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Aggressive natural killer cell leukemia (ANKL) is a rare form of natural killer (NK)-cell neoplasm with median survival of less than 2 months. Recently, the genomic mutation analysis using tumor cells reveled that the mutational profile of ANKL was similar to that of extranodal NK / T-cell lymphoma, which has relatively better prognosis than ANKL, explaining no causative mutations with a dismal prognosis. Here, using patient-derived xenograft model (PDX) mouse, we show that hepatic niche plays an important role in the ANKL biology. We established PDX mouse by intravenously injecting ANKL cells derived from patient peripheral blood or bone marrow samples to immunocompromised mice, which enables comprehensive analysis for tumor cells as well as tumor microenvironment. In total, we obtained four PDX strains derived from different patients. Time series pathological and flowcytometric analyses revealed that the ANKL cells initially engrafted and proliferated in sinusoidal or peri-portal area of the liver. This sinusoid or peri-portal distribution of ANKL in the liver was also confirmed with the patient liver specimen. To further determine the feature of ANKL in the liver, we selected liver or spleen tropic cells by serial adaptive transfer from each organ to the next mice. The liver-tropic ANKL cells proliferated more rapidly than splenic ANKL cells, which was evident by the significantly shorter survival of PDX mice injected liver-tropic cells (Figure). We performed RNA-sequencing using liver-tropic ANKL cells, spleen-tropic ANKL cells and NK-cells derived from healthy donors. These three types of cells showed distinct populations in principal component analysis. To further clarify the interaction between ANKL and liver niche, we performed additional RNA sequencing using total liver of mouse with or without bearing leukemic cells. In the cell-cell interaction analysis, we used two computational methods, mixed-species RNA-seq (Komura, et al. BMC Genomics 2016), which can distinguish transcripts derived from human (cancer) with mouse (non-cancer niche cells), and NicheNet (Browaeys, et al. Nat Methods 2020), which is a computational algorithm to model intercellular communication by linking ligands to target genes. These two methods allowed us to investigate the interaction between liver niche ligands and ANKL receptors. Among the listed ligand-receptor interactions, we focused on the macrophage migration inhibitory factor (MIF) and its receptor, CD74 axis. While CD74 was upregulated in ANKL cells compared with normal NK cells, MIF was highly expressed in the liver mainly liver sinusoid and Kupffer cells. Although we failed to culture primary ANKL cells in vitro, ANKL cells treated with MIF showed improved viability in vitro compared with untreated cells. Deletion of CD74 on the ANKL cells using CRISPR-Cas9 system attenuated the tumor formation in the liver as well as in bone marrow and spleen of PDX mouse compared with the wild type ANKL cells. These findings highlight that the liver, non-canonical hematopoietic organ in adults, is a principal niche where the liver specific components are required for survival and proliferation of ANKL cells. MIF-CD74 axis might play an important role in the communication between ANKL and hepatic niche. Figure 1 Figure 1. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding; Sanofi: Research Funding; MSD: Honoraria.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Introduction: Approximately 60% of Large B cell Lymphoma (LBCL) patients that receive CD19 CAR T cell therapy with axicabtagene ciloleucel (axi-cel) experience lymphoma progression (Locke et al. Lancet Oncol. 2019) and the likelihood of response to subsequent therapy is low (Spiegel, Dahiya et al. ASCO 2019). Target loss of CD19 is observed in less than a third of patients experiencing relapse. Alternative mechanisms of resistance to axi-cel are poorly understood. Lymphoma patients with elevated serum markers of systemic inflammation, such as ferritin and IL-6, have worse outcomes following axi-cel (Locke, Neelapu et al. Mol.Ther.2017; Faramand et al. ASH 2018). We hypothesized that suppressive monocytic myeloid derived suppressor cells (M-MDSCs), which are associated with worse chemotherapy outcomes in LBCL (Azzaoui et al. Blood 2016), and tumor driven inflammation may be present and responsible for decreased efficacy of axi-cel in LBCL. Methods: LBCL patients undergoing axi-cel treatment were enrolled onto prospective sample collection protocols. Patients were stratified for analysis into ongoing responders (complete response or partial response) or relapsed (progressive disease) after a minimum of 3 months follow-up (range 3 - 15 months). M-MDSCs, defined as a Lin-, CD11b+, CD33+, CD15-, CD14+, HLA-DRlow population, were sorted from leftover apheresis material after collection for axi-cel manufacture. M-MDSC ability to suppress proliferation of autologous T cells stimulated with CD3/CD28 coated beads was measured by 3H thymidine incorporation. Circulating peripheral blood M-MDSCs, quantified by % of live cells by flow cytometry, were measured at the time of apheresis and serially after axi-cel infusion until day 30. In vitro mouse experiments utilized a CD19-CD28 CAR and cytokine-induced bone marrow MDSCs (Thevenot et al. Immunity 2014). Cytokines were measured by ELISA and cytotoxicity against CD19 bearing cell lines used xCELLigence real-time cell analysis, as we have done previously (Li et al. JCI Insight 2018).Tumor biopsies were taken within 1 month prior to infusion of axi-cel. Limited gene expression profiling of tumor microenvironment (TME) genes used the Nanostring IO360 panel (770 genes). Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Results: First, we demonstrated that M-MDSCs sorted from patient apheresis material suppressed the proliferation of autologous T cells (n=6). We next enumerated M-MDSCs in the peripheral blood (n = 32). M-MDSC numbers initially decreased after lymphodepleting chemotherapy but recovered to baseline levels by day +10. The level of M-MDSCs following CAR T cell therapy strongly correlated with pre-CAR T baseline levels (R = 0.871, p Conclusions: Systemic inflammatory myeloid cytokines, circulating M-MDSCs in the blood and chronic IFN in the TME all associate with LBCL relapse after axi-cel CAR T cell therapy. Our observations support that CAR T cells can be suppressed by baseline patient and tumor-related factors and strategies to overcome these factors should be targeted to improve patient outcomes. MDJ and HZ contributed equally. Disclosures Jain: Kite/Gilead: Consultancy. Bachmeier:Kite/Gilead: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Mullinax:Iovance: Research Funding. Davila:Celgene: Research Funding; GlaxoSmithKline: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; Atara: Research Funding; Bellicum: Consultancy; Adaptive: Consultancy; Anixa: Consultancy. Locke:Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy.

An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Introduction: Of patients receiving CD19 CAR T cell therapy for large B cell lymphoma (LBCL), approximately 1 in 10 experience severe cytokine release syndrome (CRS) and 1 in 3 experience severe neurotoxicity. While CAR T cells trigger the onset of these toxicities, CRS and neurotoxicity are thought to occur as a consequence of activated myeloid cells amplifying cytokine and catecholamine release, thereby stimulating inflammation both systemically and at the blood-brain barrier. However, patient and tumor-related factors that account for differences in the amount of toxicity remain poorly understood. Methods: Serum cytokine levels were measured on an ELLA point of care device prior to lymphodepleting chemotherapy and throughout inpatient treatment with CD19 CAR T cell therapy (axicabtagene ciloleucel) for LBCL. Catecholamine levels were measured as we have previously reported. Tumor biopsies were taken within 1 month prior to infusion of CAR T cells. RNA expression was measured by RNAseq and/or a Nanostring IO360 panel consisting of 770 genes found in the tumor microenvironment (TME) in cancer. Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Mouse CAR T cell studies utilized mouse CD19-targeted CAR T cells derived from C57BL/6 splenocytes and cultured in vitro with myeloid cells and target cells to evaluate cytotoxicity and/or cytokine secretion. Elicited mouse macrophages were collected from peritoneal fluid 4 days after IP injection of 3% Brewer's thioglycollate medium. In vivo studies with mouse CD19-targeted CAR T cells were performed in IL2Ra-/- mice given cyclophosphamide as a pre-conditioning chemotherapy followed by adoptive transfer and analyses for CAR T cell and B cell persistence, as well as cytokines. Results: Of 58 patients undergoing CD19 CAR T cell therapy for LBCL, 8 (14%) had severe (grade 3 or higher) CRS and 16 (28%) had severe (grade 3 or higher) neurotoxicity. At baseline, peripheral blood levels of IL-6, IFN-γ, IL-15 and ferritin were significantly higher in patients who would subsequently experience severe CRS and severe neurotoxicity. Confirming our recent animal model of CRS we determined that peak serum catecholamine levels were higher in patients experiencing severe CRS. To identify if myeloid cells potentiate cytokine release we co-cultured CAR T cells with CD19 target and macrophages obtained from elicited mouse peritoneum. When these macrophages were added, IL-6 release from CAR T cells significantly increased compared to when macrophages were absent. Next, we studied the baseline TME in LBCL CAR T patients. Of 36 patients, 10 (27%) experienced severe neurotoxicity following CAR T cell therapy. By cell type score, the severe neurotoxicity group had a lower expression of genes associated with T cells overall and specifically Tregs. Also significantly lower in the severe neurotoxicity group were T cell genes including multiple subunits of CD3, CD3ζ, FOXP3, ICOS, CD62L and others. Association of increased T cell infiltration in the