Patent Application: US-201715665443-A

Abstract:
the present invention is directed to methods and compositions for treating cancer , including , hematologic malignancies , such as b - cell malignancies , with anti - tumor antibody - tumor suppressor fusion proteins in order to selectively restore tumor suppressor gene function to cancer cells in which such tumor suppressor gene function has been lost . the present invention is also directed to methods and compositions for diagnosing cancer and for predicting and assessing response to treatment .

Description:
fls are among the most common types of non - hodgkin &# 39 ; s lymphoma ( nhl ). they are characterized by the translocation t ( 14 ; 18 )( q32 ; q21 ) and increased expression of bcl2 ( bende , smit and van noesel 2007 ). amplification of c - myc , loss of p53 and deletions of chromosome 6q have all been associated with progression and shortened survival in fl ( nanjangud , et al . 2007 ). up to 20 % of fls sustain large and hemizygous deletions of chromosome 6q11 - 27 , suggesting the presence of one or more tumor suppressor genes in this region ( offit , et al . 1993 ) ( gaidano , et al . 1992 ). clinically , fl shows persistent growth and eventual progression . outcomes have improved with the addition of the anti - cd20 antibody rituximab to standard chemotherapeutic regimens , but transplantation remains the only curative option for fl ( relander , et al . 2010 ). we used an unbiased loss - of - function screen to complement genomic analyses of tumors . using an rnai library tailored to the 6q deletions seen in fl , we identified a secreted form of the epha7 receptor ( epha7 ecd ) ( holmberg , clarke and frisén 2000 ) as a tumor suppressor . hemizygous loss of epha7 occurs in 12 % of fls , and the gene is differentially silenced in up to 72 % of fls . in vivo knockdown of epha7 accelerates lymphoma development in mouse models of fl . conversely , the purified epha7 ecd protein has striking anti - tumor effects on xenografted human lymphoma cells . moreover , by fusing epha7 to an anti - cd20 antibody , we are able to target epha7 &# 39 ; s tumor suppressive activity to cd20 + lymphoma cells in vivo . thus , we identify a surprising role for epha7 ecd as an antitumor protein with significant therapeutic potential in lymphoma , and we describe a new strategy , use of anti - tumor antibody - tumor suppressor fusion proteins , to selectively restore tumor suppressor gene function to cancer cells in which such function has been lost . genomic analysis of 6q deletions in fl and burkitt &# 39 ; s lymphoma ( bl ) we conducted a systematic functional genomics study into the molecular pathogenesis of fl ( fig1 a ). first , we analyzed 64 fls representing pathological grades i - iii ( grade i , 21 ; ii , 23 ; iiia , 16 ; iiib , 8 ) by array - comparative genomic hybridization ( acgh ). we observed 92 common ( i . e ., present in more than 10 % of fls tested ) regions of deletion ( crd ); 38 were tumor - specific and not found in the reference dna , four were physiological and 50 were copy number variations ( cnvs ) ( fig1 b ). consistent with previous cytogenetic studies ( offit , et al . 1993 ) ( gaidano , et al . 1992 ) ( hauptschein , et al . 1998 ), we found that deletions affecting chromosome 6q11 - 27 occurred in 23 % of fls ( 15 / 64 cases ). individual cases showed a heterogeneous pattern of 6q loss ( fig2 ). cumulative analyses revealed crds that ranged from 5 kilobases ( kb ) ( crd11 ) to 27 megabases ( mb ) ( crd4 ) and harbored between one and 78 genes ( fig1 c and 1 d ). almost all the deletions were hemizygous , and small regions of apparent homozygous loss within crd4 and crd11 did not affect any genes . analysis of six hiv - associated burkitt &# 39 ; s lymphomas ( bls ) revealed partially overlapping 6q deletions in two cases ( fig1 d , fig3 ). thus , while the size and complex patterns of hemizygous 6q deletions in fl suggest the presence of multiple tumor suppressor genes in this region , genomic data alone do not directly pinpoint a specific tumor suppressor gene . unbiased rnai screen identifies epha7 as a tumor suppressor gene in 6q11 - 27 given the complexity of 6q deletions in fl , we wondered whether an unbiased deletion - specific loss - of - function screen could point to potential tumor suppressor genes . we constructed a library of 260 short hairpin rnas ( shrnas ) targeting 84 genes ( 1 - 7 shrnas per gene ) in a murine stem cell virus ( mscv )- based , green fluorescent protein ( gfp )- expressing vector . we used non - transformed murine pro - b lymphocytes ( fl5 - 12 cells ) engineered to express increased levels of bcl2 as a surrogate in vitro system and screened for shrnas that protect cells from cytokine ( interleukin - 3 ; il - 3 ) depletion ( mavrakis , et al . 2010 ) ( fig4 a ). briefly , we partially transduced fl5 - 12 cells overexpressing bcl2 with the pooled 6q deletion library or empty vector and monitored for enrichment of cells expressing gfp ( and shrnas ) following il - 3 depletion ( fig4 b ). sequencing identified the shrnas present in the enriched population ( fig4 c ), and individual re - testing confirmed a protective effect for shrnas targeting the tumor necrosis factor , alpha - induced protein 3 ( tnfaip3 ; a20 ) and epha7 genes ( fig4 d , fig5 and 6 ). hence , our rnai screen identifies a known tumor suppressor ( tnfaip3 ) ( compagno , et al . 2009 ) and points to epha7 as a new candidate tumor suppressor gene . in vivo knockdown of epha7 accelerates lymphoma development in mouse models of fl next , we tested the effect of epha7 in a mouse model of fl . briefly , the vavp - bcl2 model recapitulates the genetics and morphology of human fl ( egle , et al . 2004 ). we transduced vavp - bcl2 transgenic hematopoietic progenitor cells ( hpcs ) with retroviral shrna constructs and transplanted these genetically engineered cells into irradiated recipients ( wendel , et al . 2004 ) ( fig4 e ). ninety percent of control animals remained tumor free for more than 100 days ( vector ; n = 11 ). c - myc and p53 have established roles in fl transformation ( nanjangud , et al . 2007 ), and enforced c - myc expression ( p & lt ; 0 . 01 ; n = 7 ) and p53 knockdown ( median survival 60 days , p & lt ; 0 . 01 ; n = 9 ) both accelerated lymphomagenesis in vivo . epha7 knockdown had an effect on tumor latency similar to that of p53 knockdown ( p & lt ; 0 . 01 ; n = 18 ) ( fig4 . knockdown of tnfaip3 alone or in combination with knockdown of epha7 in the vavp - bcl2 model showed modest effects on lymphoma latency [ p ( tnfaip3 vs . vector ) = 0 . 28 ; n = 3 and p ( tnfaip3 + epha7 vs . epha7 ) = 0 . 