Supporting InformationUckun et al. 10.1073/pnas.1007896107Materials and Methods.Leukemic Cells. Highly enriched populations of Ficoll-Hypaque-separated surplus leukemia cells isolated from bone marrow spe-cimens of 6 infants (PT1-PT6) and 3 children (PT7-PT9) withnewly diagnosed acute B-precursor leukemia (BPL) as well assurplus leukemia cells isolated from peripheral blood specimensof 3 adults with hairy cell leukemia (HCL) (PT10-PT12) wereused in the described experiments with approval of the ParkerHughes Institute (PHI) Institutional Review Board (IRB) underthe exemption category (45 CFR Part 46.101; Category #4: Ex-isting Data, Records Review, and Secondary Use of PathologicSpecimens) in accordance with DHHS guidelines. Controlsincluded (a) the Burkitt’s/B-ALL leukemia/lymphoma cell linesRAJI (ATCC No. CCL-86), DAUDI (ATCC No. CCL-213),and RAMOS (ATCC No. CRL-1596), (b) B-cell precursorALL cell lines REH (Pre-Pre-B, ATCC No. CRL-8286),NALM-6 (Pre-B, DSMZ No. ACC-128), normal fetal liver B-cellprecursor cell lines FL8.2þ (CD2þCD19þ Pro-B/T) and FL8.2−(CD2−CD19þ Pro-B) (1, 2), and (c) the EBV-transformed B-lym-phoblastoid cell line BCL-4 from a nonleukemic individual. Inaddition, surplus leukemia cells isolated from bone marrow speci-mens of 31 infants (<1 year of age) with newly diagnosed ALL,who were treated on the CCG Infant ALL Protocol CCG-1953and 23 children (>1 year of age) with newly diagnosed high riskALL, who were treated on the CCG High Risk ALL ProtocolCCG-1961 (Eligibility: age ≥10 years or age 1–9 years with pre-senting WBC ≥50;000∕μL) as well as 7 children with newly diag-nosed standard risk ALL, who were treated on the CCG StandardRisk ALL protocol CCG-1952 (Eligibility: age 1–10 years andWBC < 50;000∕μL) were used for gene expression profiling(3) with approval of the PHI IRB under the exemption category(45 CFR Part 46.101; Category #4: Existing Data, Records Re-view, and Secondary Use of Pathologic Specimens) in accordancewith DHHS guidelines. The secondary use of existing cryopre-served leukemic cell specimens for subsequent molecular studiesdid not meet the definition of human subject research per 45 CFR46.102 (d and f) because it does not include identifiable privateinformation, as confirmed by the IRB (CCI) at Children’s Hos-pital Los Angeles (CHLA).

Western Blot Analysis of CD22 Expression. Western blot analysis ofwhole cell lysates for CD22 expression was performed by immu-noblotting using N-20, a polyclonal goat IgG CD22 antibodyrecognizing the N-terminus of the human CD22 molecule (SantaCruz, Catalog #7031), C-20, a C-terminal anti-CD22 antibody(Santa Cruz, Catalog#7029), and the ECL chemiluminescencedetection system (Amersham Life Sciences), as describedpreviously (4, 5).

Apoptosis Assays.Leukemic cells from 3 infant BPL patients (PT1,PT5, PT6) with CD22ΔE12 as well as DAUDI Burkitt’s leukemiaand FL8.2− normal fetal liver pro-B cells were treated with theanti-CD22 monoclonal antibody HB22.23 (6) at 1.0 and/or10 μg∕mL final concentrations. To detect apoptotic fragmenta-tion of DNA, cells were harvested 24 hours after exposure toanti-CD22 antibodies. DNA was prepared from Triton-X-100 ly-sates for analysis of fragmentation (7, 8). In brief, cells were lysedin hypotonic 10 mmol∕L Tris-HCl (pH 7.4), 1 mmol∕L EDTA,0.2% Triton-X-100 detergent; and subsequently centrifuged at11;000 × g. To detect apoptosis-associated ladder-like DNA frag-mentation, supernatants were electrophoresed on a 1.2% agarose

gel, and the DNA fragments were visualized by ultraviolet lightafter staining with ethidium bromide.