TME with low neurotoxicity raised the possibility that suppressive T cell subsets play a role in limiting toxicity post-CAR T cell therapy. To test this hypothesis, we injected CD19-targeted CAR T cells into an immune competent mouse model of Treg depletion (IL2Ra-/-) with established CD19+ leukemia. Treg deficient mice experienced a massive cytokine release after CAR T infusion and died prematurely due to CAR T toxicity compared to control mice with Tregs intact. Conclusions: Our observations suggest that the incidence of severe toxicity following CD19 CAR T cell therapy is influenced by baseline characteristics that are present prior to the infusion of CAR T cells. These include systemic inflammation characterized by high cytokine levels and a TME notable for a lack of infiltrating T cells. We posit a model whereby inflammation primes myeloid cells that are further activated upon CAR T cell infusion to release toxic amounts of cytokines and catecholamines. T cell subsets in the TME may modulate CAR T cells at the site of antigen encounter and prevent excessive CAR T activation. Reducing systemic inflammation or encouraging T cell infiltration into tumor prior to CAR T infusion are potential strategies for lowering the toxicity associated with CAR T therapy. Disclosures Jain: Kite/Gilead: Consultancy. Chavez:Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Novartis: Honoraria; Spectrum/Astrotech: Honoraria; Celgene/Juno: Honoraria; Kite/Gilead: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; AstraZeneca: Honoraria. Bachmeier:Kite/Gilead: Speakers Bureau. Mullinax:Iovance: Research Funding. Locke:Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy; Kite: Other: Scientific Advisor. Davila:Anixa: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Celgene: Research Funding; Atara: Research Funding; Bellicum: Consultancy.

Activation-induced cytidine deaminase (AID) is essential for class switch recombination and somatic hypermutation. Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that class switch recombination on B-cell activation is apparently inhibited by IM through down-regulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor.

[Introduction] CD19 chimeric antigen receptor (CAR) T cell therapies have been approved by the FDA for children and young adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and adults with r/r large B-cell lymphoma. Recent reports about long-term follow-up of CD19 CAR T cell therapy in B-ALL (Maude et. al. NEJM 2018, Park et. al. NEJM 2018) suggest that the median event-free survival of children and young adult patients is longer than that of adult patients (Over 11 months versus 6.1 months). The reason for the difference between survival of pediatric and adult patient is unclear, but we hypothesize it is due to age-related changes in the T cells collected from patients. Therefore, we compared the function of CAR T cells derived from young or aged mice. [Methods] Young C57BL/6J (B6) mice (6-12 weeks) and aged B6 mice (³ 72 weeks) were used as donors for CAR T cell preparation. Four types of mouse specific CD19 CAR encoded GFP fusion proteins were evaluated with all having the same anti-CD19 scFv and CD8 hinge and transmembrane domains but differing in their intracellular domain (m19Δz: lacks the CD3ξ signaling domain, m19z: CD3ξ signaling domain only, m1928z: CD28 and CD3ξ signaling domains, m19-humBBz: 4-1BB and CD3ξ signaling domains). [Results] T cells isolated from the spleen of aged B6 mice were significantly fewer than those of young B6 mice. However, CAR transduction efficiency, viability and yield were similar between young and aged CAR T cells for each CAR group. All groups of aged CAR T cells predominate with CD8+ and effector-like phenotypes at the expense of CD4+ and memory-like phenotypes after CD19+ artificial antigen presenting cell (aAPC) stimulation (Fig. 1A-1B). Furthermore, compared to CAR T cells derived from young mice, aged CAR T cells (m19z, m1928z and m19BBz) exhibited superior cytotoxicity in a real-time cell analysis for CD19+ aAPC killing (Fig. 1C). Using our immune competent in vivo murine model, aged CAR T cells were short-lived and expanded poorly despite their superior in vitro cytotoxicity. To evaluate for potential mechanisms involving preferential production of effector-like CAR T cells from aged mice we performed gene-expression, as well as single cell secretory polyfunctional analyses. While the polyfunctional strength index (PSI) of CD8+ aged CAR T cells was higher for aged CAR T cells, the increased score was due mostly to abundant secretion of a chemokine (Fig. 1D). Furthermore, the RNA-DESeq analysis demonstrated increased expression of chemokines and perturbation of the EOMES/TBET transcription factor axis. RNA-DESeq also suggested that young CAR T cells were highly active in cell proliferation and cell differentiation whereas aged CAR T cells upregulated gene expression pathways that regulated responses to stimulus and exocytosis. [Conclusions] CAR T cells derived from aged mice