Multiple <i>BTG1</i> deletion-positive clones are present in specific BCP-ALL subtypes.

<p>(A) Recurrence of multiclonal <i>BTG1</i> deletions. A sensitive PCR method was used to screen for eight different deletion breakpoints (deletion I–VIII) in <i>BTG1</i> MLPA deletion positive (+) cases (n = 65), and to screen for the three most frequent deletion breakpoints (deletion III, V and VIII) in <i>BTG1</i> MLPA deletion negative (−) cases (n = 89). (B) <i>BTG1</i> deletion frequency in the two major cytogenetic subgroups of BCP-ALL (Hyperdiploid and <i>ETV6</i>-<i>RUNX1</i>). Presence of a <i>BTG1</i> deletion in the predominant clone was determined by MLPA on the entire cohort of BCP-ALL cases (n = 722), and was compared to deletions detected as a minor clone in MLPA-negative cases (n = 89) by deletion-spanning PCR. Distributions are similar, being depleted from hyperdiploid cases and enriched in <i>ETV6</i>-<i>RUNX1</i>-positive cases as compared to the total group.</p

Expression of <i>BTG1</i> truncated read-through transcripts in BCP-ALL cells with <i>BTG1</i> deletions.

<p>(A) Schematic representation of the wild-type human <i>BTG1</i> gene, existing of two partly coding exons, and five different <i>BTG1</i> transcripts due to <i>BTG1</i> gene deletions. Exons are represented by black (coding) or white (non-coding) bars. Indicated are the RT-PCR primers that were used to detect expression of the wild-type <i>BTG1</i> transcript (primers A and B), or one of the <i>BTG1</i> truncated read-through transcripts for deletion II (primers A and C), deletion III (pimers A and D), deletion IV (primers A and E), deletion V (primers A and F), or deletion VIII (primers A and G). (B) RT-PCR analyses on total RNA isolated from the BCP-ALL cell lines Nalm6 and RS4;11 (<i>BTG1</i> wild-type) and REH, SUP-B15 and 380, each with distinct monoallelic <i>BTG1</i> deletions. (C) RT-PCR analyses on primary BCP-ALL samples in which a single <i>BTG1</i> deletion (Pt1, Pt2, Pt3 and Pt5), multiple <i>BTG1</i> deletions (Pt4 and Pt6) or no <i>BTG1</i> deletions were detected with genomic PCR (Pt7). Type of deletions (III, V, or VIII) and outcome of MLPA (p: deletion-positive; n: deletion-negative) are indicated. <i>BTG1</i> read-through transcripts were verified by sequencing (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002533#pgen.1002533.s007" target="_blank">Table S5</a>), except for Pt3-deletion V, which was an unrelated DNA sequence. (D) Quantitative real-time RT-PCR data representing relative expression levels of <i>BTG1</i> measured 5′ (primers exon 1/2) and 3′ of the <i>BTG1</i> breakpoint hotspot (primers exon 2). Expression levels were normalized to <i>HPRT</i> levels, and compared to the expression level in Nalm6, which was set to 1. The data shown represent the average of two independent cDNA reactions and triplicate qRT-PCR reactions.</p

Authentic RSSs and candidate RSSs flanking breakpoints of <i>BTG1</i> microdeletions.

<p>Mismatches from consensus are underlined;</p>a<p>RSSs flanking V(D)J gene segments;</p>b<p>Sequence shown is in reverse complement orientation;</p>c<p>Functional cryptic RSSs at proto-oncogene breakpoints <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002533#pgen.1002533-Marculescu1" target="_blank">[18]</a>.</p

<i>BTG1</i> deletions in relapsed cases.

a<p>Determined by sequence analysis of breakpoint spanning PCR product.</p>b<p>The deletion breakpoint could not be detected (N.D.) using the eight breakpoint-spanning PCR assays (I–VIII).</p>c<p>The breakpoint does not cluster within <i>BTG1</i> exon 2, but is located 2 kb downstream in the 3′UTR.</p>d<p>Homozygous deletion.</p

<i>BTG1</i> microdeletion occurrence within cytogenetic subgroups of ALL.