Genomic PCR Analysis of CD22 Gene in Leukemia Cells.DNA sequen-cing was carried out using the BigDye Terminator v.3.1 cyclesequencing kit (Applied Biosystems, Foster City, CA) (fig. S1to S3). Total genomic DNA was extracted from both patients’leukemia cells and cell lines using the Qiagen DNeasy Blood& Tissue kit (Catalog No. 6950) according to the manufacturer’sspecifications. An 885-bp PCR product encompassing the CD22exons 10, 11 and their exon-intron junctions was PCR-amplifiedusing the genomic PCR primers CD22-10/11)-seqF4 (5′-CA-CAGCTATACTGCCGTGAA-3′) and CD22-10/11-seqR4 (5′-AGGCAGAGTCTCAGTATGTC). The sequencing primer wasCD22-10/11-seq (5′-GCTCCTTCAAGGAGAATTAGTG-3′). A905-bp PCR product encompassing the CD22 exons 12, 13, andtheir exon-intron junctions was PCR-amplified using the genomicPCR primers CD22-seqF10 (5′-GGCATGAGGCAGACTGT-GAA-3′) and CD22-seqR10 (5′-AACCTCTGCCACCACGGAT-G-3′). The sequencing primers were CD22-12/13-seq (5′-CCAC-TCGGCAACAAGCCTCT-3′) and CD22-12-seqr (5′-GAAGG-AGCAGGTCCACTTCT). CD22 exon 14 was PCR-amplifiedusing the genomic PCR primers CD22-seqF11N (5′-CACAGC-CAGTTTCCTGACAC-3′) and CD22-R11 (5′-AGGGACCCT-GGCAGCATCTGAGAGCAAAAGTTCTTTGAAGTGGCAT-CTGA-3′). The primer sets (50 pmol∕μL) (0.7 μL of each primer,50 μL reaction volume, 150 ng genomic DNA, 0.5 μL of 10 mMdNTP, 2.5 UTaq polymerase/Invitrogen-Cat. No. 12355-036) wereused with the following thermal cycling conditions: 1 cycle for in-itial denature (5 min 95 °C), 32 cycles (30 sec 95 °C, 30 sec 58 °C,1 min 72 °C); hold at 72 °C for 5 min; indefinite hold at 4 °C.The PCR products were directly sequenced using the indicatedPCR primers in 5 μL reaction volumes containing 0.5 μL BigDyeterminator mix v3.1, 1 μL 5× sequencing buffer (Applied Biosys-tems), 1 μL 3.2 pmol primer, and 25 ng PCR product. Sequencingthermal cycling parameters were: 1 cycle (1 min, 96 °C), 35 cycles(10 sec at 96 °C, 5 sec at 50 °C, 150 sec at 60 °C); hold 180 sec at60 °C, and indefinite hold at 4 °C. The sequencing products fromeach reaction were cleaned using GenScript QuickClean 5M pur-ification kit (GenScript, MD) and analyzed on an ABI 3730XLDNA Analyzer (Applied Biosystems). Sequence obtained fromthe genomic PCR products was analyzed using SeqMan II con-tiguous alignment software in the LaserGene suite from DNAS-TAR Inc. and the MegAlign multisequence alignment software incomparison with the wild-type CD22 sequence (NCBI ReferenceSequence: NC_000019.9. Homo sapiens chromosome 19, Gen-ome Reference Consortium Human Build 37/GRCh37) primaryreference assembly, www.ncbi.nih.gov), as previously described(9, 10).

Determination of the Pre-mRNA Secondary Structure in Intron-De-rived Segments. We interrogated the CD22 sequence using theUCSC Genome browser (http://genome.ucsc.edu/) that reportedand aligned known human ESTs in the CD22 region of interest(chr19: 35,836,500-35,837,143). The splice acceptor and donorsites were deduced from this alignment and cross-referencingthe Collaborative Consensus Coding Sequence (CCDS) project(11) (Fig. S2). The assigned CCDid number ensures that codingsequences are consistently represented on the NCBI, Ensembl,and UCSC Genome Browsers (hg19_ccds Gene_CCDS12457.1for CD22). The DNA sequence for wild-type and patientsequences were converted to positive strand RNA complement

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sequences for alignment using the Clust W program (BioEdit Se-quence Alignment editor). Multiple alignment was constructedusing gap penalties in a position- and residue-specific mannersuch that all pairs of sequences were aligned separately in orderto calculate a distance matrix for each pair of sequences (FastApproximate Method), then a guide tree was calculated fromthe distance matrix and the sequences were progressively alignedaccording to the branching order in the guide tree (Neighbour–Joining method). The accessibility of target motifs to RNA bind-ing proteins was assessed after predicting of secondary structureof the positive strand of the CD22 pre-mRNAmolecule using theminimum free energy (MFE) calculations for sequences obtainedfrom intronic regions between exons 12 and 13 of the CD22 geno-mic sequence (NC_000019.9; 35,836,624-35,837,053, Homosapiens chromosome 19, GRCh37 primary reference assembly).We implemented a dynamic programming algorithm, RNAfold,provided by the Vienna RNA package (12) (http://rna.tbi.univie.ac.at/) that calculated MFE folding and equilibrium base-pairingprobabilities for the pre-mRNA segment corresponding to theintronic RNA complement (C35;836;624-C35;837;053) to explore howthe target sequences for RNA binding proteins residing in loopstructures, bulges or binding pair probabilities with low valuesvaried between the wild-type and patient secondary structures.