exhibited enhanced cytotoxicity but shorter persistence and less memory-like phenotypes. Our results suggest that the difference of clinical outcome between younger patients and older patients may be due to an age-dependent CAR T cell phenotype that is reflected by its unique gene expression pattern, secretory profile, and/or transcription factor balance. In our future directions we are extending these observations to human CAR T cells and identifying potential methods to improve the function of aged CAR T cells. Disclosures Davila: Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Introduction: One of the main complications of adoptive T cell therapy (ACT) is the en-masse activation of tumor-reactive T cells inducing a large release of cytokines followed by activation of other immune cells leading to adverse events. These are classified as a cytokine release syndrome (CRS) or neurotoxicity described as a CAR T Related Encephalopathy Syndrome (CRES). Several biomarkers have been associated with CRS and/or neurotoxicity such as LDH, ferritin and CRP. Cytokines have also been associated with CRS and and/or CRES, but present approaches rely on retrospective study of collected biomarkers. Here, we report the results of cytokine analysis using a point of care (POC) device to predict immune-related toxicities in patients with relapsed/refractory (R/R)DLBCL treated with axicabtagene ciloleucel (axi-cel). Methods: Patients with R/R DLBCL treated with commercial axi-cel were included in this study. Baseline serum samples were collected prior to lymphodepleting chemotherapy and then daily during hospitalization. To select which cytokines to monitor, we retrospectively analyzed 38 serum cytokines in a cohort of 53 patients with R/R B cell acute lymphoblastic leukemia (B-ALL) who were treated with 19-28z CAR T cells. The patients were divided into those requiring treatment with tocilizumab and/or steroids versus those who did not require treatment. We observed several cytokines, including IL-2, IL-6, IL-15 and IFNg, which were significantly elevated in patients with CRS and/or CRES requiring treatment (Figure 1a). Based on this analysis and results of published studies, eight serum proteins were selected in our study including IL-1b, IL-2, IL-6, IL-15, IFNg, TNFa, and angiopoietin-1 &2. We monitored these proteins using a POC device that allows for rapid daily monitoring with a turnaround time of two hours. We established that the results from the POC device strongly correlate with a current gold standard device(Luminex), which has a typical two day turn around time. CRS and CRES were prospectively graded using revised Lee criteria (Lee et al Blood 2014) and the CARTOX group (Neelapu et al. NRCO 2017) respectively by an experienced clinical team and confirmed by chart review retrospectively. Results: A total of 20 patients with R/R DLBCL treated with commercial axi-cel were identified. Median age 64 years ( range 43-73) with 80% male.In our cohort, grades 1-3 CRS were observed in 45%, 40% and 5% respectively. There were no observed grade 0 or grade 4 CRS. There were two patients (10%) who died in the setting of severe toxicity. Patients with grade 5 CRS had higher levels of IL-6 and angiopoietin 2/angiopoietin 1 ratio at day one, which correlated with severity of toxicity r=0.52 (p= 0.039) , and r=0.53 (p=0.033) respectively (Fig. 1b). Furthermore, patients with high grades CRS had elevated levels of IL-15 at day seven (r=0.83, p=0.006). The majority of patients (55%) had grade 1-2 CRES.There were no significant correlations between serum cytokine levels and CRES or between those who required tocilizumab/steroids vs. those who did not, likely due to the small sample size. In select cases, daily monitoring of cytokines using the POC device provided clinical insight that wasn't evident from standard biomarkers. For example, one patient who developed delayed CRS had high serum levels of IL-6 but did not have elevated levels of CRP(Fig.1c). Discussion: In this analysis of 20 patients, we observed a correlation between severe CRS and elevated serum cytokine levels of IL-6 and angiopoietin 2/angiopoietin 1 ratio at day one suggesting that these biomarkers may be utilized to predict severe toxicity in patients treated with ACT. While this study is limited by small sample size, our observations correlate with previously published biomarkers data in patients enrolled in clinical trials. To our knowledge this is the first reported cytokine data using commercial axi-cel. Monitoring of cytokines using a POC device is feasible and will be useful clinically. High risk patients may be identified early and help guide intervention in real time, for example day one elevated IL-6 levels might inform earlier use of tocilizumab. We continue to enroll patients to validate cytokines as predictive biomarkers with the goal of informing the development of preventative strategies to mitigate CAR T cell therapy immune related adverse events. Disclosures Locke: Cellular BioMedicine Group Inc.: Consultancy; Kite Pharma: Other: Scientific Advisor; Novartis Pharmaceuticals: Other: Scientific Advisor. