ALBERT

Description: Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.

Description: An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6(DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain(RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL.

Description: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and an aggressive malignancy. Galectin-3 (gal-3), the only antiapoptotic member of the galectin family, is overexpressed in DLBCL. While gal-3 can localize to intracellular sites, gal-3 is secreted by DLBCL cells and binds back to the cell surface in a carbohydrate-dependent manner. The major counterreceptor for gal-3 on DLBCL cells was identified as the transmembrane tyrosine phosphatase CD45. Removal of cell-surface gal-3 from CD45 with the polyvalent glycan inhibitor GCS-100 rendered DLBCL cells susceptible to chemotherapeutic agents. Binding of gal-3 to CD45 modulated tyrosine phosphatase activity; removal of endogenous cell-surface gal-3 from CD45 with GCS-100 increased phosphatase activity, while addition of exogenous gal-3 reduced phosphatase activity. Moreover, the increased susceptibility of DLBCL cells to chemotherapeutic agents after removal of gal-3 by GCS-100 required CD45 phosphatase activity. Gal-3 binding to a subset of highly glycosylated CD45 glycoforms was regulated by the C2GnT-1 glycosyltransferase, indicating that specific glycosylation of CD45 is important for regulation of gal-3–mediated signaling. These data identify a novel role for cell-surface gal-3 and CD45 in DLBCL survival and suggest novel therapeutic targets to sensitize DLBCL cells to death.

Description: Arsenic trioxide (ATO) induces apoptosis of plasma cells through a number of mechanisms including inhibiting DNA binding by NF-κB. These results suggest that this agent may be synergistic when combined with other active anti-myeloma drugs. To evaluate this we examined the effect of ATO alone and in combination with anti-myeloma treatments evaluated in vitro with MTT assays and using our severe combined immunodeficient (SCID)-hu murine myeloma models. First, we determined the effects of combining ATO with bortezomib or melphalan on the myeloma cell lines RPMI8226 and U266. Cell proliferation assays demonstrated marked synergistic anti-proliferative effects of ATO at concentrations ranging from 5x10−5M – 5x10−9M and melphalan concentrations ranging from 3x10−5M – 3x10−9M. Similar effects were observed when these cell lines were treated with bortezomib and varying concentrations of ATO (5x10−5 M – 5x10−10 M). We also investigated the potential of ATO to increase the efficacy of anti-myeloma therapies in our SCID-hu murine model LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Mice were treated with ATO alone at 6.0 mg/kg, 1.25 mg/kg, 0.25 mg/kg, and 0.05 mg/kg intraperitoneally (IP) daily x5/week starting 19 days post-implantation. Mice receiving the highest dose of ATO (6.0 mg/kg) showed marked inhibition of tumor growth and reduction of paraprotein levels while there was no effect observed in all other treatment groups. Next, 27 days following implantation of our LAGλ–1 intramuscular (IM) tumor, LAGλ–1 mice were treated with ATO (1.25 mg/kg) IP, bortezomib (0.25 mg/kg), or the combination of both drugs at these doses in the schedules outlined above. ATO or bortezomib treatment alone had no anti-myeloma effects at these low doses consistent with our previous results whereas there was a marked decrease in both tumor volume (57%) and paraprotein levels (53%) in mice receiving the combined therapy. The combination of melphalan and ATO was also evaluated in this model. LAGλ–1 bearing mice received therapy with melphalan IP x1/weekly at 12.0 mg/kg, 6.0 mg/kg, 0.6 mg/kg, and 0.06 mg/kg starting 22 days post-implantation and showed no anti-myeloma effects. Twenty-eight days following implantation of LAGλ–1 tumor, mice received ATO (1.25 mg/kg) or melphalan (0.6 mg/kg) alone at doses without anti-myeloma effects, or the combination of these agents at these doses. The animals treated with these drugs alone showed a similar growth and increase in paraprotein levels to control mice whereas the combination of ATO and melphalan at these low doses markedly suppressed the growth of the tumor by 〉50% and significantly reduced serum paraprotein levels. These in vitro and in vivo studies suggest that the addition of ATO to other anti-myeloma agents is likely to result in improved outcomes for patients with drug resistant myeloma. Based on these results, these combinations are now in clinical trials with promising early results for patients with drug resistant myeloma.