<p><i>BTG1</i> deletion status was determined using MLPA.</p>a<p>Because of missing values, numbers do not always add up to 722 BCP-ALL cases. Data was available for 637 cases on hyperdiploidy; 513 cases for <i>ETV6-RUNX1</i>; 648 cases for <i>BCR-ABL1;</i> 649 cases for <i>MLL</i>-rearranged.</p>b<p>The ‘other’ subgroup encompasses cases negative for <i>ETV6-RUNX1</i> or <i>BCR-ABL1</i> translocations, <i>MLL</i>-rearrangement and/or hyperdiploidy. This group includes 10 cases with <i>E2A-PBX1</i> translocation, of which none harbor a <i>BTG1</i> deletion.</p>c<p>Subgroup unknown includes all cases in which no data is available in one or more cytogenetic classifications.</p>d<p>Fisher's exact test was used when sample groups were small.</p

Increased levels of H3K4me3 at the <i>BTG1</i> locus in BCP-ALL versus T-ALL cell lines.

<p>(A) Quantitative real-time RT-PCR data representing relative expression levels of <i>BTG1</i> in T-ALL cell lines HSB2, Jurkat and KARPAS45, and BCP-ALL cell lines RS4;11, Nalm6 and CCRF-SB (<i>HPRT</i> normalized and related to HSB2 expression levels). Data shown are the average of two independent cDNA reactions and triplicate qRT-PCR reactions. (B and C) Percentage recovery after ChIP performed with H3K4me3 antibody (B) or H3K9/14Ac antibody (C) on T-ALL (HSB2, Jurkat, KARPAS45) and BCP-ALL (RS4;11, Nalm6 and CCRF-SB) cell lines. Real-time quantitative PCR was performed with primers specific for the region 1 kb upstream of the transcription start site (−1 kb prom), directly flanking the transcription start site (prox prom), the second exon near the breakpoint hotspot (exon 2) and towards the end of the 3′untranslated region (3′UTR) at the second (and last) exon of the human <i>BTG1</i> gene. Values represent two independent ChIP experiments. Student's <i>t</i>-test was performed to assess differences between the average recovery of T-ALL versus BCP-ALL samples. Asterisk (*) indicates a p-value<0.05.</p

Upregulation of SIRPα upon differentiation of t(15;17) NB4 cells and induction of cell death following its triggering.

<p>(A) NB4 cells were exposed to 1 µM ATRA and granulocytic differentiation of the cells was examined by cell surface expression of the common myeloid marker, CD11b. (B) SIRPα protein expression, determined by western blotting, is upregulated in ATRA-incubated NB4 cells. β-actin is used as a loading control. (C) Flow cytometric analysis of chSIRPα surface expression is determined by using ED9 mAb in transduced NB4 empty vector and chSIRPα expressing cells. (D) 24 hrs following ED9 (10 µg/ml) incubation, the percentage of cell death in chSIRPα and EV transduced NB4 cells was quantified by APC-Annexin V and PE-7AAD FACS staining. (E) Percentage of apoptosis after exposure to 1 µM ATRA is shown in combination with 10 µg/ml of ED9.</p

SIRPα protein expression in AML cell lines and patients.

<p>Western blot analysis was performed in (A) cell lines and (B) 20 pediatric AML patient samples. β-actin staining was used as loading control. (C) SIRPα expression is quantified relative to β-actin expression.</p

SIRPα upregulation in t(8;21) Kasumi-1 cells following treatment with inhibitors of epigenetic gene silencing.

<p>Kasumi-1 cells were incubated with 1 µM Decitabine, 0.5 mM valproic acid, 1 mM Butyrate and 300 nM Trichostatin. Endogenous SIRPα protein level, determined by Western blotting was upregulated at indicated time points. β-actin staining was used as a loading control.</p

SIRPα-derived signal synergizes with different antileukemic drugs.

<p>Inhibition of cell growth is depicted by combination of ED9 mAb (10 µg/ml) with (A) Ara-C and DNR in NB4 cells expressing chSIRPα (B) Ara-C, DNR, VP16, DAC and imatinib in Kasumi-1 cells expressing chSIRPα. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052143#s3" target="_blank">Results</a> are based on means of 3 experiments and are calculated using Calcusyn.</p