RT-PCR Analysis of CD22 Expression in Leukemia Cells. Reverse tran-scription (RT) and polymerase chain reaction (PCR) were usedaccording to published PCR assay procedures (13) to amplify a975-bp region (1858bp to 2833bp, GenBank accession codeX59350) of the CD22 transcript. Total cellular RNA was ex-tracted from cells lysed in guanidinium isothiocyanate usingthe RNeasy™ total RNA isolation kit (Qiagen, Santa Clarita,CA). cDNA was synthesized from total RNA using randomprimers and Superscript II reverse transcriptase (Gibco BRL).Oligonucleotide primers, 22-1 and 22-2 (5′-GCCCGGGGGAC-CAAGTGATG-3′ and 5′-GTGGAAGAGAACAGGGGCAG-GAGT-3′, respectively) were used to amplify the target PCRproduct encompassing the sequence corresponding to the trans-membrane and intracellular domains of CD22. The enzyme mixeLONGase [Taq polymerase plus the proofreading (30− > 50 exo-nuclease activity) Pyrococcus species GB-D polymerase, GibcoBRL] was used with the following cycling conditions: 1 cycle(2 min 94 °C, 1 min 55 °C, 1 min 72 °C); 14 cycles (1 min 94 °C,1 min 55 °C, 1 min 72 °C); 19 cycles (1 min 94 °C, 1 min 55 °C,3 min 72 °C); 1 cycle (1 min 94 °C, 1 min 55 °C, 7 min 72 °C). Ne-gative controls included PCR products from an RNA-free cDNAsynthesis and amplification reaction (negative control 1) and aDNA polymerase-free reaction (negative control 2). PCR pro-ducts were separated by electrophoresis in 1.2% agarose and vi-sualized by ethidium bromide staining. In parallel, PCR productswere transferred to nylon membranes and hybridized with anoligonucleotide probe specific for the CD22 Exon 11 sequence(5′-CCT GCC TCG CCA TCC TCA TCC-3′), as described(13). The RT-PCR products were gel eluted (Geneclean II kit,Bio 101, Vista, CA) and then cloned by TA Cloning into PCR-2.1 (Invitrogen, San Diego, CA) for restriction analysis andsubsequent sequencing. For EcoRI restriction digest analysis,the insert was released following digestion with EcoRI. TwoEcoRI fragment sizes (608-bp and 367-bp) are expected forthe insert based on GenBank Data (HSRNACD22). The insertwas sequenced by cycle sequencing with Cy5-labeled primers andThermoSequenase Fluorescent Labeled Primer Cycle Sequen-cing Kit (Amersham Pharmacia Biotech, Piscataway, NJ) usingan automated ALF express sequencer (Amersham PharmaciaBiotech) and analyzed using DNAStar LaserGene. The se-quences were compared with the published human cDNACD22 sequence obtained through GenBank (Accession codesX59350 and U62631, NCBI Reference Sequence: NP_001762.2).

Construction of Transgene, Generation of Transgenic Mice, and Docu-mentation of Transgene Expression.The pEμ(Py) plasmid which uti-lizes a polyoma early promoter regulated by immunoglobulin (Ig)heavy chain enhancer Eμ to drive B-cell specific gene expressionin transgenic (Tg) mice (14, 15) was treated with BamHI anddephosphorylated. A 0.8-kb SV40 Poly(A) fragment was releasedfrom the pKV-461 plasmid using BamHI-BgIII. The linearpEμ(Py) and SV40 Poly(A) fragments were ligated together tocreate the B-lineage specific transgene cassette designatedpEμ(Py)-SV40(Poly A). A full-length human CD22 cDNA withthe exon 12 deletion (hCD22ΔE12) was isolated from pBlue-script-CD22ΔE12 plasmid with NotI-XhoI, filled-in with Klenowpolymerase, recut with PvuI, filled-in, gel-purified, and subclonedin frame between the Eμ-Py and SV40 Poly(A) sequences of thedephosphorylated SmaI-linearized pEμ(Py)-SV40 (Poly A) usingstandard procedures (Fig. S6A). This hCD22ΔE12 transgeneconstruct was microinjected into the male pronucleus of fertilizedFVB/N mouse oocytes using standard protocols (16–18). The oo-cytes were implanted into the oviducts of pseudopregnant femalemice to generate hCD22ΔE12-Tg mice. Tg founder mice wereidentified by Southern blot analysis of EcoRI-digested genomictail DNA using a 2.3 kb EcoRI/EcoRI ½α-32P�dCTP labeled CD22probe (Fig. S6B). Tg founders were bred to age-matched wild-type mice to produce transgenic lines and pups were screenedfor the presence of the transgene by Southern blot analysis oftail-extracted DNA. Bone marrow cells from a male Tg hemizy-gous mouse were subjected to fluorescence in situ hybridization(FISH) analysis and reverse 4′-6-diamidino-2-phenylindole(DAPI) karyotyping to confirm the integration of thehCD22ΔE12 transgene into the mouse genome using a biotiny-lated human CD22 DNA FISH probe and standard procedures.Initial analysis of the mouse chromosomes indicated that the hu-man CD22 transgene was incorporated into the mouse genomeon the long arm of one chromosome 14. A chromosome 14-spe-cific P1-derived artificial chromosome (PAC) clone (PAC 445I19,Research Genetics, Inc./Invitrogen) was used to prepare a digox-igenin-labeled FISH probe for two-color FISH analysis on theVysis Quips system using Avidin-FITC for detection of thebiotinylated human CD22 probe (green) and CY-3 (red) for de-tection of the digoxigenin-labeled mouse chromosome 14 probe.Both probes were hybridized together onto a slide containing thespecified mouse chromosomes. Bone marrow cells displayed bothprobes on only one chromosome 14 with the other chromosome14 of the diploid set displaying only the red signal consistent withthe hCD22ΔE12 hemizygosity of the transgenic mouse (Fig. S6 Cand D). In another male transgenic mouse, the transgene wasdetected on the X-chromosome by dual labeling using the humanCD22 probe and a chromosome X specific FISH probe derivedfrom the PAC clone 473L8 (Research Genetics, Inc/Invitrogen)(Fig. S6 E and F). Transgenic were screened by multiplex PCR oftheir genomic DNA for the presence of the hCD22ΔE12 trans-gene and connected mIgH enhancer sequence using 5′-CCAGCCCCACCAAACCGAAAGTC-3′ (5′-primer of the mIgH en-hancer) and 5′-CCAGGGGCCGAGGAGATGC-3′ (3′-primerof hCD22ΔE12) yielding a 0.6-kb PCR product. PCR primersfor mouse β-casein exon 7, 5′GATGTGCTCCAGGCTAAAGTT-3′ and 5′-AGAAACGGAATGTTGTGGAGT-3′ providedan internal control for DNA integrity and PCR efficiency, andyielded a 0.5-kb PCR product (Fig. S6 G). The 100 μL PCR re-action consisted of 2.5 mM MgCl2, 1× PCR buffer, 0.2 mM eachdeoxynucleoside triphosphate, 0.1 μM each primer, 2.5 U TaqDNA polymerase (Gibco BRL, Grand Island, NY), and 2.0 μgof template DNA. The PCR conditions consisted of 31 cyclesof 1 min 15 sec at 94 °C, 2 min 15 sec at 60 °C, and 3 min 15 secat 72 °C (DeltaCycler II System, Ericomp). The PCR productswere resolved on a 1 × TAE agarose gel. Controls included geno-mic DNA from a founder mouse (POS. CON) as well as genomicDNA from a non-Tg control mouse (NEG. CON). Tg mice were