93 ; n = 5 )] ( fig7 ). analysis of tumors induced in vavp - bcl2 mice revealed features of fls . these included follicular structures and peanut agglutinin ( pna ) positivity , consistent with a germinal center ( gc ) b - cell phenotype . the lymphomas had overall low but heterogeneous ki67 , while apoptosis was absent . only the vavp - bcl2 / c - myc tumors grew in a diffuse pattern resembling diffuse large b - cell lymphoma ( dlbcl ) ( fig4 g , fig8 ). all the tumors expressed b - cell markers ( b220 , cd19 ), and exhibited varying degrees of t - cell infiltration ( egle , et al . 2004 ). sequencing of the immunoglobulin jh4 intron confirmed somatic hypermutation ( mandelbaum , et al . 2010 ) ( egle , et al . 2004 ) ( mcbride , et al . 2008 ). polymerase chain reaction ( pcr ) analysis of the immunoglobulin heavy chain locus in tumors confirmed their clonal origin ( fig9 ) ( egle , et al . 2004 ). immunoblots revealed epha7 expression in murine splenocytes and hpcs and partial knockdown in lymphomas ( fig9 d and 9 e , fig1 ). thus , epha7 behaves as a tumor suppressor gene in a murine model that recapitulates many aspects of the genetics and pathology of fl . we made similar observations regarding epha7 in the eμ - myc model of pre - b - cell lymphoma ( adams , et al . 1985 ). knockdown of epha7 ( n = 11 ) accelerated tumor development compared to vector ( p & lt ; 0 . 001 ; n = 60 ) ( fig1 ). epha7 is affected by hemizygous deletions in 12 % of fl , and we wondered whether epha7 might also be subject to epigenetic silencing or mutational inactivation . we noted a differential reduction of epha7 expression levels in lymphoma cells compared to gc b - cells ( fig1 a and 11 b ). we did not detect epha7 mutations in fl . however , quantitative reverse transcription - pcr ( qrt - pcr ) revealed decreased epha7 mrna levels in purified lymphoma cells . specifically , 41 of 50 fls ( 82 %) and four of six bls ( 67 %) exhibited decreased epha7 mrna levels compared to b - cells from gc or tonsils ( p ( lymphoma vs . b - cell ) & lt ; 0 . 02 ) ( fig3 a ). similarly , the epha7 protein was easily detected in normal tonsils by immunohistochemistry but completely absent in 231 of 332 fl samples ( 72 %) on a tissue microarray ( tma ) ( fig1 b and 11 c and fig1 ). mass spectrometric analysis ( massarray ) of the epha7 promoter in 32 primary fls and 16 lymphoma cell lines revealed extensive cpg island methylation consistent with epigenetic gene silencing ( fig1 d and 11 e ). analysis of a second panel of cells using the hpaii tiny fragment enrichment by ligation - mediated pcr ( help ) assay for methylation detection confirmed differential methylation of the epha7 promoter in lymphoma cells ( 9 fls , 155 dlbcls and 24 lymphoma cell lines ) vs . gc b - cells ( n = 9 ), the normal counterpart of the lymphomas tested ( fig1 ). concordantly , in vitro treatment of human lymphoma cells with 5 - aza - 2 ′- deoxycytidine caused re - expression of epha7 ( fig1 f , fig1 ). the effect was less pronounced in raji cells , which have only one copy of the epha7 gene ( fig1 ). thus , loss of epha7 expression in lymphomas is due to differential methylation of the epha7 promoter . consistent with our observations , differential silencing of epha7 has been reported in murine lymphomas and human b - lymphoblastic leukemias ( b - alls ) ( dawson , et al . 2007 ) ( kuang , et al . 2010 ). hence , evidence of differential epigenetic silencing supports the role of epha7 as a tumor suppressor gene in b - cell malignancies . b - cells express only a secreted , truncated isoform comprising the extracelluar domains of epha7 ephrin receptors are tyrosine kinases that form dimers and are activated upon contact with ephrin - expressing cells ( seiradake , et al . 2010 ) ( himanen , et al . 2010 ). the role of ephrin signaling in cancer is unclear ; both oncogenic and tumor suppressive functions have been proposed ( noren , et al . 2006 ) ( pasquale 2010 ). alternate splicing of epha7 produces a truncated protein ( designated epha7 ecd or epha7 tr ), which lacks the intracellular domains and the kinase activity of the full - length protein ( holmberg , clarke and frisén 2000 ) ( dawson , et al . 2007 ) ( valenzuela , et al . 1995 ). murine b - lymphocytes and 5 - aza - 2 ′- deoxycytidine - treated su - dhl - 10 cells express only epha7 ecd ( fig1 a , fig1 ), which is shed into the media ( fig1 a ). epha7 ecd binds to epha2 , blocking activation of epha2 and src kinases immunoprecipitation of immunoglobulin fc fragment - tagged epha7 ecd ( epha7 fc ) demonstrates binding of epha7 to the epha2 receptor in raji ( fig1 b ) and fl - derived dohh2 cells ( fig1 a ). enzyme - linked immunosorbent assay ( elisa ) reveals inhibition of epha2 phosphorylation by epha7 fc ( fig1 c ). both knockdown of epha2 and administration of epha7 fc protein block erk activation in raji cells ( fig1 d and 16 e ) and in su - dhl - 6 , dohh2 and karpas 422 cells ( fig1 c and 17 d ). unlike tumor cells , the non - transformed fl5 - 12 lymphocytes express epha7 ecd . knockdown of epha7 in fl5 - 12 cells activates erk , and this activation is reversed upon treatment of the cells with epha7 fc ( fig1 ). a phosphoprotein array identifies additional signaling effects of epha7 fc in raji cells ( fig1 a and 19 b ). we confirmed effects on erk , stats and src phosphorylation by immunoblot and note some differences between different lymphoma lines ( fig1 e , fig1 d and 19 e ). we modeled the interaction between epha7 and epha2 based on the known structure of epha2 ( seiradake , et al . 2010 ) ( himanen , et al . 2010 ) and its homology with the epha7 sequence ( 51 %) and domain structure . our model suggests an interaction through the receptors &# 39 ; sushi and ligand binding domains ( fig1 suggesting that epha7 ecd and epha7 fc function as decoy receptors , dimerizing with epha2 on the cell surface and inhibiting epha2 activation and signaling . more generally , our model suggests that epha7 ecd and epha7 fc can dimerize with other epha receptors on the cell surface and inhibit their activation and signaling as well . restoration of epha7 activity , by retroviral transduction or application of epha7 fc , has anti - proliferative effects against raji , su - dhl - 10 , dohh2 and karpas 422 cells in vitro ( fig2 and 21 ). intravenous administration of purified epha7 fc ( 20 μg / day for 3 days ) resulted in dramatic regression of xenografted raji and su - dhl - 10 tumors while vehicle ( i . e ., fc )- treated tumors continued to grow ( n = 12 ; p ( epha7fc vs . fc ) & lt ; 0 . 