ALBERT

Description: Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Key PointsEBV-coding miRNAs are transferred from infected into noninfected cells by exosome to regulate the function for the tumorigenesis. Production of EBV-coding miRNAs will be an excellent diagnostic marker to separate patients with EBV+ diffuse large B-cell lymphoma into 2 groups.

Description: Introduction Cell fate and lineage specification have been thought to be largely regulated at the level of transcription. However, regulation of differentiation at the transcriptional level alone does not appear to explain all hematopoietic cell fate decisions, suggesting the presence of other as-yet-unknown mechanisms. EBF1 is critical transcription factors in B-lymphopoiesis. Ectopic EBF1 expression promotes B cell development in multipotent progenitor cell (MPP). EBF1is deleted and downregulated in different B cell malignancy like some ALLs and Hodgkin lymphoma. MicroRNAs are noncoding RNAs that are vital to many cell functions and that post-transcriptionally repress target mRNAs. Each miRNA is thought to have several target mRNAs, and computational predictions suggest that more than a third of all human genes are targets of miRNAs. However, in cell fate determination, it has been thought to act as only a fine tuner. Recently we reported that miR-126 drives MLL-AF4 ALL cells, exhibiting both myloid and B cell surface markers, towards B cell without upregulating of EBF1, E2A, and PAX5, which are critical transcriptional factors in B cell lineage commitment. Moreover, miR-126 could induce partial B lymphopoiesis in EBF1 deficient hematopoietic cells (HPCs) (Okuyama.K et al. PNAS). In the present study, in order to challenge the canonical notion that cell fate is governed solely by transcriptional factors, we investigated whether miRNA have full potentiality to induce B cell development in EBF1 deficiency. Material and methods We introduce several different miRNA in EBF1-/-hematopoietic progenitor (Lin-) cells and cultured them on TSt-4 stromal cells in IMDM medium containing stem cell factor, IL-7, and Flt3 ligand (10 ng/mL each). Then comprehensive Gene-Expression analysis, genomic PCR for VDJ recombination analysis and flowcytometric analysis for B-cell lineage markers were performed. Result Among the miRNA we analyzed, miR-195 induced expression of CD19, an established B lineage marker, and upregulated of B220 , B cell associated marker, comparing with control vector expressed EBF1 KO HPCs (Figure 1a). Then we investigated the molecular marker of B committed cells,“ VDJ recombination”. In miR-195 transduced EBF1KO cells, V to DJ recombination were completed, while the control was not (Figure 1b). Based on the results obtained from surface and molecular markers, miR-195 transduced EBF1KO cells were identified as B committed cells suggesting that miR-195 can rescue B lymphopoiesis in EBF1 deficiency. Finally our microarray data shows that overexpression of miR-195 in EBF1 KO cells upregulates B cell lineage commitment genes such as Pax5, Bcl11a, VpreB1, Cd79a, lamda5 and E2A which are downsteam targets of EBF1 and downregulates the myeloid lineage specific genes which are also antagonized by EBF1 in B cell lineage specification (Figure 2a). Next in order to clarify its mechanism, we studied the target genes of miR-195. Tgfβr3, Smad7, Acvr2a, Lats, HGF, and Pbx3 were identified as the targets of miR-195 by luciferase assay in 293T cells (Figure 2b). TGF beta family and the pathway, has been reported to suppress B lymphopoiesis. We hypothesized that TGF beta family genes such as Tgfβr3, Smad7, and Acvr2a, are responsible for transcription factor independent B cell differentiation by miR-195. Further analysis is now under investigation. Conclusion MiR-195 induces CD19 expression, completion of VDJ recombination and upregulation of B cell related genes in EBF1-/-HPCs while neither PAX5 nor E2A are able to upregualte B cell related genes or induce CD19 expression in the absence of EBF1. It suggests that miR-195 is more potent than E2A and Pax5 in this system and acts more than a fine tuner in B cell lineage specification. In the canonical notion, cell fate is determined solely by transcriptional factors. Our study challenges this notion, proposing that miRNA might have potential to be an alternative of transcriptional factors in some condition (Figure 2c). As we all know, impaired differentiation by deregulation of transcriptional factors is one of “hit” to leukemia development. Accordingly miRNA can potentially rescue this deregulation to become promising therapeutic target. Disclosures: Ando: Alexion: Research Funding.