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examined for expression of the hCD22ΔE12 transgene transcriptin splenocytes by RT-PCR analysis using human CD22 primersspanning exon 12 (F1: 5′-CCAGCCCCACCAAACCGAAA-AGTC-3′; R1: 5′CCAGGGGCCGAGGAGATGC-3′). Controlsincluded the PCR products from intact human CD22 cDNA(Clontech) as well as non-Tg FVB mice. The PCR products weresubjected to Southern blot analysis with an end-labeled oligonu-cleotide probe specific for the human CD22 Exon 11 sequence(5′-CCT GCC TCG CCATCC TCATCC-3′) (Fig. S6 H). A plas-mid containing hCD22ΔE12 served as a positive control for thetransgene and human CD22 cDNA (Clontech) was included as anadditional control for the Southern blot. Western blot analysis ofsplenocytes from hCD22ΔE12-Tg mice (but not transgene nega-tive control mice) using N-20, a polyclonal anti-CD22 antibodyrecognizing the N terminus of the human CD22 molecule (SantaCruz, Catalog #7031) revealed the presence of a truncatedCD22, which was not reactive with C-20, a C-terminal anti-CD22antibody (Santa Cruz, Catalog#7029), reminiscent of the Wes-tern blot results obtained with human infant leukemia cells(Fig. S6 I and J). General procedures for Southern blot analysis,PCR, RT-PCR, Western blot analysis, and karyotyping were pre-viously published (19–23). Single-cell suspensions of splenocytesand bone marrow cells obtained from electively sacrificed6–7 wk old Tg mice and their wild-type controls were purgedof erythrocytes by hypotonic lysis and immunophenotyped by di-rect fluorescence staining and flow cytometry using anti-CD19-phycoerythrin (PE), anti-B220/CD45R-PE, and anti-IgM-FITC,using published procedures (19–23). The PHI Animal Care andUse Committee (IACUC) approved Mouse experiments, andall animal care procedures conformed to the Guide for the Careand Use of Laboratory Animals of the National Research Council(National Academy Press, Washington, DC, 1996).

Gene Expression Profiling of Splenocytes from hCD22ΔE12-Tg Miceand Primary Leukemic Cells from Patients with Newly Diagnosed In-fant vs. Pediatric ALL. Expression profiling of mouse splenocytesfor 588 genes in six functional groups was performed using theAtlas Mouse cDNA Expression arrays from Clontech Labora-tories Inc. (Cat. Number 634539) according to the manufacturer’sspecification using previously detailed standard procedures(3, 24, 25). Density readings from the phosphor images were nor-malized to 5 housekeeping genes (glyceraldehyde-3-phosphatedehydrogenase (G3PDH; GADPH), myosin I, ornithine decar-boxylase (ODC), phospholipase A and hypoxantine-guaninephosphoribosyltransferase (HPRT)). The expression values ofthe housekeeping genes were within the dynamic range of theexpression values of the genes on the array (34–233 units afterbackground subtraction). The average background subtracted ex-pression values for the 5 housekeeping genes ranged from 26.2 to45.7 across the 10 samples. The tenth percentile mean and stan-dard deviation pixel intensity values for the genes represented onthe array were considered to be ‘blank’ spots, and were used tocalculate the ‘presence’ and ‘absence’ calls. Genes were consid-ered ‘present if the mean expression value of the gene was greaterthan 3 standard deviations from the mean expression value of theblank spots. All expression levels were log to the base 2 trans-formed for statistical comparisons. The normalization procedurewas examined by performing bivariate plots between two samplessuch that the expression values were equally dispersed around theline of unity and this was a necessary prerequisite to determinedifferentially expressed genes between hCD22ΔE12-Tg andcontrol FVB mice. T-tests with degrees of freedom correctionfor unequal variances (Excel formula) were performed onnormalized values to identify discriminating genes betweenhCD22ΔE12-Tg and FVB control mice and True Discovery Rateswere calculated using observed and expected number of changesat three p-values (0.01, 0.02, 0.05). Gene expression changes werevisualized using mean centered and standardized expression va-