04 ) ( fig1 g , fig2 ). residual eph7 fc - treated raji tumors exhibited extensive apoptosis , disrupted architecture and reduced erk phosphorylation ( fig1 h - j ). systemic administration of epha7 fc in a prevention study ( 20 μg / day intravenously for 3 days ) was well tolerated and significantly delayed development of raji lymphomas ( epha7 fc , n = 5 ; fc , n = 5 ; p & lt ; 0 . 05 ) ( fig1 k ). epha7 fc treatment of other tumor cell lines expressing high levels of cell - surface epha2 , including some breast cancer cell lines , resulted in significant inhibition of epha2 signaling as assessed by erk phosphorylation . anti - lymphoma activity of anti - cd20 - epha7 ecd fusion antibody surpasses that of anti - cd20 antibody or epha7 ecd next , we tested whether fusing epha7 ecd to an anti - cd20 antibody ( rituximab ) could further enhance the therapeutic potential of epha7 ecd ( fig1 l , fig2 a ). the fusion antibody ( anti - cd20 - epha7 ) retains properties of both proteins . it recognizes cd20 + lymphoma cells and blocks epha2 and erk phosphorylation ( fig1 m , fig2 b and 23 c ). the fusion antibody was also more efficient than anti - cd20 alone in slowing proliferation of , and killing , raji or dohh2 cells in vitro ( fig1 n and 16 o , fig2 ). in vivo treatment with either anti - cd20 or anti - cd20 - epha7 ( 1 μg / day intravenously for 5 days ) was well tolerated . in raji xenografts (& gt ; 1 cm 3 at time of treatment ), administration of low - dose anti - cd20 antibody produced partial responses or slowed progression compared to vehicle ( fc ). only the fusion antibody produced complete responses ( ex vivo tumor weight 0 - 30 mg ) in 3 of 7 animals ( p = 0 . 039 , fisher &# 39 ; s exact for all three groups ) ( fig1 p , fig2 ). thus , anti - tumor antibodies can be used to selectively restore tumor suppressor function to cancer cells in which such function has been lost . dna from fresh frozen or optimal cutting temperature compound - embedded tissue was isolated by the standard phenol - chloroform extraction method . dna was quantified using a nanodrop spectrophotometer ( thermo fisher scientific inc . ; www . nanodrop . com ), its purity assessed by the ratio of absorptions at 260 nm vs . 280 nm and its integrity visualized on a 1 % agarose gel with ethidium bromide . prior to labeling each dna sample and hybridizing it to an agilent ( agilent technologies ; www . agilent . com ) 244k oligonucleotide array , digestion efficiency was checked by incubating 1 μg of dna with 1 μl of haeiii restriction enzyme ( 10 u / μl ) at 37 ° c . for 2 hours and running the undigested and digested dna ( 100 ng each ) on a 1 % agarose gel in parallel with a 1 kb dna ladder . human male dna obtained from promega corporation ( www . promega . com ; catalog # g147a ) served as the reference dna . labeling and hybridization were performed according to protocols provided by agilent . the slides were analyzed at 5 μm resolution using the agilent g2565 microarray scanner system and agilent feature extraction software ( v9 . 1 ). with the exception of fig1 , which uses the ucsc march 2006 human reference sequence ( hg18 / ncbi build 36 ), all genomic positions described in this study refer to ucsc may 2004 human reference sequence ( hg17 / ncbi build 35 ) ( http :// genome . ucsc . edu / cgi - bin / hggateway ). the modified circular binary segmentation ( cbs ) algorithm was used to identify segmental gains and losses along the autosomes . change - points were defined as segments , corresponding to p - values & lt ; 0 . 05 . a crd was defined as a gain and / or loss of two contiguous probes observed in & gt ; 10 % of the cases . a total of 92 crds were identified . of these , four were physiological changes ( b - and t - cell receptors ), 50 were cnvs and the remaining 38 were tumor - specific . the database of genomic variants ( dgv ), a catalogue of structural variations curated by the centre for applied genomics ( tcag ) ( http :// projects . tcage . ca / variation /) was used to identify and exclude cnvs . at the time of data analysis , the dgv comprised 8 , 083 entries that mapped to 3 , 933 genomic loci [ variation . hg17 . v2 . txt ; sep . 5 , 2007 ( build 35 / hg17 )]. cgh data was further processed using agilent &# 39 ; s feature extraction software to quantitate the images . for normalization , we used a custom gc - normalization algorithm , which does a loess norm on both the total intensity of the probes and the local gc content in the genomic region surrounding the probes . the normalized data were segmented using dnacopy , the standard cbs algorithm available from bioconductor ( http :// www . bioconductor . org / help / bioc - views / release / bioc / html / dnacopy . html ). each sample was then normalized to its own per - sample noise by dividing the segment means by the derivative noise ( the mean absolute values of the difference between adjacent probes on the arrays ). the next step used the rae algorithm ( taylor , et al . 2008 ) to do a multi - sample analysis of the entire cohort . the rae algorithm computes per - sample - calibrated thresholds , which can be used to compare signal levels across samples . the threshold function converts the log 2 ratio signals from the cbs output via two logistic functions to a loss [− 1 , 0 ] and gain [ 0 - 1 ] indicator output that is then averaged to give the genome - wide gain / loss recurrence frequency ( plotted in fig1 ). for the chromosome 6q loss analysis , we used a global threshold of − 0 . 5 for loss and 4 . 5 for homozygous deletion . fig3 shows the segment boundaries as computed by cbs with the vertical axis indicating the magnitude of the segment mean . see taylor , et al . ( 2008 ) for additional details . genomic dna was extracted from the lymphomas arising in transgenic vavp - bcl2 mice and from the lymphomas derived from transplanted vavp - bcl2 hsc . for dna extraction , frozen tissue was submerged in liquid nitrogen then pulverized . the resulting powder was collected and transferred to a microfuge tube on ice . dna was purified using the gentra puregene cell kit ( qiagen ; www . qiagem . com ) and diluted in water , and dna quality was assessed by visualization after electrophoresis in a 1 % agarose gel . dj recombination in murine tumors was analyzed by nested pcr as described ( yu and thomas - tikhonenko 2002 ), and samples were analyzed using an agilent 2100 bioanalyzer and dna 1000 kit . for analysis of somatic hypermutation analyses , dna samples were amplified as described ( mcbride , et al . 2008 ), and the pcr products were directly sequenced exactly as reported ( mandelbaum , et al . 