LETTERS
`
`Vol 463 | 7 January 2010 | doi:10.1038/nature08638
`
`Chronic active B-cell-receptor signalling in diffuse
`large B-cell lymphoma
`R. Eric Davis1*, Vu N. Ngo1*, Georg Lenz1*, Pavel Tolar3, Ryan M. Young1, Paul B. Romesser1,4, Holger Kohlhammer1,
`Laurence Lamy1, Hong Zhao1, Yandan Yang1, Weihong Xu1, Arthur L. Shaffer1, George Wright5, Wenming Xiao6,
`John Powell6, Jian-kang Jiang7, Craig J. Thomas7, Andreas Rosenwald8, German Ott9,
`Hans Konrad Muller-Hermelink8, Randy D. Gascoyne10, Joseph M. Connors10, Nathalie A. Johnson10,
`Lisa M. Rimsza11,12, Elias Campo13, Elaine S. Jaffe2, Wyndham H. Wilson1, Jan Delabie14, Erlend B. Smeland15,
`Richard I. Fisher12,16, Rita M. Braziel12,17, Raymond R. Tubbs12,18, J. R. Cook12,18, Dennis D. Weisenburger19,
`Wing C. Chan19, Susan K. Pierce3 & Louis M. Staudt1
`
`A role for B-cell-receptor (BCR) signalling in lymphomagenesis
`has been inferred by studying immunoglobulin genes in human
`lymphomas1,2 and by engineering mouse models3, but genetic and
`functional evidence for its oncogenic role in human lymphomas is
`needed. Here we describe a form of ‘chronic active’ BCR signalling
`that is required for cell survival in the activated B-cell-like (ABC)
`subtype of diffuse large B-cell lymphoma (DLBCL). The signalling
`adaptor CARD11 is required for constitutive NF-kB pathway
`activity and survival in ABC DLBCL4. Roughly 10% of ABC
`DLBCLs have mutant CARD11 isoforms that activate NF-kB5,
`but the mechanism that engages wild-type CARD11 in other
`ABC DLBCLs was unknown. An RNA interference genetic screen
`revealed that a BCR signalling component, Bruton’s tyrosine
`kinase, is essential for the survival of ABC DLBCLs with wild-type
`CARD11. In addition, knockdown of proximal BCR subunits
`(IgM, Ig-k, CD79A and CD79B) killed ABC DLBCLs with wild-
`type CARD11 but not other lymphomas. The BCRs in these ABC
`DLBCLs formed prominent clusters in the plasma membrane with
`low diffusion, similarly to BCRs in antigen-stimulated normal
`B cells. Somatic mutations affecting the immunoreceptor tyrosine-
`based activation motif (ITAM) signalling modules6 of CD79B and
`CD79A were detected frequently in ABC DLBCL biopsy samples but
`rarely in other DLBCLs and never in Burkitt’s lymphoma or mucosa-
`associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one
`functionally critical residue of CD79B, the first ITAM tyrosine, was
`mutated. These mutations increased surface BCR expression and
`attenuated Lyn kinase, a feedback inhibitor of BCR signalling.
`These findings establish chronic active BCR signalling as a new
`pathogenetic mechanism in ABC DLBCL, suggesting several thera-
`peutic strategies.
`DLBCL is a heterogeneous diagnostic category consisting of mole-
`cularly distinct subtypes that differ in gene expression, oncogenic
`
`aberrations and clinical outcome7,8. The ABC DLBCL subtype relies
`on constitutive NF-kB signalling to block apoptosis, but the germinal-
`centre B-cell-like (GCB) subtype does not9. Recurrent CARD11 muta-
`tions in ABC DLBCL provided genetic evidence that NF-kB signalling
`is central to its pathogenesis5. However, most ABC DLBCLs have wild-
`type CARD11 yet nonetheless rely on CARD11 to activate NF-kB
`signalling4,9.
`In normal B cells, CARD11 is engaged during antigenic stimu-
`lation of BCR signalling. Antigen specificity of the BCR is provided
`by surface immunoglobulin, but signalling is mediated by two assoc-
`iated proteins, CD79A (Ig-a) and CD79B (Ig-b)10. The CD79A–CD79B
`heterodimer is a scaffold for the assembly and membrane expression of
`the BCR and also initiates downstream signalling to the NF-kB,
`phosphatidylinositol-3-OH kinase, extracellular signal-regulated kinase
`(ERK) mitogen-activated protein (MAP) kinase and NF-AT pathways.
`Engagement of the BCR by antigen induces Src-family kinases to phos-
`phorylate tyrosines in the ITAM motifs of CD79A and CD79B. The
`tyrosine kinase Syk is activated by binding to the phosphorylated
`ITAMs, triggering a signalling cascade that involves the tyrosine kinase
`Bruton’s tyrosine kinase (BTK), phospholipase Cc and protein kinase
`Cb (PKC-b). PKC-b phosphorylates CARD11, causing it to recruit
`BCL10 and MALT1 into a multiprotein ‘CBM’ complex that activates
`IkB kinase (IKK), thereby initiating NF-kB signalling.
