decreased vimentin levels. MACC1 overexpression was significantly correlated with poor 5-year overall survival, metastasis-free survival, and disease-free survival (P<0.05) but not with poor relapse-free survival (P>0.05). Univariate analyses revealed that MACC1, E-cadherin, and vimentin levels along with T and N tumor classifications and cancer staging are significant prognostic factors of NPC (P<0.05).ConclusionOur findings showed the association between MACC1 and EMT in NPC malignancy and support the role of MACC1 as a prognostic biomarker and molecular target for NPC treatment.

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.

Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

Study on the attenuated effect of Ginkgolide B on ferroptosis in high fat diet induced nonalcoholic fatty liver disease

Ginkgolide B (GB), a main constituent of Ginkgo biloba extracts, reduces hepatic lipid accumulation and ameliorates nonalcoholic fatty liver disease (NAFLD) in obese mice, but the potential mechanism is unclear. Here we investigated the attenuated effects of GB on the disorder of lipid metabolism, oxidative stress and iron deposition in NAFLD and its potential mechanism associated with ferroptosis. Our preliminary research focused on high fat diet (HFD)-induced ApoE−/−mice gavaged with GB (20 and 30 mg kg-1•d-1, approximately equal to the human dose of 2 and 3 mg kg-1•d-1, respectively) and palmitic acid and oleic acid (PA/OA)-induced HepG2 cells treated with GB (4, 8, 16 μg/mL), respectively. Hepatic injury was assessed via biochemical, histopathological and immunohistochemical evaluations. In order to examine the mechanism of GB on ferroptosis-regulated pathway, we analyzed the expression levels of ferroptosis-related proteins, including nuclear factor erythroid 2 (Nrf2), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), transferrin receptor-1 (TFR1) and ferritin heavy chain-1 (FTH1) in vivo and vitro experiments by Western blotting. In order to further verify the correlation pathway of ferroptosis, after Nrf2 short hairpin RNA interference, we analyzed the effects of GB on Nrf2 pathway. Both HFD-fed mice and PA/OA-induced HepG2 cells displayed ferroptosis-based panel of biomarkers such as iron overload with the up-regulation of TFR1 and the down-regulation of FTH1, lipid peroxidation and inhibition of Nrf2 activity, which further induced GPX4 and HO-1 levels. Remarkably, after Nrf2 interference, GB treatment significantly increased Nrf2 expression, indicating that GB exerted anti-ferroptosis effects by activation of Nrf2 pathway. Our results are preliminarily illustrated that GB treatment has a specific effect on lipid accumulation and oxidative stress caused ferroptosis in NAFLD, possibly through Nrf2 signaling pathway.