lues from the 10 samples represented on a cluster figure. We em-ployed a one-way agglomerative hierarchical clustering techniqueto organize expression patterns using the average distance linkagemethod. For enrichment analysis, we utlized a publically availableweb tool to identify overrepresented functional annotations usinga curated, standardized set of description terms (http://amigo.geneontology.org/cgi-bin/amigo/term_enrichment). Gene ontol-ogy term for ‘GO:0048523 negative regulation of cellular process’constituted 178 genes on the Clontech Mouse array of which asignificant proportion (23 genes compared to 25 genes/410 genesunaffected, Fishers Exact Test, 2-tailed, p ¼ 0.008) were differen-tially regulated in hCD22ΔE12-Tg mice.

Expression profiling of primary leukemia cells from 31 infantsand 30 noninfant pediatric patients with newly diagnosed ALL for588 genes in six functional groups was performed using the Atlashuman cDNA expression arrays from Clontech Laboratories Inc.(Cat No 634511) according to the manufacturer’s specificationusing previously detailed standard procedures and describedabove for the mouse array (3). Pixel processing of the digitalimages was also performed using the procedure outlined forthe mouse array. To compare the gene expression levels fromthe ALL patients, a normalization procedure was applied usingthe raw signal intensities. In brief, the mean signal intensity withineach spot was subtracted from the subgrid median of the back-ground signal. For thresholding, the intensities of the duplicatespots for each gene were averaged and floored to a signal valueof 2 units. Density readings from the phosphorimages were nor-malized to 5 housekeeping genes (glyceraldehyde 3-phosphatedehydrogenase (GAPDH); brain-specific tubulin alpha 1 subunit(TUBA1); HLA class I histocompatibility antigen C-4 alphasubunit (HLAC); cytoplasmic beta-actin (ACTB); ubiquitin).All expression levels were log to the base 2 transformed. T-testswith degrees of freedom correction for unequal variances (Excelformula) were performed on normalized values to identify discri-minating genes between patient subsets.

To visualize the gene expression relationships between genesacross samples for both hCD22ΔE12-Tg mice and newly diag-nosed leukemia patients, we performed a one-way agglomerativehierarchical clustering technique to organize expression patternsusing the average distance linkage method using mean centered,standardized intensity values after log 2 transformation andnormalization procedure outlined above. Most consistent discri-minating genes in both the human and the mouse cDNA expres-sion arrays were cross-referenced to the Oncomine™ ResearchData Base (http://www.oncomine.org/) for leukemia and lympho-ma studies. We used a meta-analysis to interrogate each of thesignature genes for its previously reported expression valuesand associations in other B-lineage leukemia (10 studies; 11 com-parisons) (26–31) (or lymphoma studies (5 studies; 15 compari-sons) (26, 28, 31–33) in the Oncomine database. For each genethe fold difference and T-test p-value are reported for log-trans-formed, normalized expression levels. In order to control fornormalization artifacts in the evaluation of significant differencesbetween our study and other published studies, we comparedgene expression profiles using the same set of housekeepinggenes. Specifically, the distribution of the average fold-differencevalues for the five housekeeping genes were examined fromthe leukemia and lymphoma studies reported on the Oncominedatabase for outliers that affected the comparison with our study(GAPDH, HPRT1, ODC, PLA2G1B, MYH6 representing thehuman orthologs of the housekeeping genes used for normaliza-tion on the mouse array and showed expression values within thedynamic range of the measured pixel intensities). We focused thecomparisons on mouse ortholog genes significantly down-regu-lated in hCD22ΔE12-Tg mice for groups of genes assigned ascyclins, CDK inhibitors, tumor suppressors and G-proteins as as-signed by Clontech. Fishers exact test (2-tailed, P ≤ 0.05 deemedsignificant) was performed comparing the proportion of signifi-

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cantly assigned changes using the housekeeping genes and thosedifferentially expressed in both hCD22ΔE12-Tg mice and infantALL patients.

SCID Mouse Model of Infant BPL. In the SCID mouse xenograft ex-periments, female CB.17 SCID mice (6–8 weeks of age; Taconic/Germantown, NY) were inoculated intravenously with 0.5 mL of

a cell suspension containing 1 × 106 primary infant BPL cells. AllSCID mice were electively killed at 60 days unless they died orbecame moribund earlier due to their disseminated leukemia. Atthe time of their death or killing, mice were necropsied to confirmleukemia-associated marked hepatomegaly and/or splenomegaly(34, 35).

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Fig. S1. Genomic PCR Analysis of the Human CD22 Gene Exons 10-14. 885-bp PCR product covering the CD22 exons 10, 11 and their exon-intron junctions (A.1,B.1), a 905-bp PCR product encompassing the CD22 exons 12, 13, and their exon-intron junctions (A.1, A.2. B.2), and 964-bp PCR product covering exon 14 andits exon/intron junctions (A.2) were PCR-amplified, as described in Materials and Methods. PCR products from 6 infant BPL patients (PT1-PT6) and controlsincluding two pediatric BPL patients (PT7 and PT8), the Burkitt’s/B-ALL leukemia/lymphoma cell lines RAJI and DAUDI, BPL cell lines REH (Pre-Pre-B) and NALM-6(Pre-B), normal fetal liver B-cell precursor cell line FL8.2− (Pro-B), and the EBV-transformed B-lymphoblastoid cell line BCL-4 are shown. Empty lanes are in-dicated with a dash. Normal size PCR products were obtained in each of the 6 infant BPL cases, as indicated by the arrowheads, providing strong evidenceagainst deletions within CD22 exon 12 or its intron junctions as a possible cause for the expression of CD22ΔE12 mRNA species in these patients.