`A potential role for BCR signalling in ABC DLBCLs with wild-type
`CARD11 was revealed by an RNA interference screen. Two short
`hairpin RNAs (shRNAs) targeting the BCR pathway component
`BTK were highly toxic for an ABC DLBCL line with wild-type
`CARD11 (OCI-Ly10) but not for one with mutant CARD11 (OCI-
`Ly3), nor for GCB DLBCL and multiple myeloma lines (Fig. 1a and
`Supplementary Fig. 1). In subsequent survival assays, a BTK shRNA
`was toxic for four ABC DLBCL lines with wild-type CARD11 but not
`for OCI-Ly3 or six GCB DLBCL lines (Fig. 1b). BTK kinase activity
`
`1Metabolism Branch, 2Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. 3Laboratory of
`Immunogenetics, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA. 4Howard Hughes Medical Institute – National
`Institutes of Health Research Scholars Program, Bethesda, Maryland 20892, USA. 5Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
`National Institutes of Health, Bethesda, Maryland 20892, USA. 6Bioinformatics and Molecular Analysis Section, Division of Computational Bioscience, Center for Information
`Technology, National Institutes of Health, Bethesda, Maryland 20892, USA. 7NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of
`Health, 9800 Medical Center Drive, Rockville, Maryland 20850, USA. 8Department of Pathology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany. 9Department of Clinical
`Pathology, Robert-Bosch-Krankenhaus, and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, 70376 Stuttgart, Germany. 10British Columbia Cancer Agency, Vancouver,
`British Columbia, Canada V5Z 4E6. 11Department of Pathology, University of Arizona, Tucson, Arizona 85724, USA. 12Southwest Oncology Group, 24 Frank Lloyd Wright Drive, Ann
`Arbor, Michigan 48106, USA. 13Hospital Clinic, University of Barcelona, 08036 Barcelona, Spain. 14Pathology Clinic, Rikshospitalet University Hospital, N-0310 Oslo, Norway.
`15Institute for Cancer Research, Rikshospitalet University Hospital and Center for Cancer Biomedicine, Faculty Division of the Norwegian Radium Hospital, University of Oslo, N-0310
`Oslo, Norway. 16James P. Wilmot Cancer Center, University of Rochester School of Medicine, Rochester, New York 14642, USA. 17Oregon Health and Science University, Portland,
`Oregon 97239, USA. 18Cleveland Clinic Pathology and Laboratory Medicine Institute, Cleveland, Ohio 44195, USA. 19Departments of Pathology and Microbiology, University of
`Nebraska Medical Center, Omaha, Nebraska 68198, USA.
`*These authors contributed equally to this work.
`
`88
`
`©2010
`
`Coalition for Affordable Drugs IV LLC - Exhibit 1023
`Macmillan Publishers Limited. All rights reserved
`
`

`
`NATURE | Vol 463 | 7 January 2010
`
`LETTERS
`
`0
`2
`4
`6
`8 10 12 14
`Days of BTK shRNA induction
`
`BTK
`shRNA
`
`BTK
`rescue
`construct
`
`Allele-specific
`kinase inhibitor
`(PP1 analogue)
`
`–+ ––+
`
`Wild type
`Wild type
`ASKA mutant
`ASKA mutant
`Kinase dead
`
`++ +++
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Live cells (% of day 2)
`
`c
`
`12
`10
`8
`6
`4
`2
`Days of BTK shRNA induction
`
`Cell
`DLBCL
`line
`subtype
`OCI-Ly3
`ABC
`HBL-1
`ABC
`TMD8
`ABC
`U2932
`ABC
`OCI-Ly10 ABC
`BJAB
`GCB
`OCI-Ly19
`GCB
`GCB
`SUDHL-6
`SUDHL-10
`GCB
`GCB
`SUDHL-4
`GCB
`OCI-Ly7
`
`CARD11
`status
`Mutant
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`
`140
`120
`100
`80
`60
`40
`20
`0
`
`Live cells (% of day 2)
`
`b
`
`BTK shRNA 1
`
`BTK shRNA 2
`
`ABC DLBCL
`
`GCB DLBCL
`
`Multiple
`myeloma
`
`CARD11
`Mutant
`
`OCI-Ly3
`
`OCI-Ly10
`
`OCI-Ly7
`
`OCI-Ly19
`
`KMS12
`
`H929
`
`WT
`
`WT
`
`WT
`
`WT
`
`WT
`
`012345
`
`–1
`
`(log2(shRNA uninduced/induced))
`
`shRNA bar-code depletion
`
`a
`
`Figure 1 | BTK is a critical kinase for survival of ABC DLBCL cells. a, RNA
`interference screen in lymphoma and multiple myeloma cell lines. An
`shRNA library targeting 442 kinases was screened in the indicated cell lines
`as described4. Shown is the selective toxicity of two BTK shRNAs after three
`weeks in culture. Results are shown as means 6 s.d. for four independent
`transductions. WT, wild type. b, Selective toxicity of a BTK shRNA for ABC
`DLBCLs with wild-type CARD11. DLBCL cell lines were infected with a
`retrovirus that expresses BTK shRNA 1 together with green fluorescent
`
`protein (GFP). Shown is the fraction of GFP-positive cells relative to the
`GFP-positive fraction on day 2. c, BTK kinase activity is required for survival
`of ABC DLBCL cells. OCI-Ly10 cells were transduced with cDNAs encoding
`wild-type or mutant BTK (kinase-dead allele or analogue-sensitive kinase
`allele (ASKA)29). Wild-type but not kinase-dead BTK rescued cells with
`endogenous BTK knockdown. The ASKA isoform-specific kinase inhibitor
`1-NM-PP1 (2 mM) killed cells bearing the BTK ASKA allele.
