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Regulation of Normal B-Cell Differentiation and Malignant B-Cell Survival by OCT2

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Regulation of Normal B-Cell Differentiation and Malignant B-Cell Survival by OCT2 Regulation of normal B-cell differentiation and PNAS PLUS malignant B-cell survival by OCT2 Daniel J. Hodsona,b, Arthur L. Shaffera, Wenming Xiaoa,1, George W. Wrighta, Roland Schmitza, James D. Phelana, Yandan Yanga, Daniel E. Webstera, Lixin Ruia, Holger Kohlhammera, Masao Nakagawaa, Thomas A. Waldmanna, and Louis M. Staudta,2 aLymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and bDepartment of Haematology, University of Cambridge, Cambridge, CB2 0AH, United Kingdom Contributed by Louis M. Staudt, February 21, 2016 (sent for review January 12, 2016; reviewed by Kees Murre and Robert G. Roeder) The requirement for the B-cell transcription factor OCT2 (octamer- NP-OVA immunization (14) and another reporting normal ger- binding protein 2, encoded by Pou2f2) in germinal center B cells minal center formation after influenza challenge (15). OCA-B– has proved controversial. Here, we report that germinal center B deficient mice have normal B-cell development but are unable to cells are formed normally after depletion of OCT2 in a conditional mount a germinal center response (16–18). Thus, current evidence knockout mouse, but their proliferation is reduced and in vivo suggests that OCT2 and OCA-B have important functions in the differentiation to antibody-secreting plasma cells is blocked. This later stages of B-cell differentiation, but the precise role, if any, finding led us to examine the role of OCT2 in germinal center- for OCT2 in the germinal center reaction is unclear. derived lymphomas. shRNA knockdown showed that almost all Germinal centers form when a mature B cell encounters an- diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to tigen in the context of CD4 T-cell help and are characterized by the expression of OCT2 and its coactivator OCA-B. Genome-wide intense B-cell proliferation and hypermutation of Ig genes (19). chromatin immunoprecipitation (ChIP) analysis and gene-expres- B cells with improved affinity for the immunizing antigen as a sion profiling revealed the broad transcriptional program regu- result of Ig hypermutation are selected and eventually differen- lated by OCT2 that includes the expression of STAT3, IL-10, ELL2, tiate into either memory B cells or long-lived plasma cells. Dif- XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of fuse large B-cell lymphoma (DLBCL), the most common type of OCT2 is not a requirement for cellular addiction in DLBCL. How- non-Hodgkin lymphoma, is derived from B cells that have trans- ever, we detected amplifications of the POU2F2 locus in DLBCL tu- ited the germinal center (19). The germinal center B-cell–like mor biopsies and a recurrent mutation of threonine 223 in the DNA- (GCB) subtype of DLBCL retains expression of germinal center binding domain of OCT2. This neomorphic mutation subtly alters B-cell–restricted genes, whereas the activated B-cell–like (ABC) the DNA-binding preference of OCT2, leading to the transactivation DLBCL subtype appears to be derived from postgerminal center of noncanonical target genes including HIF1a and FCRL3. Finally, by plasmablastic cells (20). Both OCT2 and OCA-B are highly introducing mutations designed to disrupt the OCT2–OCA-B inter- expressed in normal germinal center B cells and in almost all cases face, we reveal a requirement for this protein–protein interface that of DLBCL (21, 22). A role for OCA-B in DLBCL was proposed ultimately might be exploited therapeutically. Our findings, com- based on the identification of a DLBCL-specific super-enhancer bined with the predominantly B-cell–restricted expression of near the OCA-B promoter, but this study did not investigate OCT2 and the absence of a systemic phenotype in our knockout whether OCA-B acts by binding to OCT2 or to the related and mice, suggest that an OCT2-targeted therapeutic strategy would ubiquitously expressed POU domain factor octamer-binding be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity. Significance cancer biology | lymphoma | germinal center Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma and is incurable in roughly ctamer-binding protein 2 (OCT2), a B-cell–restricted tran- POU2F2 30% of cases. Here we demonstrate the addiction of both Oscription factor encoded by the gene , binds to an major subtypes of DLBCL to the expression of the transcription ′ ′ octamer DNA motif 5 -ATGCAAAT-3 (1). It belongs to the factor OCT2 (octamer-binding protein 2) and its co-activator POU domain family of transcription factors that uses both a OCA-B. We clarify the role of OCT2 in normal germinal center POU homeodomain and a POU-specific