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Park:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Shire: Consultancy. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background Sinusoidal obstruction syndrome (SOS) is a significant, potentially life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). In particular, severe SOS frequently causes multiple organ failure and results in poor prognosis. Although early and accurate diagnosis of SOS is vital to intervene early and improve its prognosis, the diagnostic accuracy of clinical criteria, such as the Seattle criteria and Baltimore criteria, is unsatisfactory. Liver biopsy is a gold standard for SOS diagnosis and should be considered to confirm the diagnosis. However, this procedure confers the risk of major bleeding, potentially leading to fatal outcomes, due to low platelet counts or coagulation disorders in patients with SOS. Therefore, a novel, noninvasive diagnostic approach for SOS is needed. The hepatic venous pressure gradient (HVPG) representing portal pressure has been reported to be the most accurate for SOS diagnosis, but its measurement procedure is invasive. Per rectal portal scintigraphy (PRPS), which is a nuclear medicine examination, can estimate portal pressure safely using the portal shunt index (SI) calculated from radioactivity curves of the liver and heart. Only one case report has shown that PRPS was useful to detect portal hypertension in a patient with SOS after allo-HCT (Okamura H, et al. Acta Haematol 2013). To investigate the usefulness of PRPS for SOS diagnosis, we retrospectively analyzed patients who had hepatic disorder and/or ascites after allo-HCT and underwent transjugular liver biopsy and PRPS. Methods We examined consecutive patients who underwent transjugular liver biopsy with HVPG measurement and were assessed using PRPS around the same time at our institution between April 2011 and May 2017. When performing transjugular liver biopsy, we also measured the HVPG. All causes of hepatic disorder and/or ascites were pathologically determined based on liver biopsy, if applicable, with autopsy or needle necropsy findings. In PRPS, 370 MBq of Tc-99m pertechnetate was administered to the upper rectum. The SI was calculated using the ratio of liver to heart counts integrated for 24 seconds immediately after the appearance of the liver or heart time-activity curve. We assessed the diagnostic performances of the HVPG and SI for SOS using receiver-operating characteristic (ROC) curves and calculated the optimal cutoff points using the Youden index. Analyses were performed using GraphPad Prism® version 5.02 and SPSS® version 24. Results Eleven cases were evaluable. The median onset of liver damage and/or ascites was on day 27 after allo-HCT: early-phase (within 30 days) in 7 cases and late-phase (after 30 days) in 4 cases. All patients were histologically diagnosed: 5 with SOS and 6 with non-SOS. The median interval from PRPS to transjagular liver biopsy was +4 days (range, -13 to 15 days). SI values from PRPS correlated with HVPG values in 11 pairs of the measurements (Spearman correlation coefficient 0.92, P < 0.001, Figure 1A). The areas under the ROC curves of the HVPG and SI for SOS diagnosis were 0.85 (95% confidence interval (CI), 0.61-1.0) and 0.77 (95% CI, 0.45-1.0), respectively. The best cutoff values of the HVPG and SI were calculated as 10 mmHg and 56%, respectively. Table 1 shows the sensitivity, specificity, and positive and negative predictive values for the HVPG, SI, and Seattle criteria for SOS diagnosis among all 11 cases. As a subgroup analysis, in 7 patients who developed hepatic disorder and/or ascites in the early phase after allo-HCT, both HVPG and SI were significantly higher in patients with SOS than in those without SOS (mean HVPG: 13.3 mmHg in SOS vs. 4.3 mmHg in non-SOS, P = 0.01, Figure 1B; mean SI: 58% in SOS vs. 26% in non-SOS, P = 0.03, Figure 1C). Furthermore, in these 7 patients, both the HVPG and SI made it possible to completely discriminate between SOS and non-SOS using cutoff points calculated based on the Youden index (8 mmHg for the HVPG and 44% for the SI). Conclusion We found that the SI from PRPS represented portal pressure, even in patients after allo-HCT, and that the diagnostic accuracy of PRPS for SOS was not only superior to that of the Seattle criteria, but also comparable to that of the HVPG. In addition, especially in the early phase after allo-HCT, SI may be useful to discriminate between SOS and non-SOS. This noninvasive examination could offer a new diagnostic strategy, along with SOS clinical criteria. Disclosures Hino: Otsuka Pharmaceutical Co., Ltd.: Research Funding; Novartis: Research Funding. Nakamae:Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding.

Ten patients with adenosine deaminase deficiency (ADA−) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA− severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA−SCID.