`
`was required for the rescue of ABC DLBCL lines from the toxicity of
`BTK knockdown (Fig. 1c).
`The role of BTK in BCR signalling prompted us to investigate the
`reliance of ABC DLBCLs on other BCR pathway components. A
`
`CD79A shRNA killed all four ABC DLBCL lines with wild-type
`CARD11 but not the line with mutant CARD11 or the GCB
`DLBCL lines (Fig. 2a). In contrast, a CARD11 shRNA killed all
`ABC DLBCL lines, and a control shRNA was non-toxic. In the line
`
`a
`
`shCD79A
`
`shSyk
`
`b
`
`shIgM
`
`10
`
`12
`
`2
`
`4
`
`8
`
`10
`
`140
`120
`100
`80
`60
`40
`20
`0
`140
`120
`100
`80
`60
`40
`20
`0
`
`2
`
`4
`
`6
`8
`10
`Control shRNA
`
`12
`
`2
`
`4
`6
`8
`10
`Days after transduction
`d
`
`12
`
`TMD8
`
`6
`shIg-κ
`
`DLBCL
`Cell
`subtype
`line
`ABC
`OCI-Ly3
`ABC
`HBL-1
`ABC
`TMD8
`U2932
`ABC
`OCI-Ly10 ABC
`GCB
`BJAB
`OCI-Ly19
`GCB
`SUDHL-6
`GCB
`SUDHL-10
`GCB
`HT
`GCB
`SUDHL-4
`GCB
`OCI-Ly7
`GCB
`
`CARD11
`status
`Mutant
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`WT
`
`2
`
`4
`6
`8
`Days after transduction
`
`10
`
`ABC DLBCL
`HBL-1
`
`OCI-Ly10
`
`GCB DLBCL
`BJAB
`
`ABC DLBCL
`GBC DLBCL
`Burkitt's lymphoma
`Mantle-cell lymphoma
`
`3 2 1 0
`
`f
`
`BCR clusters per cell
`
`ABC
`ABC
`ABC
`GCB
`
`10–2
`
`100
`10–1
`0
`BCR diffusion (mm2 s–1)
`
`0.05
`
`0.10
`
`0.15
`
`IgM
`
`Membrane
`
`OCI-Ly10
`TMD8
`HBL-1
`BJAB
`
`1.0
`0.9
`0.8
`0.7
`0.6
`0.5
`0.4
`0.3
`0.2
`0.1
`0
`10–4 10–3
`
`Cumulative probability
`
`e
`
`p-Akt
`
`Akt
`
`p-ERK
`
`ERK
`
`p-IκB-α
`IκB-α
`
`Actin
`
`CD79A
`
`140
`120
`100
`80
`60
`40
`20
`0
`140
`120
`100
`80
`60
`40
`20
`0
`
`2
`
`4
`
`6
`8
`shCARD11
`
`2
`
`4
`6
`8
`10
`Days after transduction
`
`12
`
`140
`120
`100
`80
`60
`40
`20
`0
`140
`120
`100
`80
`60
`40
`20
`0
`
`Live cells (% of day 2)
`
`c
`
`ABC DLBCL
`cell line
`shCD79A
`induction
`
`TMD8
`+–
`
`HBL1
`–
`+
`
`OCI-Ly10
`–
`+
`
`Figure 2 | Chronic active BCR signalling in ABC DLBCL lines. a, Survival of
`DLBCL cell lines after shRNA-mediated knockdown of BCR signalling
`components CD79A, Syk and CARD11. b, Knockdown of immunoglobulin
`heavy or light chain is toxic for ABC DLBCLs with chronic active BCR
`signalling. c, Phosphoproteins in multiple signalling pathways depend on
`chronic active BCR signalling. The indicated ABC DLBCL cell lines were
`transduced with an shRNA targeting CD79A and phosphorylated, or total
`proteins were assessed by western blotting before and after shRNA induction
`
`for 48 h. d, Clustering of IgM in the plasma membrane was observed only in
`ABC DLBCL lines with chronic active BCR signalling, using TIRF
`microscopy. Plasma membrane density was revealed by membrane dye R18.
`e, Decreased diffusion of surface IgM in ABC DLBCL lines with chronic
`active BCR signalling compared with the GCB DLBCL line, as quantified by
`TIRF microscopy. f, Immobile BCR clusters are characteristic of lines
`representing ABC DLBCL but not other lymphoma types.
