ALKBH5-mediated N6-methyladenosine modification of TRERNA1 promotes DLBCL proliferation via p21 downregulation

Long noncoding RNAs (lncRNAs) have crucial functions in the tumorigenesis and metastasis of cancers. N6-methyladenosine (m6A) modification of RNA is an important epigenetic regulatory mechanism in various malignancies. Nevertheless, the mechanism of m6A-modified lncRNA in diffuse large B cell lymphoma (DLBCL) has remained poorly defined. In the present study, we showed that lncRNA TRERNA1 was associated with the poor prognosis of DLBCL patients. TRERNA1 with internal m6A modification was highly correlated with the demethylase ALKBH5 expression. We further demonstrated that TRERNA1 was a potential downstream target of ALKBH5-mediated m6A modification by m6A-RNA sequencing and m6A-RIP assays. Decreased m6A methylation of TRERNA1 regulated by ALKBH5 was shown to regulate cell proliferation in vitro and in vivo. The results of mechanism analyses revealed that TRERNA1 recruited EZH2 to epigenetically silence the expression of the cyclin-dependent kinases inhibitor p21 by H3K27me3 modification of its promoter region. In addition, ALKBH5 further inhibited p21 expression. Taken together, our results elucidate the functional roles and epigenetic alterations of TRERNA1 through m6A modification in DLBCL. TRERNA1, the expression of which is upregulated by ALKBH5, acts as a scaffold that decreases p21 expression. The results of the present study provide novel targets for the diagnosis and treatment of DLBCL.

AVL9 promotes colorectal carcinoma cell migration via regulating EGFR expression

Background Despite advanced treatments could inhibit progression of colorectal carcinoma (CRC), the recurrence and metastasis remain challenging issues. Accumulating evidences implicated that AVL9 played a vital role in human cancers, but it’s biological function and mechanism in CRC remain unclear. Aim To investigate the biological role and mechanism of AVL9 in colorectal carcinoma. Results AVL9 expression was significantly upregulated in tumor tissues than that in matched normal tissues both at mRNA and protein levels. High expression of AVL9 was closely correlated with M status, stages and poor prognosis of colorectal carcinoma (CRC) patients. Functionally, AVL9 overexpression promoted cell migration rather than cell proliferation in vitro, whereas AVL9 knockdown exhibited the contrary results. Mechanistically, AVL9 regulated EGFR expression, and knockdown of EGFR restrained AVL9-induced cell migration. Conclusion These findings demonstrated that AVL9 contributed to CRC cell migration by regulating EGFR expression, suggesting a potential biomarker and treatment target for CRC.

CD8 Treg-Mediated Suppression of Naive CD4+ T Cell Differentiation into Follicular Helper T Cells

<b><i>Background:</i></b> The regulatory CD8⁺ T (CD8⁺ Treg) cells play an important role in immune tolerance and have been implicated in several human autoimmune diseases. In this context, follicular helper T (T_FH) cells contribute by controlling the antibody production. In mice, CD8⁺ Treg cells control the number and function of T_FH cells however the role of human CD8⁺ Treg cells on the differentiation of naive CD4⁺ T cells into T_FH cells has not been studied. <b><i>Objectives:</i></b> Here, we evaluated the ability of human CD183⁺ CD8⁺ Treg cells to suppress T_FH cell differentiation in vitro. <b><i>Methods:</i></b> Activated CD183⁺CCR7⁺CD45RA⁻CD8⁺ Treg and CD183⁺CD25^highICOS⁺CD8⁺ Treg cells were sorted and cocultured with naïve CD4⁺ T cells under T_FH differentiation condition. The differentiation of T_FH cells was evaluated by flow cytometry. <b><i>Results:</i></b> Our results showed that activated CD183⁺CD8⁺ Treg cells upregulated the expression of Forkhead box P3 transcription factor, inducible T-cell co-stimulator (ICOS), and CD25 compared to CD183⁻CD8⁺ T cells. The CD183⁺CD25^highICOS⁺CD8⁺ Treg cells suppressed T_FH cell differentiation and CD4⁺ T cell proliferation in vitro which was not observed when CD183⁺CCR7⁺CD45RA⁻CD8⁺ Treg were cocultured with naïve CD4⁺ T cells under T_FH cell differentiation condition. <b><i>Conclusion:</i></b> These results suggest that CD25^highICOS⁺CD183⁺CD8⁺ Treg cells may regulate autoimmune and inflammatory responses mediated by T_FH cells.

Hyaluronic acid fuels pancreatic cancer cell growth

Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not preclude the growth of human tumor xenografts in mice, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. We found that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating N-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles.

Vascular Response on a Novel Fibrin-Based Coated Flow Diverter

Purpose Due to thromboembolic complications and in-stent-stenosis after flow diverter (FD) treatment, the long-term use of dual antiplatelet treatment (DAPT) is mandatory. The tested nano-coating has been shown to reduce material thrombogenicity and promote endothelial cell proliferation in vitro. We compared the biocompatibility of coated (Derivo Heal) and non-coated (Derivo bare) FDs with DAPT in an animal model. Methods Derivo® bare (n = 10) and Derivo® Heal (n = 10) FD were implanted in the common carotid arteries (CCAs) of New Zealand white rabbits. One additional FD, alternately a Derivo bare (n = 5) or Derivo Heal (n = 5), was implanted in the abdominal aorta (AA) for assessment of the patency of branch arteries. Histopathological examinations were performed after 28 days. Angiography was performed before and after FD implantation and at follow-up. Results Statistical analysis of the included specimens showed complete endothelialization of all FDs with no significant differences in neointima thickness between Derivo® bare and Derivo® Heal (CCA: p = 0.91; AA: p = 0.59). A significantly reduced number of macrophages in the vessel wall of the Derivo Heal was observed for the CCA (p = 0.02), and significantly reduced fibrin and platelet deposition on the surface of the Derivo Heal was observed for the AA. All branch arteries of the stented aorta remained patent. Conclusion In this animal model, the novel fibrin-based coated FD showed a similar blood and tissue compatibility as the non-coated FD.

MiR-155-5p suppresses SOX1 to promote proliferation of cholangiocarcinoma via RAF/MEK/ERK pathway

Background Accumulating evidence has demonstrated the close relation of SOX1 with tumorigenesis and tumor progression. Upregulation of SOX1 was recently shown to suppress growth of human cancers. However, the expression and role of SOX1 in cholangiocarcinoma (CCA) is not well characterized. Methods Expression levels of SOX1 in CCA tissues and normal bile duct tissues were examined using public GEO database. Western blot and immunohistochemistry were used to confirm the expression levels. Cell proliferation assay (CCK-8) and colony formation assay were performed to assess proliferation of CCA cells. A mouse model of subcutaneous transplantable tumors was used to evaluated proliferation of CCA in vivo. The putative regulating factor of SOX1 were determined using Targetscan and dual-luciferase reporter assay. Results SOX1 was downregulated in CCA tissues. Overexpression of SOX1 significantly inhibited cell proliferation in vitro and suppressed tumor growth in vivo. miR-155-5p directly targeted the 3′-untranslated region (3′UTR) of SOX1 and inhibited expression of SOX1, resulting in the activation of RAF, MEK and ERK phosphorylation, and thus CCA proliferation. However, restoration of SOX1 expression in miR-155-5p overexpressing cell lines decreased the phosphorylation level of RAF, MEK and ERK, as well as the proliferation of CCA cells. Conclusion MiR-155-5p decreased the expression of SOX1 by binding to its 3′UTR, which activated the RAF/MEK/ERK signaling pathway and promoted CCA progression.

Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.