Elevated serum TARC levels precede classic Hodgkin lymphoma diagnosis by several years.

Tumor cells in classic Hodgkin lymphoma produce high quantities of the thymus- and activation-related chemokine (TARC). We measured TARC levels in prediagnostic serum samples and found strikingly increased values in the vast majority of patients, as early as 6 years before diagnosis.

The lncRNA KTN1-AS1 co-regulates a variety of Myc-target genes and enhances proliferation of Burkitt lymphoma cells.

Long non-coding RNAs (lncRNAs) are involved in many normal and oncogenic pathways through a diverse repertoire of transcriptional and posttranscriptional regulatory mechanisms. LncRNAs that are under tight regulation of well-known oncogenic transcription factors such as c-Myc (Myc) are likely to be functionally involved in their disease-promoting mechanisms. Myc is a major driver of many subsets of B cell lymphoma and to date remains an undruggable target. We identified three Myc-induced and four Myc-repressed lncRNAs by use of multiple in vitro models of Myc-driven Burkitt lymphoma and detailed analysis of Myc binding profiles. We show that the top Myc-induced lncRNA KTN1-AS1 is strongly upregulated in different types of B cell lymphoma compared with their normal counterparts. We used CRISPR-mediated genome editing to confirm that the direct induction of KTN1-AS1 by Myc is dependent on the presence of a Myc E-box-binding motif. Knockdown of KTN1-AS1 revealed a strong negative effect on the growth of three BL cell lines. Global gene expression analysis upon KTN1-AS1 depletion shows a strong enrichment of key genes in the cholesterol biosynthesis pathway as well as co-regulation of many Myc-target genes, including a moderate negative effect on the levels of Myc itself. Our study suggests a critical role for KTN1-AS1 in supporting BL cell growth by mediating co-regulation of a variety of Myc-target genes and co-activating key genes involved in cholesterol biosynthesis. Therefore, KTN1-AS1 may represent a putative novel therapeutic target in lymphoma.

Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eµ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Detection of circulating tumor DNA in plasma of patients with primary CNS lymphoma by digital droplet PCR.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL. METHODS: We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively. RESULTS: MYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196. CONCLUSION: The low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.

Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphoma.

The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient-sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC-driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR-15a. TSC1 knockdown results in elevated mTORC1-dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC-driven cancers.

Rosetting T cells in Hodgkin lymphoma are activated by immunological synapse components HLA class II and CD58.

A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II-matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.

Longitudinal monitoring of BKPyV miRNA levels in kidney transplant recipients with BKPyV-related pathology reflects viral DNA levels and remain high in viremia patients after clearance of viral DNA.

INTRODUCTION: It is unclear whether polyomavirus BK (BKPyV) microribonucleic acid (miRNA) measurement has additional diagnostic and predictive value in kidney transplant recipients (KTR) as compared to current methods of monitoring BKPyV DNA loads. PATIENTS AND METHODS: A retrospective, longitudinal study was performed in 30 KTR with BKPyV viruria (n = 10), BKPyV viremia (n = 10), or BKPyV-associated neuropathy (BKPyVAN) (n = 10). Bkv-miR-B1-3p and 5p and BKPyV DNA load were measured in urine and plasma and compared using receiver operating characteristic (ROC) curves. RESULTS: Levels of Bkv-miR-B1-3p and 5p and BKPyV DNA correlated strongly. Overall, mostly analog courses of urinary and plasma miRNA and DNA loads were observed. Areas under the ROC curves were not significantly different between miRNAs and DNA. Only, in contrast to BKPyV DNA load, BKPyV miRNA levels increased from 6 to 12 months in the viremia group, while in the BKPyVAN group, a decline was seen in both DNA and miRNA. CONCLUSIONS: In this study, we could not demonstrate an additional value of BKPyV miRNA detection compared to BKPyV DNA monitoring in the early phase after kidney transplantation. We did observe significant differences between the viremia and the BKPyVAN groups during follow-up. This study was performed with a small number of patients and therefore results should be verified in a larger patient cohort. Furthermore, future studies with larger patient groups are necessary to elucidate final clinical value of these data.

Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway.

Cytochrome P450 enzymes (CYP450s) exert mighty catalytic actions in cellular metabolism and detoxication, which play pivotal roles in cell fate determination. Preliminary data shows differential expression levels of CYP27C1, one of the "orphan P450s" in human lung cancer cell lines. Here, we study the functions of CYP27C1 in lung cancer progression and drug endurance, and explore its potential to be a diagnostic and therapeutic target for lung cancer management. Quantitative real-time PCR and immunoblot assays were conducted to estimate the transcription and protein expression level of CYP27C1 in human lung cancer cell lines, which was relatively higher in A549 and H1975 cells, but was lower in H460 cells. Stable CYP27C1-knockdown A549 and H1975 cell lines were established, in which these cells showed enhancement in cell proliferation, colony formation, and migration. In addition, aberrant IGF-1R/Akt/p53 signal transduction was also detected in stable CYP27C1-knockdown human lung cancer cells, which exhibited greater tolerance towards the treatments of anticancer agents (including vinorelbine, picropodophyllin, pacritinib, and SKLB610). This work, for the first time, reveals that CYP27C1 impacts lung cancer cell development by participating in the regulation of the IGF-1R/Akt/p53 signaling pathway, and the level of CYP27C1 plays indispensable roles in dictating the cellular sensitivity towards multiple anticancer agents.

Soluble PD-L1 is a promising disease biomarker but does not reflect tissue expression in classic Hodgkin lymphoma.

Individually, tissue and soluble markers involved in the programmed cell death protein 1/programmed death-ligand (PD-1/PD-L) axis have been described as biomarkers with clinical value in classical Hodgkin lymphoma (cHL). In the context of the success of immune checkpoint blockade therapy in cHL, it is interesting to discover whether plasma levels of proteins in the PD-1/PD-L axis are a reflection of expression by the corresponding tissue. Paired tissue and plasma samples of cHL patients were collected and analysed for PD-1, PD-L1 and PD-L2 levels. In addition, vascular endothelial growth factor (VEGF) and CD83, molecules regarded to influence the expression of PD-1, PD-L1 and/or PD-L2, were included. PD-L1 was upregulated in the plasma of cHL patients compared to healthy controls and correlated well with several clinical parameters. Strong PD-L1 expression in the tumour microenvironment contributed to high soluble (s)PD-L1 levels, although there was no direct correlation between plasma PD-L1 levels and total expression of PD-L1 in corresponding cHL tissue. Interestingly, we observed a positive correlation between VEGF and PD-1 levels in both tissue and plasma. In conclusion, although PD-L1 is a promising soluble biomarker in cHL, its levels do not reflect the total tissue expression. Future studies focusing on PD-L1 as a predictor for immune checkpoint treatment response, should include both biopsy and plasma samples.

Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity.

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins.

Interim thymus and activation regulated chemokine versus interim 18 F-fluorodeoxyglucose positron-emission tomography in classical Hodgkin lymphoma response evaluation.

Serum thymus and activation regulated chemokine (TARC) levels reflect classical Hodgkin lymphoma (cHL) disease activity and correspond with treatment response. We compared mid-treatment interim TARC (iTARC) with interim 18 F-fluorodeoxyglucose positron-emission tomography (iPET) imaging to predict modified progression-free survival (mPFS) in a group of 95 patients with cHL. High iTARC levels were found in nine and positive iPET in 17 patients. The positive predictive value (PPV) of iTARC for a 5-year mPFS event was 88% compared to 47% for iPET. The negative predictive value was comparable at 86% for iTARC and 85% for iPET. Serum iTARC levels more accurately reflect treatment response with a higher PPV compared to iPET.