`
`©2010
`
`Macmillan Publishers Limited. All rights reserved
`
`89
`
`

`
`LETTERS
`
`NATURE | Vol 463 | 7 January 2010
`
`HBL-1, the knockdown of surface CD79A expression by different
`shRNAs caused a proportional decrease in surface IgM, implying that
`the toxicity of CD79A knockdown was due to a loss of surface BCR
`(Supplementary Fig. 2a). CD79B shRNAs were also toxic to ABC
`DLBCLs, and the degree of CD79B knockdown was proportional
`to the decrease in surface BCR and to toxicity (Supplementary Fig.
`2b, c). To investigate the role of the immunoglobulin receptor, we
`developed shRNAs targeting IgM and Ig-k (Supplementary Fig. 3).
`These shRNAs were also selectively toxic to ABC DLBCLs with wild-
`type CARD11, establishing a direct role for immunoglobulin in this
`signalling (Fig. 2b).
`The NF-kB pathway is activated by BCR signalling in ABC DLBCLs
`because knockdown of BTK, CD79A, CD79B and CARD11 decreased
`the expression of NF-kB target genes and inhibited IKK (Supplemen-
`tary Fig. 4). BCR signalling also activates the phosphatidylinositol-3-
`OH kinase and ERK MAP kinase pathways in these cells, because
`CD79A knockdown inhibited phosphorylation of Akt and ERK in
`addition to IkB-a (Fig. 2c).
`A subsequent focused shRNA screen suggested that other BCR
`signalling components contribute to chronic active BCR signalling,
`including Syk, BLNK, phospholipase Cc2 and PKC-b (Supplemen-
`tary Fig. 5). A Syk shRNA killed two ABC DLBCL lines with wild-type
`CARD11 (HBL-1 and TMD8) but not two others (OCI-Ly10 and
`U2932), and also had no effect on OCI-Ly3 or GCB DLBCL lines
`(Fig. 2a), despite comparable knockdown (Supplementary Fig. 6a).
`Not only was OCI-Ly10 insensitive to Syk knockdown but it also died
`with ectopic expression of wild-type but not kinase-dead Syk
`(Supplementary Fig. 6b). A previous study with a Syk inhibitor,
`R406, concluded that most DLBCLs rely on tonic BCR signalling11.
`However, R406 killed Syk-independent GCB and ABC DLBCL lines
`(including OCI-Ly10), suggesting that its toxicity in these lines may
`be due to inhibition of other kinases and not BCR signalling
`(Supplementary Fig. 6c).
`We next used total internal reflection fluorescence (TIRF) micro-
`scopy to reveal BCRs on the surface of lymphoma lines. In normal
`mouse B cells, TIRF microscopy revealed that antigen exposure
`causes BCRs to form clusters with decreased diffusion, leading to
`BCR signalling12. All five ABC DLBCL lines had prominent BCR
`clusters that were not present in 16 other lines derived from GCB
`DLBCL, Burkitt’s lymphoma or mantle-cell lymphoma (Fig. 2d, f).
`BCR clusters were also observed in biopsies from three patients with
`ABC DLBCL (Supplementary Fig. 7a). Moreover, the BCRs in ABC
`DLBCLs diffused less rapidly than those in other lymphoma lines
`(Fig. 2e, f). We observed a correlation between BCR clusters and
`phosphotyrosine accumulation in ABC DLBCL lines, suggesting that
`these structures may be actively signalling (Supplementary Fig. 7b).
`Taken together, these findings establish a continuing requirement for
`proximal BCR signalling in ABC DLBCLs with wild-type CARD11.
`Because these lines also depend on CARD11, like antigen-activated
`normal B cells, we refer to this phenomenon as ‘chronic active’ BCR
`signalling. We wish to distinguish chronic active BCR signalling from
`‘tonic’ BCR signalling. Tonic BCR signalling promotes cell survival in
`all mature mouse B cells13,14, but mice deficient in CBM components
`have relatively normal numbers of mature follicular B cells15. It there-
`fore seems likely that CARD11 is not essential for tonic BCR signalling
`but is required for chronic active BCR signalling. Moreover, chronic
`active BCR signalling is characterized by BCR clustering, which is not
`observed in resting mouse B cells that depend on tonic BCR signalling12.
`To provide genetic evidence of BCR signalling in the pathogenesis
`of ABC DLBCL, we resequenced genes in the BCR pathway in DLBCL
`cell lines and biopsies. We identified missense mutations affecting the
`first tyrosine of the CD79B ITAM motif in two cell lines, HBL-1
`(Y196F) and TMD8 (Y196H) (Fig. 3a). Both lines were heterozygous
`for this mutation, but more than 90% of the CD79B messenger RNA
`in HBL-1 was derived from the mutant allele (data not shown). These
`mutations prompted us to resequence the CD79B ITAM region in
`225 DLBCL biopsies. In 18% (29 out of 161) of ABC DLBCLs, the
`
`90
`
`first ITAM tyrosine was replaced by a variety of amino acids as a
`result of point mutations; in one case, this residue was removed by a
`three-base-pair deletion (Fig. 3a, b). Less common were missense
`mutations in other ITAM residues and deletions that disrupted all
`or part of the motif. Of 64 GCB DLBCLs, only one had a mutation
`affecting the first ITAM tyrosine and one other had a different ITAM
`mutation (L199Q). Overall, the frequency of CD79B ITAM muta-
`tions was significantly higher in ABC DLBCL (21.1%) than in GCB
`DLBCL (3.1%) (P 5 8.9 3 1024). CD79B ITAM mutations were not
`present in 20 Burkitt’s lymphoma and 16 gastric mucosa-associated
`lymphoid tissue (MALT) lymphoma biopsies. In six cases of ABC
`DLBCL, analysis of non-malignant
`tissue established that
`the
`CD79B mutations were somatically acquired by the malignant cells
`(Supplementary Fig. 8).
