Key Points• EBV infection leads to PRMT5 overexpression and global epigenetic changes that are essential to drive B-lymphocyte transformation.• Highly selective PRMT5 inhibitors represent a novel, first-in-class drug that restores critical regulatory checkpoints in lymphoma cells.Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV 1 lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. (Blood. 2015;125(16):2530-2543

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmu-notherapy. Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatu-zumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malig-nancies. Because rituximab and milatu-zumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochon-drial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab-mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-B pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL. (Blood. 2011; 117(17):4530-4541) Introduction Mantle cell lymphoma (MCL) is a B-cell malignancy with a variable histology and clinical course, distinguished by the characteristic translocation t(11;14)(q13, q32) that results in overexpres-sion of cyclin D 1 and consequent dysregulation of cell-cycle control. 1 In addition, MCL exhibits alterations in cell survival pathways, including constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling 2 and nuclear factor-B (NF-B). 3 Despite the hallmark genetic translocation in MCL, the clinical course of MCL is variable with some patients experiencing indolent disease, 4 whereas others exhibit rapid progression. 5 MCL patients have a median overall survival (OS) of approximately 3 years, and no consensus exists for standard first-line therapy. 6-9 Although aggressive therapies including chemoimmunotherapy 10,11 or stem cell transplantation 12,13 have been shown to improve outcomes, no therapy offers the potential for cure. Given the absence of curative therapy and the limited number of options for patients with relapsed/refractory MCL, novel treatment approaches are essential. Rituximab (Genentech), a chimeric anti-human CD20 monoclo-nal antibody (mAb), has been used in multiple strategies to treat patients with MCL. 14 As a single agent, rituximab has been tested in patients with newly diagnosed and relapsed/refractory MCL with response rates (RR) of 27% to 38% and a median response duration of 6 to 12 months. 15,16 Interestingly, the RR obtained in untreated patients was not higher than in relapsed...

De novo CD5+ diffuse large B-cell lymphomas (DLBCL) are a distinct subgroup of DLBCL with poor prognosis. However the role of rituximab-containing therapy and salvage stem cell transplantation in this patients’ population remain to be defined. We retrospectively reviewed clinical features and outcomes of 102 patients with de novo CD5+ DLBCL treated with rituximab-containing therapy at 9 different institutions. By Hans’ criteria, 64 patients had activated B-cell (ABC) subtype, 24 germinal center B-cell (GCB) subtype, and 14 were not evaluated. No patients had a myc translocation. Eighty-three patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP), 7 with rituximab, etoposide, cyclophosphamide, doxorubicin, vincristine, prednisone (R-EPOCH) and 6 with R-CHOP with methotrexate, 3 g/m2. The overall response rate to frontline therapy was 85%. The 3-year progression free survival (PFS) and overall survival (OS) for all patients were 40% and 65%, respectively. The 3-year PFS for ABC- and GCB-subtypes was 34% and 45%, respectively. The 3-year OS for ABC- and GCB-subtypes was 62% and 67%, respectively. The median time to second treatment failure was 3 months and 1 month for ABC- and GCB-subtypes, respectively. Twenty of 28 (71%) transplanted patients with autologous, allogeneic, or both, relapsed. This study confirms the poor prognosis of de novo CD5+ DLBCL in a large multi-center cohort despite initial rituximab-containing chemotherapy and suggests that stem cell transplantation fails to salvage the majority of these patients. Approaches to prevent recurrence and/or novel therapies for relapsed disease are needed for this subgroup of DLBCL patients.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phosphoAkt and cyclin D1 and subsequent cellcycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagiclysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti- IntroductionMantle cell lymphoma (MCL) is a B-cell malignancy that comprises 3%-8% of non-Hodgkin lymphoma cases diagnosed each year. 1 Whereas the current treatment approach of using combination chemotherapeutic regimens can lead to complete remission, virtually all MCL patients relapse and outcome remains poor, with a median survival of only 3 years. 2 The aggressive clinical behavior of MCL may be because of the complex pathophysiology of the disease, which includes cell-cycle dysregulation driven by cyclin D1 overexpression, alteration in the DNA-damage response, and constitutive activation of key antiapoptotic pathways such as PI3K/Akt and NF-B. [3][4][5][6] Given the absence of curative therapy and the limited number of options for patients with relapsed/refractory MCL, it will be essential to improve our understanding of the complex biology of this disease so that novel treatment approaches can be developed. FTY720 (fingolimod), is a synthetic analog of sphingosine that was developed as an immunosuppressive agent. 7,8 Based on the results of a recent phase 3 clinical trial, FTY720 has been approved by the US Food and Drug Administration (FDA) to treat relapsed multiple sclerosis. 9 We have recently reported that FTY720 has in vitro and in vivo activity in MCL. 10 FTY720 promotes death of MCL cell lines and primary MCL tumor cells via caspaseindependent radical oxygen species (ROS) generation, downmodulation of phospho-Akt and cyclin D1, with accumulation of cells in G 0 /G 1 and G 2 /M phases of the cell cycle. Whereas these data provided information explaining the antitumor activity of FTY720, the effects of this drug on the pathophysiology of MCL required further characterization.In the present study, we show that FTY720 inhibits autophagic flux and induces MCL cell death through lysosomal membrane permeabilization and subsequent translocation of lysosomal hydrolases in the cytosol. Because the autophagy-lysosomal pathway represents an important regulatory mechanism governing the cellular proteome, we hypo...

The B-cell receptor pathway (BCR) is aberrantly activated in select B-cell malignancies. This knowledge has allowed for the development of inhibitors of different crucial steps of this pathway. Bruton's tyrosine kinase (BTK) is a key component of BCR signaling and functions as an important regulator of multiple cell functions including differentiation, proliferation, and survival in various B-cell malignancies. Ibrutinib is a potent, selective BTK inhibitor that has shown significant activity in specific subtypes of B-cell non-Hodgkin's lymphomas (NHLs). Given the high response rates, tolerability, and acceptable toxicities, ibrutinib was recently approved by the US Food and Drug Administration (FDA) for the treatment of patients with relapsed mantle cell lymphoma and chronic lymphocytic leukemia. It is also currently being evaluated in combination with chemotherapy and as frontline therapy in B-cell NHL. This review summarizes the preclinical and clinical development of ibrutinib in the treatment of B-cell NHL.