There is no reliable laboratory indicator of the onset of chronic graft-versus-host disease (cGVHD). This study looks at whether the expression of OX40, a member of the tumor necrosis factor receptor family, is related to the development of cGVHD in patients who underwent allogeneic hematopoietic stem cell transplantation. Peripheral blood mononuclear cells from 22 patients after day 100 were subjected to multicolor flow cytometry. The percentages of both OX40+CD4+ and OX40+CD8+T cells were significantly higher in patients with cGVHD than those without (P

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm of largely unknown genetic basis, which is associated with human T-cell leukemia virus type-1 (HTLV-1) infection. To delineate a genetic landscape of somatic alterations in ATL, we have performed an integrated genetic study, in which whole-genome/exome (WGS/WES) and transcriptome sequencing (RNA-seq) was performed for a cohort of 83 paired ATL samples, followed by extensive validation using targeted sequencing of detected mutations in 370 follow-up samples. A striking feature of driver lesions in ATL was their strong enrichment in the components of T-cell receptor (TCR) / NF-κB pathway. Accounting for more than 90% of ATL cases, these lesions were characterized by the predominance of activating alterations, including hotspot missense mutations in PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%) and FYN (4%). Among these, most frequently mutated was PLCG1, which encodes phospholipase C γ1 (PLCγ1), a key regulator of the proximal TCR signaling. Besides the S345F and S520F mutations recently reported in cutaneous T-cell lymphoma, we identified an additional hotspot mutations (R48W, E1163K, and D1165H). The second most frequently mutated gene was PRKCB, encoding a member of the protein kinase C (PKC) family of proteins (PKCβ), a pivotal signaling molecule downstream of PLCγ. The frequent mutations of PKCβ were unexpected, because it is PKCθ that has been implicated in TCR signaling, whereas PKCβ has been more focused in the context of B-cell receptor signaling. Approximately 93% of the PRKCB mutations were confined to the catalytic domain with a prominent hotspot at D427, suggesting gain-of-function nature of these mutations. Consistent with this, when transduced with the D427N PKCβ mutant, HEK293T and/or Jurkat cells showed increased membrane translocation after PMA/Ionomycin-stimulation, enhanced IKK phosphorylation and p65 nuclear translocation, and augmented NF-κB transcription, compared to wild-type PKCβ-transduced cells. Thus, these PRKCB mutations are the first activating mutations of this family identified in human cancers. Downstream to PKC lies CARD11, a scaffolding protein required for antigen receptor-induced NF-κB activation. Although previously reported in B-cell lymphomas, CARD11 mutations were more common in ATL (24%). In B-cell lymphomas, mutations are largely limited to the coiled-coil (CC) domain, whereas in ATL, they were clustered not only within the CC domain, but also within the PKC-responsive inhibitory domain, showing a prominent mutational hotspot at E626. The inhibitory domain has been implicated in autoinhibition, whose deletion leads to constitutive activation of CARD11. Intriguingly, WGS identified small intragenic deletions confined to this domain (exons 14-17) in 4 cases (8%) without canonical mutations, and RNA-seq confirmed the skipping of the corresponding exons in these cases. Remarkably, CARD11 mutation significantly co-occurred with PRKCBmutations, suggesting potential functional synergism between these lesions. Actually, overexpression of wild-type CARD11 induced NF-κB activation, which was further augmented by E626K mutation. Similarly, when both CARD11 (E626K) and PRKCB (D427N) mutants were co-expressed, more enhanced NF-κB activation was observed. RNA-seq and follow-up RT-PCR screening also identified novel gene fusions in TCR / NF-κB pathway: five CTLA4-CD28 and three ICOS-CD28 fusions were observed in seven (7%) of the 105 cases examined, of whom one patient carried both chimeric fusions. WGS revealed tandem duplications of 2q33.2 segments containing CD28, CTLA4, and ICOS, compatible with the corresponding fusion transcripts. B7/CD28 co-signaling molecules, including CD28, CTLA4, and ICOS co-receptors, play pivotal roles in positive and negative regulations of TCR signaling. All the predicted chimeric proteins had the cytoplasmic part of CD28, and are expected to be expressed under the control of the regulatory element of CTLA4 or ICOS, likely leading to prolonged expression of CD28 co-stimulator. Our findings suggest that deregulated TCR / NF-κB pathway caused by genetic alterations is a hallmark of ATL pathogenesis. The predominance of gain-of-function mutations in this pathway offers good opportunities for exploiting these mutations for the targets of novel drugs to better manage patients. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

To identify candidate antigens in aplastic anemia (AA), we screened proteins derived from a leukemia cell line with serum of an AA patient and identified diazepam-binding inhibitor-related protein 1 (DRS-1). Enzyme-linked immunosorbent assay (ELISA) revealed high titers of anti–DRS-1 antibodies (DRS-1 Abs) in 27 (38.0%) of 71 AA patients displaying increased paroxysmal nocturnal hemoglobinuria (PNH)–type cells (PNH+), 2 (6.3%) of 32 PNH– AA patients, 5 (38.5%) of 13 PNH+ myelodysplastic syndrome (MDS) patients, and none of 42 PNH– MDS patients. DRS-1 gene was abundantly expressed in myeloid leukemia cell lines and in CD34+ cells derived from healthy individuals. Stimulation of T cells from an AA patient displaying high DRS-1 Abs with a putative CD4+ T-cell epitope (amino acid residues [aa's] 191-204) presented by HLA-DR15, which overlapped with a hot spot (aa's 173-198) of DRS-1 Ab epitopes, gave rise to T cells cytotoxic for L cells (murine fibroblasts) that were transfected with DRB1*1501 and DRS-1. Enzyme-linked immunospot assay demonstrated increased frequency of T-cell precursors specific to the DRS-1 peptide in other HLA-DR15+ AA patients displaying high DRS-1 Ab titers. These findings indicate that DRS-1 may serve as an autoantigen eliciting immune attack against hematopoietic stem cells in a subset of AA patients characterized by increased PNH-type cells.