`The CD79A ITAM region of the ABC DLBCL line OCI-Ly10 has a
`splice-donor-site mutation16 causing an 18-amino-acid deletion that
`removes most of the ITAM, including the second tyrosine. Though
`OCI-Ly10 was heterozygous for this mutation, more than 90% of the
`CD79A mRNA was mutated (data not shown). One ABC DLBCL
`biopsy had a similar splice-site mutation and another had mutations
`that deleted the entire CD79A ITAM (Fig. 3a). CD79A mutations
`were rare among ABC DLBCLs, occurring in 2.9% (2 out of 68) of
`biopsies.
`In mouse B cells, mutations in the CD79A or CD79B ITAM
`tyrosine residues elevate surface BCR expression by inhibiting recep-
`tor internalization17. Indeed, GCB DLBCL cells reconstituted with
`CD79A or CD79B mutants derived from ABC DLBCLs had more
`surface IgM expression than cells with wild-type isoforms, but this
`was not true of CD79 ITAM mutations that were not observed in
`samples from patients (Fig. 3c). Similarly, ABC DLBCL cells recon-
`stituted with mutant CD79B had higher surface BCR expression than
`those reconstituted with wild-type CD79B (Fig. 3d). Interruption of
`chronic active BCR signalling with the kinase inhibitor dasatinib (see
`later) increased surface BCR expression in ABC DLBCL cells with
`wild-type but not mutant CD79B (Fig. 3d). Hence, one function of
`the CD79 mutations is to maintain surface BCR expression in the face
`of chronic active BCR signalling.
`We speculated that the CD79B mutations might be genetically
`selected in ABC DLBCLs for their ability to circumvent negative
`regulatory circuits that attenuate BCR signalling. Whereas several
`Src-family tyrosine kinases can initiate BCR signalling, Lyn is unique
`in mediating negative feedback on BCR signalling18. Indeed, Lyn-
`deficient mice succumb to an autoimmune disease that has been traced
`to BCR hyperactivity19. Lyn is required for BCR internalization20,21,
`suggesting that CD79 mutations might elevate surface BCR expression
`by inhibiting Lyn. To test this, we knocked down endogenous CD79B
`expression in HBL-1 and TMD8 cells, both of which harbour a CD79B
`mutation, and complemented them with exogenous wild-type or
`mutant CD79B complementary DNAs. Immunoprecipitation of Lyn
`followed by an in vitro kinase assay showed greater Lyn kinase activity
`in cells reconstituted with wild-type CD79B (Fig. 3e). These data sug-
`gest a model in which CD79B mutations are selected in ABC DLBCLs
`to attenuate negative autoregulation by Lyn during chronic active BCR
`signalling.
`The CD79 mutants are not loss-of-function mutants because they
`prevented death of ABC DLBCL cells caused by knockdown of endo-
`genous CD79 isoforms (Supplementary Fig. 9). However, the CD79
`mutants were not functionally superior to their wild-type counter-
`parts in this assay (Supplementary Fig. 9) and did not spontaneously
`activate NF-kB when introduced into GCB DLBCL cells, unlike
`CARD11 mutants5 (data not shown). We therefore propose that
`the CD79 ITAM mutations may be selected early in the genesis of
`the malignant clone, perhaps to allow it to respond abnormally well
`to a self or foreign antigen (Supplementary Fig. 10). In this regard it is
`notable that mutations that impair CD79A or CD79B ITAM function
`in mouse B cells lead to exaggerated antigenic responses17,22,23. Future
`research should investigate the potential role of antigenic stimulation
`
`©2010
`
`Macmillan Publishers Limited. All rights reserved
`
`

`
`NATURE | Vol 463 | 7 January 2010
`
`LETTERS