Description: Abstract 1894 Background: Most of the studies of prognostic factors in acute myeloid leukemia (AML) are focused on predicting outcome after standard chemotherapy treatment. The studies that looked at the importance of these prognostic variables in predicting outcome after hematopoietic stem cell transplantation (HSCT) are very limited. Considering post-HSCT prognostic factors in stratifying AML patients may add a new level of confidence in any risk stratification of these patients. Toward this goal, we investigated the clinical relevance of CD34 expression in the primary leukemic cells on the outcome of allogeneic HSCT. Methods: Data collected from immunophenotyping of 110 patients with AML who were treated with HSCT was reviewed and the expression of CD34 on the blast population as determined by flow cytometry was correlated with clinical behavior and outcome. All the patients were treated with allogeneic HSCT. The median age of these patients was 24 (range: 14–57) and included 88 patients in the intermediate cytogenetic group and 22 in the adverse cytogenetic group. Of the 110 patients 71, (64.5%) were treated with HSCT in first remission (CR1). Results: Twenty-eight (25%) of all AML patients studied did not express CD34 on the surface of the blasts at diagnosis. Patients with CD34 negative blasts had significantly longer overall survival (OS) (P=0.003) as well as longer event free survival (EFS) (P=0.01) when all patients were considered. In the subgroup of patients who received HSCT in CR1, OS and EFS were significantly longer (P=0.017 and P=0.027, respectively) in the CD34-negative patients (N=20). Furthermore, if we consider only patients in the intermediate cytogenetic group at diagnosis, patients with CD34-negative blasts had significantly longer OS (P=0.007) and EFS (P=0.026). Even in patients with adverse cytogenetic abnormalities, OS and EFS were also significantly longer in the CD34-negative patients (P=0.01 for both). This was true when all patients were considered. The same was true when only patients transplanted in CR1 (P=0.05 for EFS in intermediate cytogenetics and P=0.05 for EFS in patients with adverse cytogenetic) were evaluated. Multivariate analysis including CD34 expression and FLT3 mutation status was carried out on a subset of patients (N=63) and showed CD34 expression was an independent prognostic factor for survival and EFS, while FLT3 mutation status became no longer a predictor. Conclusion: Our data suggests that CD34 expression on the blast cells at the time of AML diagnosis may have an adverse prognostic impact even after allogeneic HSCT. Lack of CD34 expression is a powerful independent favorable prognostic factor for AML patients if these patients are treated with HSCT after induction chemotherapy, irrespective if they were in CR1 or in CR2, and irrespective of their cytogenetic risk at diagnosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2378 Epstein-Barr virus (EBV) is a ubiquitous, gamma herpes virus that infects human epithelial cells and B lymphocytes. Over 90% of adults worldwide are infected with EBV and, collectively, this virus is associated with a broad spectrum of benign and malignant disease. EBV is a potent oncogenic virus and is capable of efficiently transforming B cells in both in vitro and in vivo models. While signaling cascades contributing toward B cell immortalization and transformation following EBV infection have been described, epigenetic events that contribute toward the B cell transformation process remain poorly characterized. EBV-transformed lymphoblastoid lines (LCL) and spontaneous B cell lymphomas that arise in the hu-PBL-SCID model of EBV-induced lymphomagenesis show abundant expression of the protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme that catalyzes symmetric dimethylation of arginine residues on histones and non histone proteins (P53, CYCLIN D1). PRMT5 partners with multiple co-repressor proteins such as HDAC2, MBD2 and DNMT3a to silence multiple regulatory and tumor suppressor gene products. All EBV-transformed B cell lines and primary tumors showed cytoplasmic and nuclear staining for PRMT5 and its associated epigenetic marks symmetric dimethyl histone 3, arginine 8 (S2Me-H3R8) and S2Me-H4R3. Resting and activated B cells did not demonstrate PRMT5 over expression or associated global epigenetic marks. Infection of primary human B cells with the B95.8 strain (but not mutant P3HR1 or inactivated EBV) led to dysregulated expression of PRMT5 as early as day 4 post infection. By day 8 post EBV infection, PRMT5 location had transitioned to the nucleus and this localization coincided with acquisition of S2Me-H4R3 and S2Me-H3R8 and loss of the asymmetric epigenetic mark 2Me-H4R3,a type I PRMT histone mark. PRMT5 over-expression was dependent on LMP-1-driven NFkB activity and transcriptional silencing of miR96 expression, a micro RNA that targets the PRMT5 3'UTR. To determine if PRMT5 over expression was essential for induction and maintenance of the transformed phenotype, we infected resting B cells with EBV and, at various time points (day 4, 7, 14 or 21), added a novel, highly selective small molecule inhibitor of PRMT5 activity, an inactive small molecule control, shRNA specific for PRMT5 or control shRNA. Absolute CD19+ cell counts and confocal microscopy experiments to monitor PRMT5 and its epigenetic marks were performed and showed that PRMT5 activity was critical for EBV-driven B cell transformation to proceed. PRMT5 inhibition of resting or activated B cells did not result in any loss of viability. Global transcriptome analysis identified several tumor suppressor genes, including the protein tyrosine phosphatase PTPROt, were silenced during EBV-driven B cell transformation. Chromatin immune precipitation (ChIP)-sequencing using monoclonal antibodies specific for PMRT5 and S2Me-H3R8 (or control IgG) confirmed the PTPROt promoter to be directly targeted by PRMT5 and PTPROt transcript was found to become silenced during EBV-driven B cell transformation. Real time PCR and RNA-seq showed PTPROt transcript to become restored with PRMT5 inhibition. PTPROt expression led to dephosphorylation and inhibition of the LYN, SYK, and Bruton's Tyrosine kinase (BTK) kinase proteins, critical proteins involved in regulation of the B cell receptor (BCR). This model provided us with direct evidence that PRMT5 activity is critical to EBV-driven B cell transformation and supports our hypothesis that PRMT5 dysregulation drives epigenetic events that directly contribute to key initiating events during B cell transformation as well as to the maintenance of the malignant phenotype. We believe this is the first example of oncogenic virus driving over expression of an epigenetic modifier that catalyzes placement of global repressive epigenetic marks that silence of regulatory and tumor suppressor genes. This data justifies pursuit of experimental therapeutic strategies focused on selective PRMT5 inhibition in cancer. Disclosures: No relevant conflicts of interest to declare.

