Downregulation Of PRDM1 Research Articles

BMP4 is insufficient to differentiate umbilical cord mesenchymal stem cells into germ cell-like cells in vitro.

Mesenchymal stem cell (MSC)-based therapies are expected to restore the fertility of infertile patients. In addition to MSC-derived paracrine effects to improve reproductive function, the differentiation of MSCs into germ cell (GC)-like cells is still a promising method to repair the injured reproductive system. The aim of this study was to examine the effect and potential mechanism of BMP4 in inducing umbilical cord MSC (UcMSC) transdifferentiation into GC-like cells. UcMSCs were isolated, cultured and identified by flow cytometry and multilineage differentiation assays. After induction with 12.5 ng/mL BMP4 for 21 days, UcMSCs were collected for further examination. Immunofluorescence was used to detect the expression of Prdm1 and Prdm14; RT-PCR and RNA sequencing were used to detect differential gene expression (DEGs). The morphology of UcMSCs became large and flat after treatment with BMP4; the expression of GC-related genes (OCT4, Prdm1, Ifitm3 and Stella) was significantly downregulated, and further immunofluorescence results also confirmed the significant downregulation of Prdm1 in UcMSCs with BMP4 induction, while the expression of Prdm14 was significantly upregulated. The results of RNA sequencing and further analysis revealed no explicit correlation between BMP4 induction and the differentiation of UcMSCs into GC-like cells based on the 662 screened DEGs in UcMSCs with or without BMP4 induction. The differentiation of MSCs into GC-like cells is rather complex, and BMP4 alone is insufficient to induce UcMSCs to differentiate into GC-like cells, regardless of protein level or gene expression level.

ZNF683 (Hobit) Marks a CD8+ T Cell Population Associated with Anti-Tumor Immunity Following Anti-PD-1 Therapy for Richter Syndrome

Richter syndrome (RS), an aggressive B cell lymphoma originating from transformation of indolent chronic lymphocytic leukemia (CLL), has been shown to be responsive to PD-1 checkpoint blockade (CPB) across clinical trials. To systematically discover the determinants of this response, we analyzed single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 RS patients enrolled in a phase I study of the anti-PD1 drug nivolumab with concurrent ibrutinib (NCT 02420912). At pre-treatment sampling, RS responders (RS-R) had a larger CD8 effector/effector memory (E/EM) population than RS non-responders (RS-NR) (p=0.04, 2-sample t-test). This population was rare in normal bone marrow (N=30, p=0.001, 2-sample t-test), displayed intermediate exhaustion, preserved cytotoxicity and was marked by expression of the transcription factor and PRDM1-homolog, ZNF683. Further implicating ZNF683-expressing CD8 T cells in CPB response, comparison of gene expression between RS-Rs and RS-NRs within transcriptionally defined CD8 T cell clusters identified higher ZNF683 expression in RS-Rs. Lastly, the kinetics of ZNF683 expression correlated with response, with relative expression per cell decreasing with RS progression. Trajectory analysis supported a T cell differentiation path from GZMK+ memory to ZNF683intermediate followed by branching towards either ZNF683high or terminal exhaustion. To establish the extent to which ZNF683high T cell signatures are detectable in a broader population of RS patients, we examined bulk RNA-seq data generated from 35 independent RS biopsy specimens and found that 7 of 35 (20%) displayed a ZNF683high signature. A similar distribution of samples with ZNF683high expression, when corrected for T cell fraction, was observed in 18 of 81 (20%) CLL samples for which non-CD19 selected transcriptomic data was available and was associated with a trend towards improved overall survival (p=0.11). We also detected a similar ZNF683high signature in a large external compendium of single cell data from tumor infiltrating lymphocytes across 18 solid and 3 hematologic tumor types and in the peripheral blood of melanoma CPB responders (p<0.001 hypergeometric test). Upon evaluation of TCGA data, we detected an association with survival in melanoma cases with high T cell expression of ZNF683 (P<0.0001). To probe the cellular impact of ZNF683, a FLAG-tagged ZNF683 construct was overexpressed in Jurkat cells. ZNF683 overexpression revealed upregulation of PRF1, ITGA1, CD244, CD226 and IL2RB and downregulation of PRDM1 and IL2. CUT&RUN was performed to identify the binding sites of ZNF683, yielding twenty-four differentially regulated peaks, among which included ENCODE-identified regulatory regions adjacent to key immune genes (TNFAIP8, BTN3A2, CD69/KLRF1, ARHGAP2, CAMK4, LMO2, IL2, TCF7). Evaluation of external ATAC-seq data generated from exhausted T cell subsets revealed PD-1high tumor-infiltrating lymphocytes to have reduced accessible chromatin across identified putative ZNF683 binding sites, including TCF7. Thus, terminal exhausted cells not only lose ZNF683 expression but also display chromatin remodeling that likely abrogates its effects. By applying the tool CISTROME-GO to integrate the CUT&RUN and RNA-seq data, we observed enrichment in pathways corresponding to antigen binding and presentation, transcription factor activity, and T cell mediated cytotoxicity and cell killing upon ZNF683 expression. To determine if the association between ZNF683 expression and response to PD-1 blockade in RS could be confirmed in peripheral blood, we analyzed RNA-seq data generated from T cells isolated from pre-treatment blood samples from 7 independent RS patients treated with PD-1 therapy. We again identified ZNF683 among the top upregulated genes in RS-R (Log2 fold change = 2.13, p=0.037), along with other genes enriched in our ZNF683high CD8 E/EM signature (BATF, CORO1A, CD38, ITGB2, GZMM), while RS-NR were enriched in NK-like genes (KLRC1, FCGR3A, KLRG1, KLRF1 and FCER1G). In conclusion, we identify ZNF683 as marking a CD8 T cell population associated with CPB response in RS blood and bone marrow and provide evidence that this transcription factor regulates key cellular pathways involved in immune anti-tumor response, with implications for understanding CPB response in RS and other malignancies.