2010 ). quantitative dna methylation analysis was carried out using massarray epityper from sequenom , inc . ( www . sequenom . com ). the massarray epityper is a tool for the detection and quantitative analysis of dna methylation using base - specific cleavage of bisulfite - treated dna and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ms ). specific pcr primers for bisulfite - converted dna were designed using sequenom &# 39 ; s epidesigner program ( www . epidesigner . com ). t7 - promoter tags were added to the reverse primer to obtain a product that could be transcribed in vitro , and a 10 - mer tag was added to the forward primer to balance the pcr conditions . unmethylated cytosine in 1 μg of tumor dna was converted into uracil by bisulfite treatment with the ez - 96 dna methylation kit ( zymo research corporation ; www . zymoresearch . com ) according to the manufacturer &# 39 ; s instructions . pcr reactions were carried out in duplicate with each of the two selected primer pairs , for a total of four replicates per sample . for each replicate , 1 ml of bisulfite - treated dna was used as template for a 5 - ml pcr reaction in a 384 - well microtiter plate , using 0 . 2 units of kapa2g fast hotstart dna polymerase ( kapa biosystems ; www . kapabiosystems . com ), 200 mm dntps and 400 nm of each primer . cycling conditions were : 94 ° c . for 15 minutes ; 45 cycles of 94 ° c . for 20 seconds , 56 ° c . for 30 seconds , 72 ° c . for 1 minute ; and one final cycle at 72 ° c . for 3 minutes . unincorporated dntps were deactivated using 0 . 3 u of shrimp alkaline phosphatase ( sap ) in 2 ml , at 37 ° c . for 20 minutes , followed by heat inactivation at 85 ° c . for 5 minutes . two ml of sap - treated reaction were transferred into a fresh 384 - well microtiter plate , and in vitro transcription and t cleavage were carried out in a single 5 - ml reaction mix , using the masscleave kit ( sequenom ) containing 1 × t7 polymerase buffer , 3 mm dithiothreitol ( dtt ), 0 . 24 ml of t cleavage mix , 22 u t7 rna and dna polymerase and 0 . 09 mg / ml rnase a . the reaction was incubated at 37 ° c . for 3 hours . after the addition of a cation exchange resin to remove residual salt from the reactions , 10 nl of epityper reaction product was loaded onto a 384 - element spectrochip ii array ( sequenom ). spectrochis were analyzed using a bruker biflex iii maldi - tof mass spectrometer ( spectroreader , sequenom ). results were analyzed using the epityper analyzer software and manually inspected for spectra quality and peak quantification . in vitro treatment with 5 - aza - 2 ′- deoxycytidine was as described ( mavrakis , et al . 2008 ). help ( hpaii tiny fragment enrichment by ligation - mediated pcr ) assay for dna methylation dlbcl specimens were obtained from patients at the bc cancer agency in vancouver or at weill cornell medical center . the use of human tissue was in agreement with research ethics boards of the vancouver cancer center / university of british columbia and weill cornell medical center . the help assay was performed as previously published ( shaknovich , figueroa and melnick 2010 ) using two probes located upstream of , and overlapping with , the transcriptional start site of the epha7 gene . products of help were hybridized to human hg17 custom promoter arrays ( roche nimblegen , inc . ; www . nimblegen . com ) covering 25 , 626 hpaii amplifiable fragments . data quality control and analysis were performed as described ( thompson , et al . 2008 ). after quality control processing , a quintile normalization was performed on each array . dna samples profiled by help were also subjected to bisulfite treatment and massarray epityper analysis as previously described . in order to cover all possible sites of digestion , primers were designed to cover the outermost hpaii sites of the selected hpaii amplifiable fragments ( haf ) as well as any other hpaii sites up to 2 , 000 base pairs upstream or downstream of the haf . correlation of massarray results with normalized data from help assay revealed a spearman &# 39 ; s rank correlation of r = 0 . 88 . the adjusted linear regression model was used to obtain the conversion formula . the study cohort for analysis of epha7 expression comprised fls consecutively ascertained at the memorial sloan - kettering cancer center ( mskcc ) between 1985 and 2000 . all cancer biopsies were evaluated at mskcc , and the histological diagnosis was based on hematoxylin and eosin ( h & amp ; e ) staining . use of tissue samples was approved by mskcc &# 39 ; s institutional review board and human biospecimen utilization committee . tmas were constructed as previously described ( scott , et al . 2007 ) except that a fully automated arrayer ( beecher instruments ata - 27 ) was used . tmas were pre - treated with cell conditioning solution 1 ( ventana medical systems , inc . ; www . ventanamed . com ), incubated with epha7 rabbit polyclonal antibody from abgent ( www . abgent . com ) at 1 : 50 dilution for 60 minutes and then stained with secondary anti - rabbit antibody from vector laboratories , inc . ( www . vectorlabs . com ) at 1 : 200 dilution for 60 minutes . cores were scored as 0 , 1 or 2 where 0 = no staining ; 1 = focal , weak staining ; and 2 = moderate - to - strong staining in more than 50 % of tumor cells . to construct the anti - cd20 - epha7 antibody , we amplified the epha7 ecd coding sequence by pcr from human genomic dna . the pcr product was cloned into pah6747 , which contains an igg1 constant region with an anti - cd20 heavy chain variable region ( dr . sherie morrison , ucla ). for antibody production , the anti - cd20 - epha7 heavy chain construct derived from pah6747 and an anti - cd20 light chain ( pag10818 ) construct ( dr . sherie morrison , ucla ) were co - transfected into 293t cells , and the media in which the cells were growing was replaced every other day . anti - cd20 - epha7 was purified from this conditioned media via affinity chromatography using recombinant protein a . briefly , the harvested media containing anti - cd20 - epha7 was dialyzed into 20 mm sodium phosphate ( ph 7 ), passed over a 1 ml hitrap rprotein a ff column ( ge healthcare life sciences ; www . gelifessciences . com ) and eluted with 100 mm glycine - hcl ( ph 2 . 7 ). eluant was collected in glass fraction tubes and immediately neutralized with 75 μl 1m tris - hcl ( ph 9 . 0 ) per ml of eluant . the antibody - containing peak fractions were pooled , dialyzed into phosphate - buffered saline and sterile filtered . purity was determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ), and antibody - fusion product concentration was determined with a spectrophotometer ( 280 nm ) using an extinction coefficient of 1 . 