Fig. S2. Normal genomic sequence of exon 12 and its surrounding intron/exon junctions. NCBI Reference Sequence: NC_000019.9. Homo sapiens chromosome19, Genome Reference Consortium Human Build 37 (GRCh37) primary reference assembly (www.ncbi.nih.gov). A 905-bp genomic sequence that spans betweenthe intronic sequence 151-bp upstream of exon 12 (g.35,836,354G) and 120-bp downstream of exon 13 (g.35,837,258T) was amplified using the sense primerCD22-seqF10 (5′-GGCATGAGGCAGACTGTGAA-3′) and the antisense primer CD22-seqR10 (5′-AACCTCTGCCACCACGGATG-3′). The beginning and ends of exons12 and 13 are numbered corresponding to the CD22 genomic sequence (NC_000019.9). We interrogated the CD22 sequence using the UCSC Genome browser(http://genome.ucsc.edu/) that reported and aligned known Human ESTs in the CD22 region of interest (chr19: 35,836,500-35,837,143). The splice acceptor anddonor sites were deduced from this alignment and cross-referencing with the collaborative consensus coding sequence (CCDS) project (11). The assigned CCDidnumber ensures that coding sequences are consistently represented on the NCBI, Ensembl, and UCSC Genome Browsers (hg19_ccds Gene_CCDS12457.1 forCD22). The coding sequence is shown in upper case and noncoding intronic sequence is depicted in lower case. The splice donor and acceptor sites are under-lined. Sense and antisense genomic PCR primers are indicated by the direction of the arrows, right or left, respectively.

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Fig. S3. Infant B-precursor leukemia cells have homozygous intronic mutations of CD22 gene. (A) Consensus sequence alignment for intronic segment be-tween exons 11 and 12 starting at position NC_000019.9: c.2208−83G = g.35,836,421G and ending at position NC_000019.9: c.2208−1G = g.35,836,503G plus ashort segment of exon 12 between c.2208 and c.2214 in primary leukemic cells from 4 infant BPL patients (PT1, PT3, PT5, PT6) showing wild-type sequence withno mutations in each case. (B) Consensus sequence alignment of exon 12 genomic sequence in primary leukemic cells from 4 infant BPL patients (PT1, PT3, PT5,PT6) showing wild-type sequence with no mutations in each case. The genomic sequence starting at position NC_000019.9: c.2208−15T = g.35,836,490T andending at position NC_000019.9: c.2327+26C = g.35,836,649C) is shown for each patient in comparison to the wild-type (WT) sequence. (C) Consensus sequencealignment for intronic segment between exons 12 and 13 starting at position NC_000019.9: c.2327+29G = g.35,836,652G and ending at position NC_000019.9:c.2328−1G = g.35,837,053G. There were 4 mutations in PT1 sequence, 6 mutations in PT3 sequence, 2 mutations in PT5 sequence, and 6 mutations in PT6sequence. The positions of the genomic DNA sequence changes are indicated in RED. A separate list is provided in Table S2.

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Fig. S4. RNA sequence alignment for pre-mRNA corresponding to the intronic sequence between exons 12 and 13. The genomic DNA sequence for the wild-type consensus sequence and patient sequences (NC_000019.9; 35,836,624-35,837,053, Homo sapiens chromosome 19, GRCh37 primary reference assembly)were converted to the sequences of the positive strand RNA complement for alignment using the Clust W program (BioEdit Sequence Alignment editor).Multiple alignment was constructed using gap penalties in a position- and residue-specific manner such that all pairs of sequences were aligned separatelyin order to calculate a distancematrix for each pair of sequences (Fast ApproximateMethod), then a guide tree was calculated from the distancematrix and thesequences were progressively aligned according to the branching order in the guide tree (Neighbour–Joining method). The results of this alignment procedureshowed an introduction of a gap in the wild-type sequence at position 106 to accommodate the insertion mutation for PT6. The bases are color coded tovisualize residue-specific mutations in the alignment of the patient sequences with the wild type. Residues corresponding to positions 148 to 166 in the alignedsequence could not be accurately determined for PT3. The RNA sequence deviations from the wild-type forward strand complement RNA sequence(C35;836;624-C35;837;053) for the depicted pre-mRNA segment are as follows: PT1: 129 U > C, 148 C > U, 204 G > C, 237U > C; PT3: 125A > −, 141C > G,146A > G, 186U > −; PT5: 233G > C, 237U > C; PT6: 103G > C, 106− > C, 114G > U, 143G > −, 186U > −, 237U > C.