Description: Heme biosynthesis in erythroid cells is intended primarily for the formation of hemoglobin. As in every cell, this synthesis requires a multi-step pathway that involves eight enzymes including the erythroid-specific δ-aminolevulinate synthase (ALAS2, the first regulated enzyme that converts glycine and succinyl CoA into ALA) and the ubiquitous ferrochelatase (FECH, the final enzyme). Heme biosynthesis also requires membrane transporters that are necessary to translocate glycine, precursors of heme, and heme itself between the mitochondria and the cytosol. Defects in normal porphyrin and/or heme synthesis and transport cause four major erythroid inherited disorders, which may or may not be associated with dyserythropoiesis (e.g., sideroblastic, microcytic anemia and/or hemolytic anemia): "X-linked" sideroblastic anemia (XLSA) and X-linked dominant protoporphyria (XLDPP) are two different and opposing disorders but related to altered gene encoding ALAS2 only. Defective activity of this enzyme due to mutations in the ALAS2 gene causes the XLSA phenotype, including microcytic, hypochromic anemia with abundant ringed sideroblasts in the bone marrow. Vice versa, gain-of-function mutations of ALAS2 are responsible of the XLDPP characterized by predominant accumulation of the hydrophobic protoporphyrin (PPIX, the last heme precursor) in the erythrocytes without anemia or sideroblasts. Furthermore, the glycine transporter (SLC25A38) and Glutaredoxin 5 genes are reported to be involved in human non-syndromic sideroblastic anemia. Congenital erythropoietic porphyria (CEP) is the rarest autosomal recessive disorder due to a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. CEP leads to excessive synthesis and accumulation of type I isomers of porphyrins in the reticulocytes, followed by intravascular hemolysis and severe anemia. The ALAS2 gene may act as a modifier gene in CEP patients (Figueras J et al, Blood. 2011;118(6):1443-51). Erythropoietic protoporphyria (EPP) results from a partial deficiency of FECH and leads similarly to XLDPP, to deleterious accumulation of PPIX in erythroid cells. Most EPP patients present intrans to a FECH gene mutation an IVS3-48C hypomorphic allele due to a splice mutation. Abnormal spliced mRNA is degraded which contributes to the lowest FECH enzyme activity and allowed EPP phenotype expression. We have identified an antisense oligonucleotide (ASO) to redirect splicing from cryptic to physiological site and showed that the ASO-based therapy mediates normal splice rescue of FECH mRNA and reduction by 60 percent of PPIX overproduction in primary cultures of EPP erythroid progenitors. Therapeutic approaches to target both ALAS2 inhibition and heme-level reduction may be useful in other erythroid disorders such as thalassemia (where reduced heme biosynthesis was shown to improve the clinical phenotype) or the Diamond-Blackfan anemia (DBA). Indeed, in some DBA patients, an unusual mRNA splicing of heme exporter FLVCR has been found, reminiscent of Flvcr1-deficient mice that develop a DBA-like phenotype with erythroid heme accumulation. Thus, FLVCR may act as a modifier gene for DBA phenotypic variability. Recent advances in understanding the pathogenesis and molecular genetic heterogeneity of heme-related disorders have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias and some porphyrins and heme transporters, new understanding of the pathogenesis of the erythropoietic disorders, and new and experimental treatments such as chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for erythropoietic porphyrias. Disclosures: No relevant conflicts of interest to declare.