Positive Regulatory Domain I-binding Factor 1 Mediates Peripheral Nerve Injury-induced Nociception in Mice by Repressing Kv4.3 Channel Expression.

The transcriptional repressor positive regulatory domain I-binding factor 1 (PRDM1) is expressed in adult mouse dorsal root ganglion and regulates the formation and function of peripheral sensory neurons. The authors hypothesized that PRDM1 in the dorsal root ganglion may contribute to peripheral nerve injury-induced nociception regulation and that its mechanism may involve Kv4.3 channel transcriptional repression. Nociception was induced in C57BL/6 mice by applying chronic constriction injury, complete Freund's adjuvant, or capsaicin plantar injection. Nociceptive response was evaluated by mechanical allodynia, thermal hyperalgesia, cold hyperalgesia, or gait analysis. The role of PRDM1 was evaluated by injection of Prdm1 knockdown and overexpression adeno-associated viruses. The interaction of PRDM1 at the Kv4.3 (Kcnd3) promoter was evaluated by chromatin immunoprecipitation assay. Excitability of dorsal root ganglion neurons was evaluated by whole cell patch clamp recordings, and calcium signaling in spinal dorsal horn neurons was evaluated by in vivo two-photon imaging. Peripheral nerve injury increased PRDM1 expression in the dorsal root ganglion, which reduced the activity of the Kv4.3 promoter and repressed Kv4.3 channel expression (injured vs. uninjured; all P < 0.001). Knockdown of PRDM1 rescued Kv4.3 expression, reduced the high excitability of injured dorsal root ganglion neurons, and alleviated peripheral nerve injury-induced nociception (short hairpin RNA vs. Scram; all P < 0.05). In contrast, PRDM1 overexpression in naive mouse dorsal root ganglion neurons diminished Kv4.3 channel expression and induced hyperalgesia (PRDM1 overexpression vs. control, mean ± SD; n = 13; all P < 0.0001) as evaluated by mechanical allodynia (0.6 ± 0.3 vs. 1.2 ± 0.2 g), thermal hyperalgesia (5.2 ± 1.3 vs. 9.8 ± 1.7 s), and cold hyperalgesia (3.4 ± 0.5 vs. 5.3 ± 0.6 s). Finally, PRDM1 downregulation in naive mice reduced the calcium signaling response of spinal dorsal horn neurons to thermal stimulation. PRDM1 contributes to peripheral nerve injury-induced nociception by repressing Kv4.3 channel expression in injured dorsal root ganglion neurons.