Treatment of patients with MYC rearrangement positive large B-cell lymphoma with R-CHOP plus lenalidomide: results of a multicenter HOVON phase II trial.

Patients with MYC-rearrangement positive large B-cell lymphoma (MYC+ LBCL) have an inferior prognosis following standard first-line therapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) as compared to patients without MYC rearrangement. Although intensive chemotherapy regimens yield higher remission rates, toxicity remains a concern. Lenalidomide is an oral immunomodulatory drug which downregulates MYC and its target genes thereby providing support using lenalidomide as additional therapeutic option for MYC+ LBCL. A phase II trial was conducted evaluating the efficacy of lenalidomide (15 mg day 1-14) in combination with R-CHOP (R2CHOP) in newly diagnosed MYC+ LBCL patients identified through a nationwide MYC-FISH screening program. The primary endpoint was complete metabolic response (CMR) on centrally reviewed 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)-computer tomography (CT)-scan at end-of-treatment. Secondary endpoints were overall survival (OS), disease-free survival (DFS) and event-free survival (EFS). Eighty-two patients with stage II-IV MYC+ LBCL were treated with 6 cycles of R2CHOP. At EOT, 67% (confidence interval (CI) 58-75%) of the patients reached CMR. With a median follow-up of 25.4 months, 2-year estimates (95% CI) for OS, DFS, EFS were 73% (62-82%), 75% (63-84%) and 63% (52-73%) respectively. In this prospective trial for newly diagnosed MYC+ LBCL patients, we found that administering R2CHOP was safe, and yields comparable CMR and survival rates as in studies applying more intensive chemotherapy regimens. Hence, these findings offer new prospects for MYC+ LBCL patients and warrant comparison in prospective randomized clinical trials. This trial was registered at www.clinicaltrialsregister.eu (#2014-002654-39).

Argonaute 2 RNA Immunoprecipitation Reveals Distinct miRNA Targetomes of Primary Burkitt Lymphoma Tumors and Normal B Cells.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AGO2-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

Targeted sequencing of circulating cell-free DNA in stage II-III resectable oesophageal squamous cell carcinoma patients.

BACKGROUND: The aim of this study was to investigate the potential of cell-free DNA (cfDNA) as a disease biomarker in oesophageal squamous cell carcinoma (ESCC) that can be used for treatment response evaluation and early detection of tumour recurrence. METHODS: Matched tumour tissue, pre- and post-surgery plasma and WBCs obtained from 17 ESCC patients were sequenced using a panel of 483 cancer-related genes. RESULTS: Somatic mutations were detected in 14 of 17 tumour tissues. Putative harmful mutations were observed in genes involved in well-known cancer-related pathways, including PI3K-Akt/mTOR signalling, Proteoglycans in cancer, FoxO signalling, Jak-STAT signalling, Chemokine signalling and Focal adhesion. Forty-six somatic mutations were found in pre-surgery cfDNA in 8 of 12 patients, with mutant allele frequencies (MAF) ranging from 0.24 to 4.91%. Three of the 8 patients with detectable circulating tumour DNA (ctDNA) had stage IIA disease, whereas the others had stage IIB-IIIB disease. Post-surgery cfDNA somatic mutations were detected in only 2 of 14 patients, with mutant allele frequencies of 0.28 and 0.36%. All other somatic mutations were undetectable in post-surgery cfDNA, even in samples collected within 3-4 h after surgery. CONCLUSION: Our study shows that somatic mutations can be detected in pre-surgery cfDNA in stage IIA to IIIB patients, and at a lower frequency in post-surgery cfDNA. This indicates that cfDNA could potentially be used to monitor disease load, even in low disease-stage patients.

Tumour necrosis as assessed with 18F-FDG PET is a potential prognostic marker in diffuse large B cell lymphoma independent of MYC rearrangements.