Introduction: Canonical notion demonstrates that Cell fate is determined by transcriptional factor. Accordingly, the lineage specific transcriptional factors have been investigated for various kinds of cells. Especially, in the hematopoietic system, the extensive research for lineage specific transcriptional factors had elucidated the transcriptional factors which regulated lineage commitment. B cell commitment and development requires the activities of multiple transcription factors, including the early B cell factor (EBF), PAX5, and E2A. These transcription factors regulate B cell development in a stage specific manner. The hematopoietic progenitor cells which are deficient for any of them, cannot commit B cells. Among them EBF1 is presumed to be more potent. Rescue early pro B cell to induce the expression of several key proteins including RAG that enable gene rearrangement to occur by opening of IgH locus. We found that the B cell developmental arrest caused by EBF1 deficiency can be rescued by a single non coding RNA. These B cells which are deficient EBF1 but showed the expression of CD19, B cell lineage specific surface marker and VDJ recombination, molecular markers of B cell commitment in vitro B cell differentiation system cocultured with Tst4 cells, stromal cell lines. We further investigated the quality and differentiation potential of these B lineage commitment cells in the in vivo mouse model and elucidated the mechanism of this phenomenon. Material and methods: We collected EBF1−/− fetal liver hematopoietic progenitor (Lin−) cells and cultured them on TSt-4 stromal cells after infecting with non coding RNA and control vector in IMDM medium containing cytokines and then injected it into NOG mice. Collect bone marrow (BM), thymus and spleen from those mice. Then comprehensive Gene-Expression analysis, real time PCR for VDJ recombination analysis was performed and checked surface marker by flowcytometry. Result: We analyzed BM and spleen of non coding RNA infected EBF1 KO cells injected mice and found the expression of CD19 in BM as well as in spleen and upregulated of B220 also, comparing with control vector expressed cells. Furthermore, surface IgM expression of CD19 positive cells in the spleen is upregulated compared with the cells in the BM (Figure 1). Several target genes of the non-coding RNAs were identified by use of cDNA array analysis and luciferase reportor assay. Among them, several genes were involved in TGF beta pathway. As TGF beta family and the pathway, has been reported a critical factor which is negatively regulating B lymphopoiesis (Figure 2). We hypothesized that TGF beta family genes such as Tgfβr3, Acvr2a, are responsible for B cell differentiation for which EBF1 is dispensable .We cultured EBF KO cells for 14 days with and without TGF beta 1,2,3 antibody and Activin A antibody on TST4 cells. We found that increase mean intensity (MFI) of B220 into antibodies positive cocultured cells (Figure 3) to suggest, the suppression of TGF beta pathway is partially responsible for B cell differentiation under EBF1 deficiency. Conclusion: Canonical notion of cell fate determination of B cells defines that EBF1 is an indispensible factor for B lymphopoiesis. However, from our previous and present study it is proved that without EBF1 B cell development can progress to pro B to pre B cell and Immature B cell stage and “VDJ recombination” occur in the absence of EBF. Furthermore, in vivo mouse model, EBF1 deficient hematopoietic progenitor cells differentiated into IgM positive cells. Therefore we can conclude that EBF is dispensable of VDJ recombination, opening of IgH locus, binding of RAG protein and B cell differentiation to the mature stage. One of the mechanisms is possibly due to the stimuli from microenvironment, such as TGF beta family and pathway. Furthermore, the detail mechanism of IgH locus opening, epigenetic changes and chromatin remodeling around the IgH locus in the absence of EBF is under investigation. Disclosures Chanda: Japan Society for the Promotion of Science(JSPS): Research Funding.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated. To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in > 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. Disclosures No relevant conflicts of interest to declare.