Transcription factor NF-κB promotes acute lung injury via microRNA-99b-mediated PRDM1 down-regulation

Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1.

Genomic and Transcriptomic Characterization of IgM Multiple Myeloma Identifies a Pre-Germinal Center Plasma Cell Disorder with Immature B-Cell Transcription-Factor Signature

Genomic and Transcriptomic Characterization of IgM Multiple Myeloma Identifies a Pre-Germinal Center Plasma Cell Disorder with Immature B-Cell Transcription-Factor Signature

Induction of Follicular-Like CXCR5+CD8+T Cells by TGF-β1/IL-23 Is Limited During HIV Infection

Follicular CD4+ T cells are the main HIV reservoirs due to, among other factors, the low frequency of CD8+ T cells in lymphoid follicles. Follicular CXCR5+ CD8+ T cells are associated with HIV control, but their differentiation conditions are yet undefined. In this study, we explored the in vitro effect of transforming growth factor (TGF)-β1, interleukin (IL)-12, and IL-23 on the induction of CXCR5, the follicle homing receptor, in human circulating CD8+ T cells from seronegative, and treated HIV-infected individuals. The combination of TGF-β1 plus IL-23 induced the highest expression of CXCR5 in purified CD8+ T cells. These CXCR5+ CD8+ T cells also expressed a transcriptional and phenotypic profile similar to that of follicular CD4+ T cells, such as the upregulation of BCL6, inducible costimulator and CD40L, and downregulation of PRDM1. These cells responded in vitro to CXCL13 and had low expression of CCR7. In addition, after polyclonal stimulation, they produced IL-21, interferon-γ, and de novo perforin. However, in comparison with seronegative individuals, CD8+ T cells from HIV-infected patients had a lower response to TGF-β1/IL-23, a defect that was restored with the blockade of the programmed cell death 1 inhibitory receptor. Thus, TGF-β1 plus IL-23 induce follicular-like CXCR5+ CD8+ T cells in seronegative individuals, but in HIV-infected patients there is a limited response which could impair the generation of this cell population.

JAK3/STAT3 oncogenic pathway and PRDM1 expression stratify clinicopathologic features of extranodal NK/T‑cell lymphoma, nasal type.

The inactivation of tumor suppressor gene positive regulatory domain containing I (PRDM1) and activation of signal transducer and activator of transcription 3 (STAT3) have been detected in the majority of extranodal NK/T-cell lymphoma, nasal type (EN-NK/T-NT) cases. In the present study, their association with and effects on the clinicopathologic features of EN-NK/T-NT are described. PRDM1 was revealed to be expressed in 19 out of 58 patients (32.8%) with EN-NK/T-NT, and phosphorylated STAT3 was overexpressed in 42 out of 58 (72.4%). Oncogenic pathways were investigated by NanoString encounter technology in 5 PRDM1(+) and 5 PRDM1(−) EN-NK/T-NT specimens. Multiple oncogenic pathways involved in cell apoptosis, cellcycle (CC) and angiogenesis were discriminately activated in EN-NK/T-NT cases, and in PRDM1(+) cases in particular. The sustained activation of the Janus kinase 3 (JAK)/STAT3 pathway was more pronounced. In addition, missense mutations in the SRC homology 2 domain of STAT3 were detected in 7 out of 37 EN-NK/T-NT cases (18.92%), and the acquired mutation was related to the activation of the JAK3/STAT3 pathway. The downregulation of PRDM1 and upregulation of phospho-STAT3 (Tyr705) were associated with angiocentric infiltration of EN-NK/T-NT (P=0.039). Notably, the prognosis of patients in the PRDM1(+)/STAT3 [mutated (mut-)] group was considerably improved than that of patients in the STAT3(mut+)/PRDM(−) group (P=0.037). In addition, the inhibition of NK/T cell lymphoma cell lines by Stattic and tofacitinib could suppress cell proliferation by inducing cell apoptosis or arresting the CC. The present results revealed that the JAK3/STAT3 oncogenic pathway and PRDM1 expression could stratify clinicopathologic features of EN-NK/T-NT. The inhibition of the JAK3/STAT3 pathway may serve as a treatment option for EN-NK/T-NT.