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Fig. S5. RT-PCR analysis of CD22 transcripts from infant B-precursor leukemia cells uncovers an exon 12 splicing defect. (A) Restriction map of CD22 cDNA.Arrows designate location of RT-PCR oligonucleotide primers, 22-1 and 22-2, that were used to amplify a 975 bp product encompassing the sequence encodingthe transmembrane and cytoplasmic domains of CD22. (B) RT-PCR analysis of FL8.2− fetal liver-derived nonleukemic B-cell precursors showed the anticipated975-bp single PCR product, whereas infant BPL cells from PT1, PT3, and PT5 yielded a distinct second PCR product of approximately 800-bp size. (C) An oli-gonucleotide specific for exon 11 was end-labeled with γ-32P-ATP and hybridized to Southern blots of CD22 PCR products shown in B. The positions of the CD22RT-PCR products are indicated with arrow heads. (D) EcoRI restriction analysis of cloned CD22 RT-PCR products from control FL8.2− cells yielded two fragmentsof the expected sizes of 600-bp and 350-bp. (E and F) EcoRI restriction analysis of cloned CD22 RT-PCR products from primary infant BPL cells from PT1 (E) andPT3 (F) yielded abnormal fragment pairs of 500-bp (instead of 600-bp) + 350-bp in the majority of the clones. (G) Sequence analysis demonstrated that thesmaller approximately 800-bp RT-PCR product shown in (B) results from deletion of exon 12. Depicted are automated sequence chromotographs from PT1showing sequence traces spanning exon 11–exon12 junction in a clone with wild-type (WT) CD22 sequence in one clone (Clone 1) and exon 11–exon 13 junctionin 4 clones (clones 2,3,5,6) of 7 with CD22 exon 12 deletion (CD22ΔE12). One single clone (clone 8) showed a 76-bp deletion within exon 9 between c.1846 andc.1921 instead of CD22ΔE12, which likely represents a PCR artifact. (H) The CD22mRNA sequence generated by deletion of exon 12. (I) Translation of CD22ΔE12sequence into protein results in a truncating frameshift starting at aa 736 (CD22-β isoform, GenBank Accession Codes #U62631 and X59350) with the additionof 15 novel amino acids (RCRVLRDAETSPGLR) not seen in wild-type sequence followed by a TGA stop codon.

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Fig. S6. Generation of mice transgenic for human CD22ΔE12 gene. (A) Schematic of the transgene construct. (B) Southern blot analysis of genomic DNA fromfounder mice. M: size markers, 1-kb DNA ladder. The transgene construct was included as a positive control. Genomic DNA from a nontransgenic mouse wasincluded as a negative control. (C) Dual color FISH analysis of metaphase chromosomes from bone marrow cells of an hCD22ΔE12-Tg mouse showing humanCD22ΔE12 transgene (green) on one chromosome 14 of the diploid set (red). (D) Reverse DAPI karyotyping of chromosomes from C. (E) Dual color FISH analysisof metaphase chromosomes from bone marrow cells of an hCD22ΔE12-Tg male mouse showing hCD22ΔE12 transgene (green) on sex chromosome X (red). (F)Reverse DAPI karyotyping of chromosomes from E. (G) Genomic CD22 transgene PCR analysis of splenocytes from hCD22ΔE12-Tg mice. Controls includedgenomic DNA from a founder mouse (POS. CON) as well as genomic DNA from a non-Tg control mouse (NEG. CON). A 5′ primer of the mIgH enhancerand a 3′ primer of hCD22ΔE12 were used to amplify a 0.6-kb PCR product. PCR primers for mouse β-casein exon 7 yielded a 0.5-kb PCR product as an internalcontrol for DNA integrity and PCR efficiency. (H) RT-PCR analysis of splenocytes from transgenic mice for the expression of the hCD22ΔE12 transgene transcriptusing human CD22 primers spanning exon 12. Controls included the PCR products from intact human CD22 cDNA (Clontech) as well as nontransgenic FVB mice(NEG. CON). The PCR products were subjected to Southern blot analysis with an end-labeled oligonucleotide probe specific for the human CD22 Exon 11sequence. A plasmid containing human CD22ΔE12 served as positive control for the transgene and human CD22 cDNA (Clontech) was included as an additionalcontrol for the Southern blot. (I) Western blot analysis of splenocytes from CD22ΔE12 transgenic mice using a CD22 antibody recognizing the N terminus of thehuman CD22 molecule. (J) Western blot analysis of splenocytes from CD22ΔE12 transgenic mice using a CD22 antibody recognizing the C terminus of thehuman CD22 molecule.

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Table S1. In vivo growth of infant BPL cells in SCID mice in relationship to expression of a truncated CD22 coreceptor protein

Patient Age (Months)/Sex MLL-AF4 Status Expression of Truncated CD22 Development of Overt Leukemia in SCID Mice

PT1 4/F + + 10∕10PT2 8/M + − 0∕10PT3 6/M − + 8∕10PT4 2/F + − 0∕10PT5 2/F + + 10∕10PT6 1/F + + 10∕10

The expression of MLL-AF4 fusion transcript was determined by RT-PCR (13). Groups of 10 SCID mice were injected i.v. with a 500 μL inoculumof 1 × 106 primary leukemia cells (suspended in PBS) from each of 6 infant BPL patients. Mice were electively sacrificed at 60 days unless theydied or became moribund earlier due to disseminated leukemia. At the time of their death or killing, mice were necropsied to confirmleukemia-associated marked hepatomegaly and/or splenomegaly.