43 . elisa using anti - human igg ( h + l ) ( jackson immunoresearch laboratories , inc . ; www . jacksonimmuno . com ; # 709 - 005 - 149 ) and horseradish peroxidase - conjugated - anti - human igg fc ( jackson immunoresearch laboratories , inc . ; # 709 - 035 - 098 ) antibodies was used to verify protein purity and integrity . cell culture , cell viability and proliferation assays , vectors and pooled shrna library screen fl5 - 12 murine lymphocytes were stably transduced with bcl2 ( fl5 - 12 / bcl2 ); il - 3 depletion studies and viral transductions were as described ( mavrakis , et al . 2008 ). cell viability was assessed using the guava viacount assay ( millipore corporation ; www . millipore . com and lds751 cell - permeant nucleic acid stain ( invitrogen ; www . invitrogen . com ) as previously described ( mavrakis , et al . 2008 ). the retroviral constructs utilized are based on mscv and include bcl2 ( wendel , et al . 2004 ) and individual or pooled shrna constructs ( dickins , et al . 2005 ). pooled shrna screening technology has been described ( mavrakis , et al . 2010 ). briefly , the shrna library was constructed by pooling the individually cloned shrnas . the screen design is depicted in fig4 a . fl5 - 12 / bcl2 cells were transduced at low multiplicity of infection with the library pool containing 262 shrnas and subjected to 7 days of il - 3 depletion . after recovery of viability , samples were collected for dna isolation . integrated shrnas were amplified by pcr , subcloned into the pgem - t easy vector ( promega ) and identified by shrna sequencing . individual ‘ hits ’ from the screen were re - tested in the same in vitro assay and confirmed using multiple shrnas against the same genes . the shrnas against pten and p53 have been previously reported ( mavrakis , et al . 2010 ) ( wendel , et al . 2006 ). the vavp - bcl2 model of fl ( egle , et al . 2004 ) and the adoptive transfer of retrovirally transduced hpcs ( wendel , et al . 2004 ) have been described . data were analyzed in kaplan - meier format using the log - rank ( mantel - cox ) test for statistical significance . h & amp ; e staining , terminal deoxynucleotidyl transferase dutp nick end labeling ( tunel ) and analyses for ki67 , cleaved caspase , b220 and other surface markers were as described by mavrakis , et al . ( 2008 ). tumor xenografts were established by subcutaneous injection of 1 × 10 6 raji or su - dhl - 10 human lymphoma cells mixed with matrigel ( bd biosciences ; www . bdbiosciences . com ) into the flanks of nod / scid ( nod . cb17 - prkdc ( scid )/ j ) mice . once tumors exceeded 1 cm 3 in size , mice were treated by three intratumoral injections of vehicle or 20 μg epha7 fc ( recombinant mouse epha7 fc chimera ; r & amp ; d systems ; www . rndsystems . com ). tumors were weighed and volumes were measured as described ( bergers , et al . 1999 ). systemic administration of epha7 fc and anti - cd20 - epha7 was by tail vein injection . immunoblots were performed from whole cell lysates or supernatant as described ( wendel , et al . 2004 ). briefly , 50 μg of protein per sample were resolved on sds - page gels , transferred to immobilon - p transfer membranes ( millipore ) and probed with antibodies against epha7 ( santa cruz biotechnology , inc . ; www . scbt . com ; # sc - 917 diluted 1 : 200 ), epha2 ( millipore , inc . # 05 - 480 diluted 1 : 1000 ), bcl2 ( santa cruz biotechnology , inc . # sc - 509 diluted 1 : 500 ), c - myc ( santa cruz biotechnology , inc . # sc - 40 diluted 1 : 200 ), phosphorylated eif4e - bp1 ( cell signaling technology , inc . ; www . cellsignal . com ; # 9451 diluted 1 : 1000 ), phosphorylated erk1 / 2 ( cell signaling technology , inc . # 9101 diluted 1 : 800 ), erk1 / 2 ( cell signaling technology , inc . # 9102 diluted : 1000 ), phosphorylated src ( cell signaling technology , inc . # 2101 diluted 1 : 1000 ), phosphorylated s6 ribosomal protein ( cell signaling technology , inc . # 2215 diluted 1 : 1000 ), phosphorylated akt ( cell signaling technology , inc . # 4058 diluted 1 : 1000 ) and tubulin ( sigma - aldrich co . ; www . sigmaaldrich . com ; # t5168 diluted 1 : 5000 ). blots were developed chemiluminescently using the amersham ecl western blotting system ( ge healthcare life sciences ). elisa for phosphorylated epha2 in raji cell lysates was performed utilizing the human phospho - epha2 duoset ic ( r & amp ; d systems # dyc4056 - 2 ) according to the manufacturer &# 39 ; s instructions . a human phosphoprotein detection array ( r & amp ; d systems # ary003 ) was probed with cell lysates according to the manufacturer &# 39 ; s instructions . in addition to the epha7 fc protein obtained from commercial sources ( r & amp ; d systems ; see above ), we produced an identical protein using a baculoviral expression system . a dna fragment corresponding to epha7 amino acids lys31 through asn525 was cloned into the bamh1 / not1 sites of the pacgp67b - based ( bd biosciences ) pma152 , a baculovirus vector with an igg fc - tag at the c - terminus of the protein - coding region ( antipenko , et al . 2003 ) ( xu , et al . 2008 ). the recombinant baculovirus constructs were co - transfected , with baculogold linearized baculovirus dna ( bd biosciences ), into sf9 insect cells . passage four recombinant baculovirus was used to infect hi - 5 cells in suspension at a density of 1 . 8 × 10 6 cells / ml in sf - 900 ii sfm protein - free insect cell culture medium ( invitrogen ). infected cells were grown at 27 ° c . and 100 rpm and harvested after 64 hours . hi - 5 cell supernatant containing the secreted epha7 fc was loaded onto a protein a sepharose column and eluted by a step - wise ph gradient fractionation in 100 mm glycine . the yield was 1 - 2 mg protein per liter of hi - 5 cell suspension ( antipenko , et al . 2003 ) ( xu , et al . 2008 ). total rna was extracted from tumor samples and cell lines using the allprep dna / rna / protein mini kit ( qiagen ). cdna synthesis , real - time - pcr and analysis by the δδ ct method were performed as described ( mavrakis , et al . 2008 ). epha7 pcr utilized tttcaaactcggtacccttca as forward primer and cattgggtggagaggaaatc as reverse primer and sybr green detection ; glyceraldehyde 3 - phosphate dehydrogenase ( gapdh ) was used as a standard ( gagtcaacggatttggtcgt forward primer , gacaagcttcccgttctcag reverse primer and sybr green detection ); and human beta glucuronidase ( gusb ; applied bio systems ; www . appliedbiosystems . com ; # 4333767f ) was used as an endogenous control . sequencing of genomic dna was performed as described ( veeriah , et al . 2010 ). genomic dna was amplified using a repli - g midi kit ( qiagen ). the exonic regions of interest ( ncbi build 36 . 