EBV-miR-BHRF1-2 targets PRDM1/Blimp1: potential role in EBV lymphomagenesis.

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in aggressive lymphomas, including diffuse large B-cell lymphoma (DLBCL). It has been shown in DLBCL and Hodgkin lymphoma that PRDM1 is downregulated by cellular microRNAs. In this study, we identify the Epstein–Barr virus (EBV) microRNA (miRNA), EBV-miR-BHRF1-2, as a viral miRNA regulator of PRDM1. EBV-miR-BHRF1-2 repressed luciferase reporter activity by specific interaction with the seed region within the PRDM1 3' untranslated region. EBV-miR-BHRF1-2 inhibition upregulated PRDM1 protein expression in lymphoblastoid cell lines (LCL), supporting a role of miR-BHRF1-2 in PRDM1 downregulation in vivo. Discordance of PRDM1 messenger RNA and protein expressions is associated with high EBV-miR-BHRF1-2 levels in LCLs and primary post-transplant EBV-positive DLBCL. Enforced expression of PRDM1-induced apoptosis and cell cycle arrest in LCL cells. Inhibition of EBV-miR-BHRF1-2 negatively regulates cell cycle and decreases expression of SCARNA20, a small nucleolar RNA that is also downregulated by PRDM1 overexpression. The interaction between EBV-miR-BHRF1-2 and PRDM1 may be one of the mechanisms by which EBV-miR-BHRF1-2 promotes EBV lymphomagenesis. Our results support the potential of EBV-miR-BHRF1-2 as a therapeutic target in EBV-associated lymphoma.

The Genetic Deletion of 6q21 and PRDM1 and Clinical Implications in Extranodal NK/T Cell Lymphoma, Nasal Type.

6q21 genetic deletion has been frequently detected in extranodal NK/T cell lymphoma, nasal type (EN-NK/T-NT), and PRDM1 is considered as candidate gene. However, direct detection of PRDM1 deletion has not been well documented. We investigated genetic alterations of 6q21 and PRDM1 in 43 cases of EN-NK/T-NT and cell lines by FISH. PRDM1 expression was evaluated by immunohistochemistry and Western blot. The correlation between genetic alteration and PRDM1 expression and the significance in clinic-pathologic were analyzed. Heterozygous deletion of 6q21 and/or PRDM1 was observed in 24 of 43 cases (55.81%) of EN-NK/T-NT including 16 cases (37.21%) for 6q21 deletion and 19 cases (44.19%) for PRDM1 deletion. Similarly, heterozygous codeletion of 6q21 and PRDM1 was identified in NK92 and NKL cells. The heterozygous deletion of 6q21 and/or PRDM1 was correlated with PRDM1 expression. However, genetic deletion of 6q21 and/or PRDM1 was not correlated with clinicopathological features of EN-NK/T-NT, while PRDM1 expression showed positive effect on the outcome of patients as those as disease site, B symptom, and clinical stage. Thus, heterozygous deletion of 6q21 and/or PRDM1 was frequently detected in EN-NK/T-NT and correlated with downregulation of PRDM1. But the prognostic role of genetic deletion needs to be further evaluated in larger cohort.

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis.EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control.MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects.In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. DisclosuresNo relevant conflicts of interest to declare.