`
`CD79B Y196 ITAM mutation
`CD79B ITAM deletion
`CD79B other ITAM mutation
`
`34/161
`
`2/64
`
`ABC
`DLBCL
`
`GCB
`DLBCL
`
`0/20
`0/16
`Burkitt's MALT
`
`a
`
`Y196H
`(10×)
`
`Y196D
`(4×)
`
`Y196C
`(5×)
`Y196S
`(2×)
`
`Y196N
`(6×)
`Y196F
`(3×)
`ΔY196
`(1×)
`
`b
`
`210
`193
`CD79B
`
`DHTYEGLDIDQTATYEDIDHTYEGLDIDQTATYEDI
`ITAM
`E197G
`L199Q
`(1×)
`(1×)
`H194Q; Δ195–197
`(1×)
`Δ205–229
`(1×)
`
`Δ196–229
`(1×)
`
`Δ193–229
`(1×)
`
`202
`185
`CD79A
`
`ENLYEGLNLDDCSMYEDIENLYEGLNLDDCSMYEDI
`ITAM
`
`Δ191–226
`(1×)
`
`Δ179–226
`(1×)
`
`Δ191–208
`(1×)
`
`10
`0
`Lymphoma
`Subtype
`
`100
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`CD79B mutation frequency (%)
`
`DMSO
`
`Dasatinib
`
`Dasatinib
`
`DMSO
`
`CD79B
`isoform
`Mutant
`Wild type
`
`2.0
`
`1.6
`
`1.2
`
`0.8
`
`0.4
`
`0
`
`Relative surface IgM (MFI)d
`
`Synthetic
`CD79B mutants
`
`CD79B Y207F
`(ITAM 2nd TYR
`mutation)
`CD79B WT
`
`CD79B Y207A
`(ITAM 2nd TYR
`mutation)
`CD79B WT
`
` 3
`
` 3
`
` 2
`
` 1
`
` 0
`
` 3
`
` 2
`
` 1
`
`Synthetic
`CD79A mutants
`
`CD79A YY190/201FF
`(ITAM TYR mutations)
`CD79A WT
`
`CD79A Y212F
`(BLNK binding
`mutation)
`CD79A WT
`
`ABC DLBCL
`CD79B mutants
`
`CD79B Y196H
`(ABC DLBCL 482)
`CD79B WT
`
`CD79B Y196C
`(ABC DLBCL 428)
`CD79B WT
`
` 3
`
` 2
`
` 1
`
` 0
`
` 3
`
` 2
`
` 1
`
`CD79B Y196F
`(ABC DLBCL 538/
`HBL-1)
`CD79B WT
`
`CD79B E197G
`(ABC DLBCL 687)
`CD79B WT
`
` 3
`
` 2
`
` 1
`
` 0
`
` 3
`
` 2
`
` 1
`
` 3
`
` 2
`
` 1
`
` 0
`
` 3
`
` 2
`
` 1
`
`ABC DLBCL
`CD79A mutants
`
`CD79A Δ179–226
`(ABC DLBCL 676)
`CD79A WT
`
`CD79A Δ191–208
`(OCI-Ly10)
`CD79A WT
`
` 3
`
` 2
`
` 1
`
` 0
`
` 3
`
` 2
`
` 1
`
`c
`
`log10(Surface IgM)
`
` 1
`
` 2
`
` 3
`
` 0
` 0
`
` 1
`
` 2
`
`CD79B reconstitution
`WT Y196F WT Y196F WT Y196F WT Y196F
`
`Expt 2
`Expt 1
`HBL-1
`
`Expt 2
`Expt 1
`TMD8
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`CD79B cells)
`
`Lyn kinase activity (% WT
`
` 0
` 0
`
` 1
`
` 2
`
` 3
`
` 0
` 0
`
` 1
`
` 2
`
` 3
`
`CD79B reconstitution
`WT
`Y196F
`–
`+
`
`+
`
`–
`
`e
`
`IP
`Lyn
`
`ATP
`WB
`p-Tyr
`(4G10)
`
`WB
`Lyn
`
` 0
` 0
` 0
` 3
` 2
` 1
` 0
`Retroviral/CD79A RNA (log10(CD8))
`
`CD79B
`reconstitution
`WT Y196F
`
`In vitro
`kinase
`
`Flag–CD79B
`
`Actin
`
`Figure 3 | CD79A and CD79B ITAM mutations in ABC DLBCL. a, CD79B
`and CD79A ITAM mutations in DLBCL biopsies and lines (case number in
`parenthesis). b, CD79B ITAM mutation frequencies in lymphoma biopsies.
`c, Mutant CD79A and CD79B isoforms increase surface IgM. The GCB
`DLBCL line BJAB was reconstituted with either wild-type or mutant
`CD79A/B proteins. Surface IgM is depicted relative to CD79 RNA levels,
`estimated with bicistronic expression of CD8. ‘Synthetic’ mutants were not
`observed in patient samples. d, CD79B mutations prevent downmodulation
`of surface BCR by BCR signalling. The ABC DLBCL line HBL-1 was
`reconstituted with wild-type or Y196H mutant CD79B and treated for 24 h
`
`with dimethylsulphoxide (DMSO) or dasatinib, a BCR signalling inhibitor.
`Surface IgM (mean fluorescence intensity; MFI) is depicted relative to the
`levels in cells with wild-type CD79B treated with DMSO. Results are shown
`as means 6 s.e.m. for two experiments. e, CD79B mutations inhibit Lyn
`kinase activity in ABC DLBCLs. The indicated ABC DLBCL lines were
`reconstituted with wild-type or Y196F mutant CD79B. Lyn kinase activity in
`immunoprecipitates (IP) was estimated by densitometric analysis of western
`blots (WB) as phospho-Lyn (using anti-phosphotyrosine antibody 4G10)
`relative to total Lyn.