OBJECTIVES: MYC gene rearrangements in diffuse large B cell lymphomas (DLBCLs) result in high proliferation rates and are associated with a poor prognosis. Strong proliferation is associated with high metabolic demand and tumour necrosis. The aim of this study was to investigate differences in the presence of necrosis and semiquantitative 18F-FDG PET metrics between DLBCL cases with or without a MYC rearrangement. The prognostic impact of necrosis and semiquantitative 18F-FDG PET parameters was investigated in an explorative survival analysis. METHODS: Fluorescence in situ hybridisation analysis for MYC rearrangements, visual assesment, semiquantitative analysis of 18F-FDG PET scans and patient survival analysis were performed in 61 DLBCL patients, treated at a single referral hospital between 2008 and 2015. RESULTS: Of 61 tumours, 21 (34%) had a MYC rearrangement (MYC+). MYC status was neither associated with the presence of necrosis on 18F-FDG PET scans (necrosisPET; p = 1.0) nor associated with the investigated semiquantitative parameters maximum standard uptake value (SUVmax; p = 0.43), single highest SUVmax (p = 0.49), metabolic active tumour volume (MATV; p = 0.68) or total lesion glycolysis (TLG; p = 0.62). A multivariate patient survival analysis of the entire cohort showed necrosisPET as an independent prognostic marker for disease-specific survival (DSS) (HR = 13.9; 95% CI 3.0-65; p = 0.001). CONCLUSIONS: MYC rearrangements in DLBCL have no influence on the visual parameter necrosisPET or the semi-quantiative parameters SUVmax, MATV and TLG. Irrespective of MYC rearrangements, necrosisPET is an independent, adverse prognostic factor for DSS. KEY POINTS: • Retrospective analysis indicates that MYC rearrangement is not associated with necrosis on 18 F-FDG PET (necrosis PET ) scans or semiquantitative 18 F-FDG PET parameters. • Necrosis PET is a potential independent adverse prognostic factor for disease-specific survival in patients with DLBCL and is not influenced by the presence of MYC rearrangements.

Heterogeneous Pattern of Dependence on Anti-Apoptotic BCL-2 Family Proteins upon CHOP Treatment in Diffuse Large B-Cell Lymphoma.

Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes.

Current Smoking is Associated with Decreased Expression of miR-335-5p in Parenchymal Lung Fibroblasts.

Cigarette smoking causes lung inflammation and tissue damage. Lung fibroblasts play a major role in tissue repair. Previous studies have reported smoking-associated changes in fibroblast responses and methylation patterns. Our aim was to identify the effect of current smoking on miRNA expression in primary lung fibroblasts. Small RNA sequencing was performed on lung fibroblasts from nine current and six ex-smokers with normal lung function. MiR-335-5p and miR-335-3p were significantly downregulated in lung fibroblasts from current compared to ex-smokers (false discovery rate (FDR) <0.05). Differential miR-335-5p expression was validated with RT-qPCR (p-value = 0.01). The results were validated in lung tissue from current and ex-smokers and in bronchial biopsies from non-diseased smokers and never-smokers (p-value <0.05). The methylation pattern of the miR-335 host gene, determined by methylation-specific qPCR, did not differ between current and ex-smokers. To obtain insights into the genes regulated by miR-335-5p in fibroblasts, we overlapped all proven miR-335-5p targets with our previously published miRNA targetome data in lung fibroblasts. This revealed Rb1, CARF, and SGK3 as likely targets of miR-335-5p in lung fibroblasts. Our study indicates that miR-335-5p downregulation due to current smoking may affect its function in lung fibroblasts by targeting Rb1, CARF and SGK3.

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma.

BACKGROUND/AIMS: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. METHODS: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. RESULTS: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. CONCLUSION: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis.

miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs-miR-23a-3p, miR-24-3p, and miR-27a-3p-were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three HL cell lines and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed increased protein levels on miR-24-3p inhibition for CDKN1B/P27kip1 and MYC. In summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs cell growth possibly via targeting CDKN1B/P27kip1 and MYC.