The downregulation of PRDM1/Blimp-1 is associated with aberrant expression of miR-223 in extranodal NK/T-cell lymphoma, nasal type

BackgroundThe mechanism for inactivation of positive regulatory domain containing I (PRDM1), a newly identified tumour suppressor gene in extranodal NK/T-cell lymphoma, nasal type (EN-NK/T-NT) has not been well defined. The aim of the present study was to investigate the expression of PRDM1 in EN-NK/T-NT and analyse its downregulation by miRNAs.MethodsPRDM1 and miRNA expression were evaluated in EN-NK/T-NT samples by immunohistochemical analysis, qRT-PCR, and in situ hybridisation. Luciferase assays were performed to verify the direct binding of miR-223 to the 3′-untranslated region of PRDM1 mRNA. In addition, the effect of miR-223 on PRDM1 expression was assessed in NK/T lymphoma cell lines by transfecting a miR-223 mimic or inhibitor to increase or decrease the effective expression of miR-223. Overall survival and failure-free survival in EN-NK/T-NT patients were analysed using Kaplan-Meier single-factor analysis and the log-rank test.ResultsInvestigation of the downregulation of PRDM1 in EN-NK/T-NT cases revealed that PRDM1-positive staining might be a favourable predictor of overall survival and failure-free survival in EN-NK/T-NT patients. However, the negative staining of PRDM1 usually presented transcripts, suggesting a possible post-transcriptional regulation. miR-223 and its putative target gene, PRDM1, exhibited opposite patterns of expression in EN-NK/T-NT tissues and cell lines. Moreover, PRDM1 was identified as a direct target gene of miR-223 by luciferase assays. The ectopic expression of miR-223 led to the downregulation of the PRDM1 protein in the NK/T-cell lymphoma cell line, whereas a decrease in miR-223 restored the level of PRDM1 protein.ConclusionsOur findings reveal that the downregulation of the tumour suppressor PRDM1 in EN-NK/T-NT samples is mediated by miR-223 and that PRDM1-positive staining might have prognostic value for evaluating the clinical outcome of EN-NK/T-NT patients.

Promoter and Exon 1 Hypermethylation of the Tumor Suppressor Gene PRDM1/Blimp-1 indicates Its Pathogenetic Role in EBV-Positive Burkitt Lymphoma,