Introduction: EBV positive diffuse large B-cell lymphoma of the elderly (EBV positive DLBCL, elderly) has been newly categorized in 2008 WHO classification. The incidence is higher in Asian countries than Western countries. The prognosis of DLBCL has been improved by the introduction of rituximab, while EBV positive DLBCL remains unknown. Recently we reported that EBV positive DLBCL of the elderly did have quit inferior prognosis than EBV negative DLBCL. The mechanism lying under the inferiority has yet been elucidated. We hypothesize that tumor microenvironment plays a role in the mechanism, as EBV related lymphoma are usually accompanied with massive infiltration of non-tumor cells. We have previously found that tumor secreted small RNAs were selectively taken by macrophages and dramatically change the character into tumor supporting phenotype in the EBV positive lymphoma. Accordingly, we focus the interaction between tumor cells and macrophages in EBV positive lymphoma microenvironment. Methods: We investigated the number of CD163-positive macrophage (CD163+M2) in the DLBCL specimen with and without EBV positivity. The effect of EBV positivity in the tumor cells on the macrophages infiltrated in the tumor were studied by use of the coculture system using human monocytic cell line (THP-1) and human Burkitt lymphoma cells lines (Akata) which has two subclones such as EBV positive and negative. PMA-induced macrophages from THP1 cells were cocultured with EBV positive or negative Akata, then the expression of the several cytokines such as TNF-a, IL-10, CXCL10, and VEGF were measured by real-time PCR. Finally, we tried to clarify the impact of the macrophages on tumor formation in vivo by using xeno-transplantation model. Hematopoietic humanized NOG mice were infected with EBV to induce EBV related lymphoproliferative disease (LPD). After the appearance of symptoms of the LPD such as body weight loss, mice were treated with Clodronate to deplete macrophages. Result: The multivariate analysis for DLBCL patients demonstrated the statistically significant association between both high scores of CD163+M2 macrophages (CD163-positive cell > 20%) and EBV positivity, and poor prognosis (p = 0.0084, 0.0020, respectively.), which implies that EBV affected the quantity of CD163+M2 macrophages in the tumor microenvironment. Co-culture of THP1 with EBV positive lymphoma cells significantly upregulated CXCL10 and VEGF, when compared with EBV negative cells. (p = 0.0073, 0.0161, respectively.). (Figure 1) Most surprisingly, EBER positive B cells almost completely disappeared by macrophage depletion by Clodronate treatment (Figure 2). These results suggest that the macrophages in the EBV positive tumor microenvironment are crucial for survival of EBV+ tumor cells. Conclusion: EBV status of the lymphoma cells affected on TAMs in the way such as up-regulation of CXCR10 and VGEF, angiogenetic factors which are presumed to support tumor survival. The in vivo depletion of macrophages by Clodronate also demonstrated that they were indispensable for EBV positive tumor cell survival. Taken together, the interaction of EBV positive tumor cells and tumor associated macrophages is of crucial importance in the biology and formation of EBV positive lymphoma. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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.

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.

Abstract 3533 microRNAs (miRNAs) control many developmental and physiological processes. However, it is assumed to work as a fine tuner in cell fate determination, which has been shown to be regulated by transcription factors. Here, we challenge this canonical notion. miR-126 is downregulated in MLL-AF4 ALL, biphenotypic leukemia expressing both B cell and myeloid markers, compared with other types of ALLs. CD19 and CD20, B cell differentiation markers, were upregulated when miR-126 is reexpressed in MLL-AF4 ALLs (Figure 1). Interestingly, we found that miR-126, in leukemic cells, induces B cell differentiation (Figure 2a) without changing the expression of E2A, EBF1, or PAX5, (Figure 2b) which are assumed to be critical regulators of B cell development indicating that miR-126 induced B cell differentiation independently of transcriptional factors, such as PAX5, E2A, and EBF1. To test the hypothesis, we tried to rescue block of B cell differentiation in EBF1 knock-out hematopoietic progenitor cells (EBF1 KO HPCs) by exogenous expression of miR-126. As a result, significant upregulation of B cell markers, such as B220, RAG1/2, and CD79a/b was induced by miR-126 in EBF1 KO HPCs which show complete block at pre-pro B cell stage.(Figure 3a) Moreover, miR-126 increased cell proliferation, indicating EBF1 KO HPCs differentiate into large preB cells. Moreover, miR-195, which is upregulated toward pro B cells then downregulated in B cell differentiation, also can partially rescue block of B cell differentiation in EBF1 KO HPCs. (Figure 3b) The block in B lymphopoiesis imposed by the absence of E2A or Pax5 can be overcome by exogenous expression of EBF1. Conversely E2A or Pax5 failed to compensate the effect of EBF1. Thereafter miR-126 and miR-195 potentiates to play more roles than E2A or Pax5 in this system and act as more than a fine tuner in B cell differentiation. Our results elucidate a novel mechanism for the regulation of cell fate independent of transcriptional factor, establish an important role for miRNAs in mammalian lineage specification, and suggest that miRNA could be a new possible therapeutic target for dedifferentiation induced by deregulation of transcriptional factors in ALL, which calls for further studies. Disclosures: No relevant conflicts of interest to declare.