Table S2. Homozygous intronic mutations of CD22 gene in infant BPL cells

Position of DNA Sequence Change

Patient Mutation No. Chr. Location in NC_000019.9Standard Human Genome Variation

Society (HGVS) Nomenclature Corresponding SNP

PT1 1 g.35,836,751 c:2327þ 128A > G rs48051192 g.35,836,770 c:2327þ 147G > A rs104065393 g.35,836,826 c:2327þ 203C > G rs104135004 g.35,836,859 c:2328 − 195A > G rs4805120

PT3 1 g.35,836,747 c:2327þ 124delT –2 g.35,836,763 c:2327þ 140G > C _3 g.35,836,768 c:2327þ 145delT _4 g.35,836,769 c:2327þ 146delG _5 g.35,836,770 c:2327þ 147G > C _6 g.35,836,808 c:2327þ 185delA _

PT5 1 g.35,836,855 c:2328 − 199C > G rs104135262 g.35,836,859 c:2328 − 195A > G rs4805120

PT6 1 g.35,836,727 c:2327þ 104InsG _2 g.35,836,736 c:2327þ 113C > A _3 g.35,836,764 c:2327þ 141delC _4 g.35,836,808 c:2327þ 185delA _5 g.35,836,726 c:2327þ 103C > G _6 g.35,836,859 c:2328 − 195A > G rs4805120

PCR-based genomic sequence analysis of CD22 gene revealed homozygous mutations within a 132-bp segment of the intronicsequence between exons 12 and 13. Details are shown in Fig. S3C.

Table S3. CD22 RT-PCR analysis of primary leukemia cells

RNA Source PCR Clones with CD22 ΔE12

B-lineage Leukemia PatientsPT1, Infant BPL 7∕10PT3, Infant BPL 8∕10PT5, Infant BPL 4∕7PT6, Infant BPL 2∕4PT7, Pediatric BPL 0∕3PT8, Pediatric BPL 1∕4PT9, Pediatric BPL 0∕4PT10, Adult HCL 2∕21PT11, Adult HCL 2∕13PT12, Adult HCL 2∕15Normal B-Precursor Cell LinesFL8.2þ, Fetal Liver Pro-B/T 0∕9FL8.2−, Fetal Liver Pro-B 0∕11

RNA samples from primary leukemic cells from 4 infants (PT1, PT3, PT5, PT6) and 3 pediatric patients with BPL(PT7, PT8, PT9) as well as 3 adults with HCL (PT10, PT11, PT12) and 2 fetal liver-derived normal B-precursor celllines were analyzed for mRNA species containing coding sequences for exons 10–14 by RT-PCR. Automatedsequencing and/or EcoRI restriction digest analysis showed that the majority of PCR clones from infant BPLpatients have a splicing defect resulting in exon 12 deletion (CD22ΔE12) that was not detected in normal B-precursor cell lines. A minority of PCR clones from adult HCL patients and one clone from a pediatric BPLpatient (PT8) also harbored CD22ΔE12.

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Table S4. Meta-analysis of CD22ΔE12 signature gene expression using the oncomine database

Comparison (versus Normal) Ref. No. APC GNB2 MDM2 SATB1

A. B-lineage LeukemiasBPL 2 −3.75 −1.55BPL 5 −1.27CLL 1 −1.33 −1.44 −3.11CLL 3 −3.95CLL 4 −1.55 −2.79CLL 6 −1.86 −5.04HCL 3 −1.35 −1.87 −2.65B. B-lineage LymphomasDiffuse Large B-Cell Lymphoma 1 −1.59 −2.22Burkitt’s Lymphoma 3 −1.54 −6.23Centroblastic Lymphoma 3 −1.87 −3.56Cutaneous Follicular Lymphoma 8 −1.38 −1.44Diffuse Large B-Cell Lymphoma 1 −1.61 −2.61Diffuse Large B-Cell Lymphoma 3 −1.48 −2.90Diffuse Large B-Cell Lymphoma 7 −1.39Diffuse Large B-Cell Lymphoma 6 −1.42 −3.65Diffuse Large B-Cell Lymphoma 8 −1.61Follicular Lymphoma 1 −1.26 −1.57Follicular Lymphoma 3 −1.32Follicular Lymphoma 6 −2.01 −2.83Germinal Center B-Cell-Like, Diffuse Large B-Cell Lymphoma 1 −1.58 −2.26Mantle Cell Lymphoma 3 −5.86Marginal Zone B-Cell Lymphoma 8 −1.73 −1.36

Four signature genes that were significantly down-regulated in Infant ALL patients and hCD22ΔE12-Tg mice wereinterrogated using the Oncomine data base for their expression in other studies comparing B-lineage leukemiasand non-Hodgkin’s lymphomas. (A) Enrichment of the gene expression signature was observed in 7 out of the 11comparisons for three different B-lineage leukemias deposited into the database. Fold differences relative to‘normal’ B-cell/B-precursor expression (T -test p-values < 0.05, negative values represent down-regulation in leukemiccells) are shown for BPL, CLL, and HCL cells. (B) Enrichment of the gene expression signature was observed in 15out of the 18 comparisons for 9 different B-lineage lymphomas deposited into the database. Fold differencesrelative to ‘normal’ expression (T -test P-values < 0.05, negative values represent down-regulation in leukemic cells)are shown for comparisons of B-lineage lymphoma cells with normal B-cells.

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