Abstract 3471PRDM1/Blimp1, a lymphoma tumor suppressor and master regulator of plasma cell differentiation, is inactivated by genomic mutation and deletion in a subset of activated B-cell-type diffuse large B-cell lymphomas (DLBCL), underscoring the importance of terminal differentiation impairment in lymphoma pathogenesis. The role of PRDM1 inactivation in Burkitt lymphoma (BL) is currently not known. Two findings, however, may suggest possible PRDM1 involvement in BL pathogenesis. First, although PRDM1 protein is consistently absent in BL, about 40% of BL cases are positive for IRF4/MUM1, suggesting asynchronous expression of these two proteins. Secondly, it has been hypothesized that microRNA-127 overexpression in EBV+ BL may promote BL development through down-regulation of PRDM1. Sequence analysis of the coding region of PRDM1 in BL cell lines and primary tumors did not demonstrate any mutational changes. To investigate whether epigenetic inactivation of PRDM1 may play a pathogenetic role in BL, BL cell lines and primary cases were bisulfite sequenced to assess the methylation status of 41 CG dinucleotides in a 601 base-pair region spanning the distal promoter (DP) and a CG island that extends from the proximal promoter to the first exon of PRDM1. These include (i) 11 BL cell lines, 9 EBV+ (4 latency I, 2 Wp-restricted, and 3 with latency III drift) and 2 EBV-; (ii) 7 EBV+ lymphoblastoid cell line (LCL); (iii) 62 primary BL cases, formalin-fixed, paraffin-embedded tissues (FFPE); (iv) 4 B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCL-U), FFPE. Naïve, germinal center (GC) and memory B cells sorted by flow cytometry were also analyzed as controls. Hypermethylation in PRDM1 promoter and exon 1 was seen in all EBV+ BL cell lines except one with latency III, but not in EBV- BL lines. None of the LCL demonstrated PRDM1 hypermethylation, implying that EBV is unlikely to mediate PRDM1 hypermethylation directly. Methylation status of PRDM1 was successfully determined in 39 BLs (30 sporadic, 9 HIV-related) and 5 BCL-Us (4 sporadic, 1 HIV-related). Hypermethylation was seen in 11 BLs and 1 BCL-U (28.6% of total). All the methylated cases were EBV(+) (p=0.004). Overall, 12 of 28 (43%) EBV(+) BL and BCL-U cases exhibited hypermethylation. PRDM1 hypermethaylation was independent of BL subtype and MUM1/IRF4 expression status. To determine if PRDM1 hypermethylation can potentially repress PRDM1 transcription, BL cell lines Ramos (PRDM1 unmethylated) and Mutu1 (PRDM1 hypermethylated) are treated with IL-21 (50ng/mL), a cytokine that can mediate terminal differentiation by inducing PRDM1, for 2 to 5 days. While PRDM1 is induced by IL-21 in Ramos, levels of PRDM1 are not significantly increased in Mutu1, even though STAT3, a downstream mediator of IL-21, is activated in the latter. This finding suggests that PRDM1 hypermethylation has the potential to repress PRDM1 transcription in the presence of PRDM1-inducing signals. This function, however, may be pathogenetically more relevant in a BL precursor cell than in the final BL tumor cell. EBV+ BL is thought to derive from an EBV-infected late germinal center (GC) B cell, which harbors c-myc translocation and begins, though abortively, memory B cell differentiation and adopts an EBV latency I form. BL consistently express BCL6, a known PRDM1 transcription repressor, and harbor very low levels of PRDM1 mRNA. This suggests that high BCL6 expression, rather than PRDM1 hypermethylation, may be the primary determinant of its repressed transcription seen in BL. Indeed, PRDM1 mRNA expression is 25 to 1200 fold higher in LCLs which harbor very low levels of BCL. Furthermore, reducing expression of MTA3, which forms a complex with BCL6 and mediates PRDM1 transcription repression, in Daudi increases PRDM1 transcript levels. Therefore, we hypothesize that PRDM1 hypermethylation in BL more likely represents a memory of an epigenetic event that functions in the earlier stage of BL pathogenesis. PRDM1 hypermethylation may function to repress its transcription in a BL precursor cell as it is exposed to inducing signals, e.g. IL-21, during GC transit. Our study expands the spectrum of B cell lymphomas in which PRDM1 plays a tumor suppressor role, and supports the importance of impairment of terminal differentiation in the pathogenesis of a subset of aggressive B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B Cell Lymphomas : A Potential Role of the MicroRNA Let-7

PRDM1/Blimp-1, a master regulator for B cell terminal differentiation, is a putative tumor suppressor in diffuse large B cell lymphomas (DLBCL). Inactivating mutations of PRDM1 have been previously identified in a subset of nongerminal center B cell-like (GCB) DLBCL. We investigated the presence of alternative mechanisms of down-regulating PRDM1 in a cohort of 25 primary DLBCL and six DLBCL cell lines. While some DLBCL, predominantly the GCB-type, showed low levels of both PRDM1alpha mRNA and protein, presumably as a result of direct transcription repression, discordant expressions between the two were identified in a subset of DLBCL without PRDM1 mutations, the primarily non-GCB type, consistent with translational down-regulation. This subset of DLBCL exhibits relatively high PRDM1alpha mRNA levels but low levels of PRDM1. Data obtained from expression analysis, luciferase reporter assays, and transfection experiments support a role of targeting of PRDM1 by microRNA let-7 family in mediating this down-regulation. Let-7, in particular let-7b, is overexpressed in DLBCL relative to normal GCB cells, suggesting that it is deregulated. Thus, abnormal epigenetic down-regulation of PRDM1 by let-7 and other microRNAs may represent an alternative mechanism of reducing normal PRDM1 function in a subset of DLBCL with relatively high PRDM1alpha mRNA expression and unmutated PRDM1. These findings provide further evidence for an important role of impairment of terminal B cell differentiation in DLBCL pathogenesis.