1 ) were broken into amplicons of 500 bp or less , and the primer3 program was used to design specific primers covering exonic regions plus at least 50 base pairs of flanking intronic sequence . m13 tails were added to facilitate sanger sequencing . pcr reactions were carried out in 384 - well plates in a duncan dt - 24 ( kbiosystems limited ; www . kbiosystems . com ) water bath thermal cycler , with 10 ng of amplified dna as template , using a touchdown pcr protocol with taq hotstart dna polymerase ( kapa biosystems ). the touchdown cycling conditions were : 95 ° c . for 5 minutes ; three cycles of 95 ° c . for 30 seconds , 64 ° c . for 30 seconds , 72 ° c . for 60 seconds ; three cycles of 95 ° c . for 30 seconds , 62 ° c . for 30 seconds , 72 ° c . for 60 sec ; three cycles of 95 ° c . for 30 sec , 60 ° c . for 30 seconds , 72 ° c . for 60 seconds ; 37 cycles of 95 ° c . for 30 seconds , 58 ° c . for 30 seconds , 72 ° c . for 60 seconds ; and one final cycle at 70 ° c . for 5 minutes . the resulting dna sequencing templates were purified using agencourt ampure ( beckman coulter , inc . ; www . beckmangenomics . com ). the purified pcr reaction products were split in two , and sequenced bidirectionally with m13 forward and reverse primers and bigdye terminator v3 . 1 cycle sequencing kit ( applied biosystems ). dye terminators were removed using the agencourt cleanseq kit ( beckman coulter , inc . ), and sequence reactions were run on an abi prism 3730xl sequencing apparatus ( applied biosystems ). mutations were detected using an automated detection pipeline at the mskcc bioinformatics core . bi - directional sequencing reads and mapping tables ( to link read names to sample identifiers , gene names , read direction and amplicon ) were subjected to a filter that excludes reads that have an average phred score of & lt ; 10 for bases 100 - 200 . passing reads were assembled against the reference sequences for each gene , containing all coding and untranslated exons including 5 kb upstream and downstream of the gene , using command line consed 16 . 0 ( gordon , abajian and green 1998 ). assemblies were passed on to polyphred 6 . 02b ( nickerson , tobe and taylor 1997 ), which generated a list of putative candidate mutations , and to polyscan 3 . 0 ( chen , et al . 2007 ), which generated a second list of putative mutations . the lists were merged together into a combined report , and the putative mutation calls were normalized to ‘+’ genomic coordinates and annotated using the genomic mutation consequence calculator . the resulting list of annotated putative mutations was loaded into a postgresql database along with select assembly details for each mutation call ( assembly position , coverage and methods supporting mutation call ). to reduce the number of false positives generated by the mutation detection software packages , only point mutations supported by at least one bi - directional read pair and at least one sample mutation called by polyphred were considered , and only putative mutations that are annotated as having nonsynonymous coding effects , occur within an exon or within 11 base pairs of an exon boundary , or have a conservation score & gt ; 0 . 699 were included in the final candidate list . indels called by any method were manually reviewed and included in the candidate list if found to hit an exon . all putative mutations were confirmed by a second pcr and sequencing reaction , in parallel with amplification and sequencing of matched normal tissue dna . all traces for mutation calls were manually reviewed . fig1 . oncogenomic study to identify tumor suppressor genes in fl . a . the study design combines genomic tumor analyses with an rnai screen and validation in murine models and in xenografts ; b . acgh analysis of 68 fls showing frequencies of genomic gain ( red ) and loss ( blue ) across the genome ; c . high resolution depiction of recurrent gains ( red ) and losses ( blue ) affecting chromosome 6q with crds indicated ; d . mapping of crds . the observed 6q deletions are typically large and hemizygous and do not readily identify a target gene . fig2 . case - by - case analysis of gains and losses across chromosome 6q in fls . a . regions of copy number loss and gain aligned with a map of chromosome 6 . losses are indicated in blue and gains in red with color intensity representing the signal strength . location of some key genes as well as of crd4 and crd9 is indicated . b . epha7 is affected in eight of 13 deletions involving crd4 ; similarly tnfaip3 is affected in nine of twelve cases harboring crd9 deletions . c . epha7 , prdm1 and tnfaip3 are frequently co - deleted . among 64 fl cases , deletions affect epha7 in eight ( 12 . 5 %), prdm1 in six ( 9 . 4 %) and tnfaip3 in nine ( 14 %). in five of 64 fls ( 7 . 8 %), all three genes are affected . the deletions are almost entirely hemizygous , and no gene is directly affected by bi - allelic loss . the size of the deletions and the complex pattern suggest multiple targets ; however the genomic data alone do not pinpoint any specific gene associated with fl . fig3 . acgh analysis of six hiv - associated bls . a . segmental analyses corrected for cnvs reveals 6q deletions in two of six cases compared to a reference . b . whole genome changes and the proportion of bls affected . the smallest common region of deletion in 6q is extends from nucleotide 84 , 341 , 552 through nucleotide 117 , 084 , 517 , which overlaps with crds 4 - 7 in fls and includes epha7 and prdm1 . fig4 . rnai screen and in vivo validation of candidate tumor suppressor genes . a . design of a pooled , deletion - specific shrna library screen in a surrogate model ( immortalized fl5 - 12 / bcl2 cells ). b . fluorescence - activated cell sorting ( facs ) analyses of cells transduced with the pooled 6q deletion library showing enrichment of cells expressing shrnas ( and the gfp reporter ) following il - 3 depletion . c . absolute number and identity of shrna sequences retrieved from the enriched population . d . epha7 and tnfaip3 map , respectively , to the crd4 and crd9 in fl ; prdm1 did not score in this screen . e . adoptive transfer enables genetic studies in the vavp - bcl2 model of fl . f . tumor latencies for animals receiving vavp - bcl2 transgenic hpcs transduced with empty vector ( black , n = 11 ), or shrnas against epha7 ( red , n = 18 ) or p53 ( green , n = 9 ) or over - expressing c - myc ( blue , n = 7 ). g . microscopic pathology and immunohistochemistry of vavp - bcl2 lymphomas expressing the indicated constructs . red arrows indicate infiltrating tumor cells . fig5 . individual validation of the screen results . fl5 - 12 / bcl2 cells partially transduced with indicated shrna / gfp or control constructs are shifted into il - 3 - deficient media and monitored for