domain to bind DNA – biology and identify the genes and pathways that it regulates (2 4). DNA binding induces a conformational change in the in malignant B cells. Our findings suggest that pharmacological POU domain that permits recruitment of the coactivator OCA-B – MEDICAL SCIENCES “ ” “ ” POU2AF1 agents designed to target OCT2 itself or the OCT2 OCA-B in- (also known as BOB-1 or OBF1, encoded by ), terface would be an effective and nontoxic therapeutic strat- which stabilizes the complex and further enhances transcrip- egy in DLBCL. tional activation (5–10). OCT2 and OCA-B are largely restricted in expression to the Author contributions: D.J.H. and L.M.S. designed research; D.J.H., A.L.S., R.S., J.D.P., Y.Y., B-cell lineage (1, 7–10). Although OCT2 initially was thought to D.E.W., L.R., and H.K. performed research; M.N. and T.A.W. contributed new reagents/ direct the B-cell–restricted expression of Ig genes by binding to analytic tools; D.J.H., W.X., G.W.W., and L.M.S. analyzed data; and D.J.H. and L.M.S. wrote octamer motifs in their promoters, OCT2-knockout cell lines the paper. Reviewers: K.M., University of California, San Diego; and R.G.R., The Rockefeller subsequently were shown to transcribe Ig genes normally (11). University. Because mice with germline deletion of Pou2f2 die shortly after The authors declare no conflict of interest. birth from an undetermined cause (12), fetal liver and bone mar- Data deposition: The sequence reported in this paper has been deposited in the Gene row chimeras have been used to investigate the function of OCT2- Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo. deficient B cells. Such mice have reduced B1 and marginal zone B 1Present address: Division of Bioinformatics and Biostatistics, National Center for Toxico- cells, and B-cell proliferation and Ig secretion are reduced when logical Research, Food and Drug Administration, Jefferson, AR 72079. the cells are stimulated in vitro (12, 13). The role of OCT2 in 2To whom correspondence may be addressed. Email: [email protected]. antigen-dependent germinal center responses is controversial, with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. one study finding a defect in the germinal center response to 1073/pnas.1600557113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1600557113 PNAS | Published online March 18, 2016 | E2039–E2046 Downloaded by guest on September 26, 2021 protein 1 (OCT1) (23). One study of follicular lymphoma described A B apparent loss-of-function mutations in POU2F2, leading to the Pou2f2 fl/wt Pou2f2 fl/fl GC GC suggestion that OCT2 may prevent the formation of lymphomas p=0.001 GC 40 (24). In the present study, we set out to clarify the role of OCT2 in 9.5 8.7 normal germinal center reactions and to determine whether OCT2 30 acts to promote or restrain the development of germinal center- CD95 derived lymphomas. CD95 20 PNA PNA Results 10 EDU pos Abnormal Germinal Center Function and Plasma Cell Differentiation C Day 14 Day 28 EDU+ (% of GC cells) in OCT2-Deficient Mice. We generated conditional Pou2f2-knock- 7 p < 0.0001 7 p < 0.0001 0 out mice from targeted ES cells created by the European Con- 6 6 fl/wt fl/fl ditional Mouse Mutagenesis Consortium. Lox P sites placed on SSC 5 5 EDU either side of exons 8–11 ensured deletion of the entire POU Pou2f2 Pou2f2 fl/fl domain (Fig. S1A). Pou2f2 mice were crossed first to FLPE 4 4 D Spleen NP30 Spleen NP4 recombinase mice (25) to excise the neomycin cassette and then 3 NP30 IgG13 NP30 IgG1 100 p = 0.03 100 p = 0.03 to ERT2-Cre mice in which the Cre recombinase is tamoxifen 7 p < 0.0001 7 p < 0.0001 80 80 inducible (26). We confirmed correct gene targeting by Southern blotting (Fig. S1 B–D) and observed efficient Cre-mediated de- 6 6 60 60 letion in germinal center B cells and in splenic B cells (Fig. S1 E 5 5 40 40 and F). Cre-mediated deletion was associated with the persis- 20 20 Pou2f2 4 4 tence of a transcript and the production of an unstable 0 0 – F G 3 NP4 IgG1 3 NP4 IgG1 protein that lacked exons 8 11 (Fig. S1 and ). Homozygous input lymphocytes BM NP30 BM NP4 80 40 deletion of Pou2f2 was not associated with any sign of ill health 5 p < 0.0003 5 p < 0.0004 p = 0.002 p = 0.0002 Serum Ig Titre (Log10 endpoint units) (Log10 Titre Serumendpoint Ig or altered behavior in mice observed for more than 2 mo after 60 fl/wt 30 deletion. Heterozygous floxed (Pou2f2 ), Cre-expressing mice were used as controls. 4 4 40 20 ASC per million million per ASC Seven days after immunization with sheep red blood cells, the 20 10 numbers of splenic B cells, T cells, and germinal center B cells NP30 IgM NP30 IgM were normal, with no difference in the proportion of germinal 3 3 0 0 center light zone (centrocyte) and dark zone (centroblast) cells fl/fl fl/fl fl/wt fl/wt (Fig.
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