`
`b
`
`ABC DLBCL
`cell line
`
`HBL-1
`
`TMD8
`
`OCI-Ly10
`
`0 1 2 4
`
`0 0.5 1 2 4
`
`0 0.5 1 2 4
`
`Dasatinib
`Rx (h)
`p-IκB-α
`IκB-α
`p-Akt
`p-ERK
`Akt
`ERK
`p-Lyn
`Lyn
`
`p-Tyrosine
`
`β-Actin
`
`0
`
`3.12
`
`25
`12.5
`6.25
`PCI-32765 (nM)
`
`50
`
`100
`
`Chronic active
`BCR signalling
`
`–+++ ––
`
`DLBCL
`subtype
`ABC
`ABC
`ABC
`ABC
`GCB
`GCB
`
`OCI-Ly10
`HBL1
`TMD8
`OCI-Ly3
`BJAB
`OCI-Ly19
`
`120
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`12.5
`
`50
`25
`200
`100
`Dasatinib (nM)
`
`400
`
`800
`
`0
`
`1.56 3.12 6.25 12.5 25
`IKK-β inhibitor (µM)
`
`a
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Live cells (% of control)
`
`Figure 4 | Therapeutic strategies to target chronic active BCR signalling.
`a, Viability of DLBCL lines assessed by assay with 3-(4,5-dimethylthiazol-2-
`yl)-2,5-diphenyltetrazolium bromide (MTT) after four days of treatment
`with various doses of dasatinib, the BTK inhibitor PCI-32765 (compound 13
`
`in ref. 25) or an IKK-b inhibitor27. b, Effect of dasatinib on phosphoprotein
`levels in ABC DLBCL cells. Three ABC DLBCL lines were treated with 50 nM
`dasatinib for the indicated durations and analysed by western blotting. Rx,
`treatment.
`
`©2010
`
`Macmillan Publishers Limited. All rights reserved
`
`91
`
`

`
`LETTERS
`
`NATURE | Vol 463 | 7 January 2010
`
`in chronic active BCR signalling and in the spontaneous BCR
`clustering that characterizes ABC DLBCLs. BCR clustering does
`not depend on the CD79B mutations (Supplementary Fig. 11), sug-
`gesting that other mechanisms contribute to this aspect of chronic
`active BCR signalling.
`We considered therapeutic strategies to exploit chronic active BCR
`signalling in ABC DLBCL. Dasatinib, a kinase inhibitor approved for
`the treatment of chronic myelogenous leukaemia, inhibits Src-family
`kinases and BTK24. Dasatinib killed ABC DLBCL lines that rely on
`chronic active BCR signalling but not the BCR-independent line
`OCI-Ly3 or GCB DLBCL lines (Fig. 4a). A selective BTK inhibitor,
`PCI-32765 (ref. 25), was also selectively toxic to cell lines with
`chronic active BCR signalling (Fig. 4a). By contrast, all ABC
`DLBCL lines were sensitive to an IKK-b inhibitor. In BCR-dependent
`lines, dasatinib decreased the phosphorylation of IkB-a, Akt, ERK
`and Lyn, as well as total protein tyrosine phosphorylation and IKK
`activity (Fig. 4b and Supplementary Fig. 12). Dasatinib toxicity may
`therefore be due to NF-kB inhibition, which causes apoptosis, and
`Akt/mTOR inhibition, which causes ‘metabolic catastrophe’26.
`Indeed, rapamycin, an mTOR inhibitor, synergized with an IKK-b
`inhibitor in killing ABC DLBCL lines with chronic active BCR signal-
`ling (Supplementary Fig. 13). Our studies suggest that the position of
`molecular lesions in the BCR and NF-kB signalling pathways could be
`used to guide the therapy of ABC DLBCL. ABC DLBCLs with wild-
`type CARD11 and chronic active BCR signalling might respond to a
`BTK inhibitor, such as PCI-32765, and possibly to inhibitors of
`Src-family kinases, PKC-b or Syk,
`in some cases. By contrast,
`CARD11-mutant tumours would need to be treated with agents
`that target downstream components of the NF-kB pathway such as
`IKK27. A precise delineation of which ABC DLBCL cases depend on
`chronic active BCR signalling awaits the development of predictive
`biomarkers and the results of clinical trials involving BCR signalling
`inhibitors.
`
`METHODS SUMMARY
`Cell lines possessing the ecotropic retroviral receptor and the tetracycline repressor
`were generated and used in RNA interference library screening, shRNA toxicity
`assays and cDNA complementation studies as described4. DLBCL cell lines were
`assigned to the ABC or GCB subtypes by gene expression profiling4 (Sup-
`plementary Fig. 14). shRNA screening results are given in Supplementary Tables
`1 and 3, and shRNA sequences are listed in Supplementary Tables 2 and 3. Specific
`shRNA-mediated mRNA and protein knockdown was documented (Fig. 2c and
`Supplementary Figs 6a and 15). IKK reporter lines were engineered to express an
`IkB-a–Photinus luciferase fusion and Renilla luciferase27. TIRF imaging of the BCR
`was based on techniques described previously12.