changes in the proportion of gfp - expressing cells . an increase in the proportion of gfp - expressing cells ( indicated with red outline ) implicates co - transduced genes in the survival and proliferation of bcl2 - expressing b - cells upon il - 3 depletion . fig6 . characterization of epha7 shrnas . a . assessment of three shrnas ( sh1 - 3 ) targeting epha7 in fl5 - 12 / bcl2 cells as described in fig5 . b . lysates of facs - sorted fl5 - 12 / bcl2 cells expressing one of three shrnas targeting epha7 ( sh1 - 3 ) or vector ( v ) blotted and probed with antibodies as indicated . ( p21 has been reported as a common off - target of shrnas .) fig7 . role of tnfaip3 in vavp - bcl2 hpc recipient animals . a . tumor latencies for animals receiving vavp - bcl2 transgenic hpcs transduced with empty vector ( black , n = 11 ), or shrnas targeting epha7 ( red , n = 18 ), tnfaip3 ( green , n = 3 ) or epha7 and tnfaip3 ( blue , n = 5 ). b . lysates of fl5 - 12 / bcl2 cells expressing vector or an shrna targeting tnfaip3 blotted and probed with antibodies as indicated . fig8 . microscopic pathology and immunohistochemistry of vavp - bcl2 lymphomas . red arrows indicate infiltrating tumor cells . fig9 . molecular characterization of murine vavp - bcl2 lymphomas . a . location of primers used to analyze clonality . b . nested pcr analysis of dj recombination reveals a single band at ˜ 140 bp , indicating clonality of this vavp - bcl2 / epha7 lymphoma . c . pcr analysis of dj recombination in lymphomas arising in transgenic vavp - bcl2 animals and in mice receiving hpcs transduced with the indicated shrna constructs confirms their clonality . d . lysates from vavp - bcl2 transgenic hpcs ( hsc ) and splenocytes ( spleen ) blotted and probed with antibodies as indicated . e . whole tumor lysates of vavp - bcl2 tumors expressing c - myc or shrnas targeting epha7 , foxo3 or p53 and probed with antibodies as indicated . fig1 . candidate tumor suppressor genes in the eμ - myc lymphoma model . a . diagram comparing the 6q deletions seen in fl and bl . b . the eμ - myc model and our hpc transplantation approach . c . tumor latencies for animals receiving eμ - myc transgenic hpcs transduced with empty vector ( black , n = 60 , including concurrent and historic controls ), with shrna against epha7 ( red , n = 11 ) or with akt ( blue , n = 8 ); p ( shepha7 or akt vs . vector ) & lt ; 0 . 05 . d . lysates from vector - or shepha7 - expressing eμ - myc lymphomas blotted and probed as indicated . e . microscopic pathology and immunohistochemistry of eμ - myc lymphomas expressing the indicated constructs . fig1 . epha7 is differentially silenced in fls and expressed in gc b - cells . a . qrt - pcr results for epha7 in purified b - cells from reactive tonsils ( t ), gc , fls and bls . results are displayed as mean ± standard deviation ; p ( tumor vs . normal ) & lt ; 0 . 05 for fl and bl . b . immunohistochemical detection of the epha7 protein in a normal tonsil . c . representative section of tma representing 322 human fls stained for epha7 . d and e . massarray analysis of epha7 promoter methylation in 32 fls ( d ) and 16 human lymphoma lines ( e ) and positive / negative controls ( ctrl ); the color scale indicates the degree of methylation with red indicating 0 % and yellow 100 %. f . qrt - pcr of epha7 mrna levels in human lymphoma cell lines treated with 5 - aza - 2 ′- deoxycytidine ( aza ); p ( aza vs . untreated ) & lt ; 0 . 01 for all cell lines ; raji cells are hemizygous for epha7 . fig1 . higher resolution epha7 immunohistochemistry of human tissues . a . epha7 stain of normal tonsil . b and c . representative negative ( b ) and positive ( c ) stains of human fls on the tma . ( the tma also includes , as controls , normal human kidney and liver and gastric cancers with known epha7 staining patterns .) fig1 . help analysis of differential epha7 promoter methylation in normal gc b - cells , fl , dlbcl and dlbcl - derived cell lines . a . location of the probes used to analyze epha7 promoter methylation . b and c . results with probe 1 comparing centroblasts ( cb ; n = 9 ) with fl ( n = 8 ) and dlbcl ( n = 155 ) ( b ) and cb with dlbcl - derived cell lines ( c ). d and e . analogous results for probe 2 . silencing is progressive from gc b - cells to fl and aggressive dlbcl and nearly complete in dlbcl lines (* indicates p & lt ; 0 . 05 ). the data are consistent with massarray and expression data and indicate differential silencing between lymphomas and gc b - cells . fig1 . characterization of epha7 ecd expressed from a 5 - aza - 2 ′- deoxycytidine - treated human lymphoma line . a . rt - pcr showing re - expression of the epha7 transcript in su - dhl - 10 cells upon treatment with 1 μg and 5 μg 5 - aza - 2 ′- deoxycytidine ( aza ). b . cdna sequence ( seq id no : 01 ) of the re - expressed rna . c . translation ( seq id no : 02 ) of the cdna sequence ( seq id no : 01 ). d . domain structure of the protein encoded by the re - expressed rna . e . predicted three - dimensional structure based on similarity with the known epha2 structure . f . gel purification of the fc - tagged ectodomain of epha7 ( epha7 fc ). fig1 . analysis of the epha7 locus and its expression in raji lymphoma cells . a . genomic qpcr to determine epha7 gene copy number in raji lymphoma cells is consistent with loss of one allele of epha7 in these cells . b . qrt - pcr of epha7 mrna confirms the absence of epha7 expression in raji cells compared to normal tonsils (* denotes p & lt ; 0 . 05 ). fig1 . epha7 fc can block oncogenic signals and suppress the growth of xenografted human lymphomas . a . lysates and conditioned media from fl5 - 12 / bcl2 cells expressing empty vector , an shrna targeting epha7 ( shepha7 ) or full length epha7 ( epha7fl ) probed with an antibody against epha7 . b . immunoprecipitation of lysates of raji cells treated with epha7 fc ( fc - tagged ectodomain of epha7 ) or with fc control , immunoprecipitated with anti - epha7 antibody and probed with antibodies to epha7 or epha2 . c . elisa for epha2 phosphorylation in raji cells treated with eph7 fc or fc . d . immunoblot of lysates of raji cells treated with fc control , epha7 fc ( 5 μg ) or a small interfering rna ( sirna ) directed against epha2 . e . immunoblot of lysates of raji cells treated with 5 μg / ml epha7 fc as indicated . f . model of the epha2 - epha7 ecd interaction based on the known structure of epha2 and its homology with epha7 ( lbd , ligand binding domain ; egf , egf - like domain ; fniii , fibronectin domain ). g . xenografted raji lymphomas grown in the flanks of nod / scid mice and treated three times on alternate days by intratumoral administration of 20 μg epha7 fc ( red circle ) or vehicle ( fc ; black circle ). h . microscopic pathology of epha7 fc - treated and mock - treated raji lymphomas stained as indicated . i . immunoblot of lysates of tumors treated with epha7 fc or vehicle ( fc ) in vivo . j . matched - pair analysis of tumor volumes of eight ( a - h ) epha7 fc - treated ( red ) and vehicle ( fc )- treated ( black ) raji lymphomas . k . intravenous ( i . v .) administration of epha7 fc ( 20 μg / day × 3 days ; red ) delays tumor development following injection of 1 × 10 6 raji lymphoma cells vs . administration of vehicle ( fc ). 1 . schematic of anti - cd20 - epha7 antibody . m . immunoblot of lysates of raji cells treated with anti - cd20 antibody ( cd20 ), with anti - cd20 - epha7 antibody ( cd20 / e7 ) or untreated ( untr .). n . proliferation of raji cells treated as indicated ; * denotes p ( cd20 / e7 vs . cd20 ) & lt ; 0 . 05 . o . apoptosis of raji cells treated as indicated at 24 hours and 48 hours (* and ** denote p & lt ; 0 . 05 ). p . weight of tumors from mice bearing raji xenografts (& gt ; 1 cm 3 ) left untreated ( untr .) or given 1 μg / day × 5 days of anti - cd20 antibody ( cd20 ) or anti - cd20 - epha7 antibody ( cd20 / e7 ). tumors ( in matrigel ) were collected two days after last treatment , weighed ex vivo and classified as complete response ( cr ; 0 - 30 mg ), partial responses ( pr ; 30 - 100 mg ) or no change / progressive disease ( nc / pd ; & gt ; 100 mg ). fig1 . epha7 signaling effects in human lymphoma cell lines . a . immunoblot of lysates of dohh2 cells treated with epha7 fc or fc , immunoprecipitated with anti - epha7 antibody and probed with antibodies to epha7 or epha2 . b . confirmation of epha2 expression in raji cells by immunoblot . c . immunoblot of lysates of raji cells treated with an sirna against epha2 for the indicated times and probed as indicated . d . immunoblot of cell lysates of su - dhl - 6 , dohh2 , and karpas 422 cells treated with epph7 fc as indicated reveals some cell type - specific differences and similarities in signaling . fig1 . signaling effects of epha7 fc and shrna - mediated epha7 knockdown . a . fl5 - 12 / bcl2 cells expressing empty vector or shepha7 probed with the indicated antibodies . b . fl5 - 12 / bcl2 cells expressing shepha7 ( fl5 - 12 / bcl2 / shepha7 ) treated with epha7 fc ( 5 μg / ml ) for the indicated times and probed as indicated . fig1 . epha7 fc affects several signaling molecules in human lymphoma cells . a . phosphoprotein array probed with lysates from raji cells treated for 15 minutes with 5 μg / ml purified epha7 fc or vehicle . b . quantification of phosphoprotein array results ; the blue line indicates values for untreated cells . c . dose - response relationship for epha7 fc - mediated erk inhibition . d and e . immunoblots of lysates of raji ( d ) and su - dhl - 10 ( e ) lymphoma cells treated for 15 minutes with 5 μg / ml purified epha7 fc or vehicle ( v ) and probed for the indicated signaling molecules . fig2 . retroviral expression of epha7 ecd in human lymphoma cells . a . qrt - pcr showing the level of retroviral expression of epha7 ecd ( epha7 tr ) mrna in raji and su - dhl - 10 cells . b . growth curve of facs - sorted populations of vector - or epha7 ecd ( epha7 tr )- expressing raji and su - dhl - 10 cells . c . progressive depletion of epha7 ecd ( epha7 tr )/ gfp - expressing raji cells from mixed populations during 72 hours in in vitro culture . fig2 . induction of apoptosis by epha7 fc in fl - derived cell lines in vitro . a . facs analysis of apoptosis induced by treatment with 5 μg epha7fc in raji , karpas 422 and dohh2 cells . b . quantification of apoptosis in a panel of lymphoma lines ( v , vehicle / fc ; e7 , epha7 fc ; p ( e7 vs . v ) & lt ; 0 . 05 ). fig2 . matched - pair analysis of xenografted su - dhl - 10 and raji human lymphoma cells treated with epha7 fc or fc . a . photograph of tumors ex situ following treatment with 20 μg epha7 fc or fc ( vehicle ). b . tumor weights comparing epha7 fc - treated and fc - treated tumors ( p & lt ; 0 . 05 for su - dhl - 10 and raji ). fig2 . purification and functional characterization of an anti - cd20 - epha7 ecd fusion antibody . a . elisa measurement of anti - cd20 and anti - cd20 - epha7 antibody production . b . facs analysis of cd20 + raji cells treated with the anti - cd20 or anti - cd20 - epha7 antibodies and a fluorescein isothiocyanate ( fitc )- labeled anti - igg antibody . c . elisa of epha2 phosphorylation in raji cells treated with fc ( untr . ), epha7 fc or anti - cd20 - epha7 (* indicates p ( either treatment vs . vehicle ) & lt ; 0 . 05 ). fig2 . anti - cd20 - epha7 induces cell death and blocks proliferation of dohh2 lymphoma cells . a . apoptosis of untreated ( untr . ), anti - cd20 ( cd20 )- or anti - cd20 - epha7 ( cd20 / e7 )- treated dohh2 cells at 24 hours and 48 hours . b . proliferation of dohh2 cells untreated ( untr . ), treated with anti - cd20 ( cd20 ) or anti - cd2 - epha7 antibody ( cd20 / e7 ). fig2 . microscopic histology and immunohistochemistry of raji xenografts . tumors were treated with anti - cd20 - epha7 antibody ( 1 μg / day × 5 days ) or vehicle and collected two hours after the final treatment . the anti - cd20 - epha7 - treated tumors reveal some increase in tunel positivity , reduced proliferation and reduced erk phosphorylation . adams , j . m ., et al . “ the c - myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice .” nature 318 , no . 6046 ( december 1985 ): 533 - 538 . antipenko , alexander , et al . “ structure of the semaphorin - 3a receptor binding module .” neuron 39 , no . 4 ( august 2003 ): 589 - 598 . bende , r j , l a smit , and c j van noesel . “ molecular pathways in follicular lymphoma .” leukemia 21 , no . 1 ( january 2007 ): 18 - 29 . bergers , gabriele , kashi javaherian , kin - ming lo , judah folkman , and douglas hanahan . “ effects of angiogenesis inhibitors on multistage carcinogenesis in mice .” science 284 , no . 5415 ( april 1999 ): 808 - 812 . chen , ken , et al . “ polyscan : an automatic indel and snp detection approach to the analysis of human resequencing data .” genome research 17 , no . 5 ( may 2007 ): 659 - 656 . compagno , mara , et al . “ mutations of multiple genes cause deregulation of nf - κb in diffuse large b - cell lymphoma .” nature 459 , no . 7247 ( june 2009 ): 717 - 721 . dawson , d . w ., et al . “ global dna methylation profiling reveals silencing of a secreted form of epha7 in mouse and human germinal center b - cell lymphomas .” oncogene 26 , no . 29 ( june 2007 ): 4243 - 4252 . egle , alexander , alan w . harris , mary l . bath , lorraine o &# 39 ; 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