`Tumour biopsies were obtained before treatment from patients with de novo
`DLBCL28, gastric MALT lymphoma and Burkitt’s lymphoma. All samples were
`studied in accordance with a protocol approved by the National Cancer Institute
`Institutional Review Board.
`
`Full Methods and any associated references are available in the online version of
`the paper at www.nature.com/nature.
`
`Received 18 April; accepted 4 November 2009.
`
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`kB activity is required for survival of activated B cell-like diffuse large B cell
`lymphoma cells. J. Exp. Med. 194, 1861–1874 (2001).
`10. Dal Porto, J. M. et al. B cell antigen receptor signaling 101. Mol. Immunol. 41,
`599–613 (2004).
`11. Chen, L. et al. SYK-dependent tonic B-cell receptor signaling is a rational
`treatment target in diffuse large B-cell lymphoma. Blood 111, 2230–2237 (2008).
`12. Tolar, P., Hanna, J., Krueger, P. D. & Pierce, S. K. The constant region of the
`membrane immunoglobulin mediates B cell-receptor clustering and signaling in
`response to membrane antigens. Immunity 30, 44–55 (2009).
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`mature B cells by inducible gene targeting results in rapid cell death. Cell 90,
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`activation. Nature Rev. Immunol. 4, 348–359 (2004).
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`B cell receptor genes B29 (Igb, CD79b) and mb1 (Iga, CD79a). Proc. Natl Acad. Sci.
`USA 100, 4126–4131 (2003).
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`signaling. Oncogene 23, 8001–8006 (2004).
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`initiation and down-regulation. Immunity 7, 69–81 (1997).
`20. Niiro, H. et al. The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32)
`regulates B cell antigen receptor internalization. J. Immunol. 173, 5601–5609
`(2004).
`21. Ma, H. et al. Visualization of Syk-antigen receptor interactions using green
`fluorescent protein: differential roles for Syk and Lyn in the regulation of receptor
`capping and internalization. J. Immunol. 166, 1507–1516 (2001).
`22. Kraus, M., Saijo, K., Torres, R. M. & Rajewsky, K. Ig-a cytoplasmic truncation
`renders immature B cells more sensitive to antigen contact. Immunity 11, 537–545
`(1999).
`23. Torres, R. M. & Hafen, K. A negative regulatory role for Ig-a during B cell
`development. Immunity 11, 527–536 (1999).
`24. Hantschel, O. et al. The Btk tyrosine kinase is a major target of the Bcr-Abl
`inhibitor dasatinib. Proc. Natl Acad. Sci. USA 104, 13283–13288 (2007).
`25. Pan, Z. et al. Discovery of selective irreversible inhibitors for Bruton’s tyrosine
`kinase. ChemMedChem 2, 58–61 (2007).
`26. Jin, S., DiPaola, R. S., Mathew, R. & White, E. Metabolic catastrophe as a means to
`cancer cell death. J. Cell Sci. 120, 379–383 (2007).
`27. Lam, L. T. et al. Small molecule inhibitors of IkB-kinase are selectively toxic for
`subgroups of diffuse large B cell lymphoma defined by gene expression profiling.
`Clin. Cancer Res. 11, 28–40 (2005).
`28. Lenz, G. et al. Stromal gene signatures in large-B-cell lymphomas. N. Engl. J. Med.
`359, 2313–2323 (2008).
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`
`Supplementary Information is linked to the online version of the paper at
`www.nature.com/nature.
`
`Acknowledgements We thank L. Honigberg for PCI-32765, L. Dang for IKK-b
`inhibitors, S. Ansher for dasatinib, and S. Tohda for the TMD8 cell line. This
`research was supported by the Intramural Research Program of the National
`Institutes of Health, the National Cancer Institute, the Center for Cancer Research,
`the National Institute of Allergy and Infectious Disease, and the National Human
`Genome Research Institute. P.B.R. was a Howard Hughes Medical
`Institute-National Institutes of Health Research Scholar.
`
`Author Contributions R.E.D., V.N.N., G.L., P.T., R.M.Y., P.B.R., H.K., L.L. and A.L.S.
`designed and performed experiments. H.Z., Y.Y. and W.X. performed experiments.
`G.W., W.X. and J.P. analysed data. J.J. and C.J.T. synthesized reagents. A.R., G.O.,
`H.K.M.-H., R.D.G., J.M.C., N.A.J., L.M.R., E.C., E.S.J., W.H.W., J.D., E.B.S., R.I.F.,
`R.M.B., R.R.T., J.R.C., D.D.W. and W.C.C. supplied samples from patients and
`reviewed pathological and clinical data. S.K.P. supervised research. L.M.S. designed
`and supervised research and wrote the manuscript.
`
`Author Information Gene expression profiling data have been deposited in the
`Gene Expression Omnibus (GEO) under accession number GSE18