File S1 - Constitutive Function of the Ikaros Transcription Factor in Primary Leukemia Cells from Pediatric Newly Diagnosed High-Risk and Relapsed B-precursor ALL Patients

Supporting information including materials and methods and supplemental figures/tables.
Figure S1, Correlation Between Transcript Expression Levels of Ikaros Target Genes in Primary Leukemic Cells from Pediatric ALL Patients. Expression values expressed as Standard Deviation units were compiled for the 7 studies and rank ordered according to the mean expression of three highly correlated transcripts (205038_at, 205039_s_at, 216901_s_at, 227344_at and 227346_at; 3 of these were common in all Affymetrix platforms - 205038_at, 205039_s_at, 216901_s_at). Heat map depicts up and down regulated transcripts ranging from red to green respectively and clustered according to average distance metric (A). T-tests were performed for the combined Standard Deviation units from the 5 datasets (2-sample, Unequal variance correction, P-values<0.05 deemed significant) to reveal 42 transcripts representing 29 genes as significantly up-regulated in samples with high Ikaros/IKZF1 expression (B). Figure S2, Ikaros Target Gene Transcript Levels in Primary Leukemic Cells from Diagnostic Bone Marrow Specimens of Newly Diagnosed Pediatric ALL Patients. We compiled the archived “The Microarray Innovations in Leukemia” (MILE) study gene expression profiling (GEP) data on primary leukemic cells (GSE13159) from 576 B-lineage ALL/BPL and 174 T-lineage ALL patients. Heat map depicts up and down regulated transcripts ranging from red to green respectively and clustered according to average distance metric (A). A cluster of 8 IK target genes (GSN, DHRS3, LAMC1, PTK2, TSPAN13, F13A1, TGFBR3, PDK1) exhibited highly significant down regulation in T-lineage ALL. Differential expression levels of IKZF1 gene (3 transcripts) and 29 IK target genes (42 transcripts) were compared in T-tests utilizing the DQN3 values (2-sample, Unequal variance correction, P-values<0.05 deemed significant). Gene expression values were transformed into standard deviation units and effect sizes were reported using differences standard deviation units between B-lineage ALL and T-lineage ALL (B). There was a significant increase in the multi-variate mean of the standard deviation scores for these 45 transcripts comparing B-lineage ALL with T-lineage ALL (MANOVA, F1,748 = 168.7, P<0.0001). Figure S3, Ikaros Target Gene Transcript Levels in Primary Leukemic Cells from Diagnostic Bone Marrow Specimens of Newly Diagnosed Pediatric ALL Patients who Subsequently Experienced a Relapse. Expression levels of IK target genes were also examined for primary leukemic cells from matched-pair diagnosis vs. relapse specimens of B-lineage ALL/BPL patients using archived GEP datasets from 2 independent studies (GSE 3912, GSE18497). Gene expression values for leukemia cells obtained from 59 B-lineage ALL patients at diagnosis (GSE3912, N=32 and GSE18497, N=27) were compared to the gene expression values for leukemia cells obtained from 17 T-lineage ALL patients at diagnosis (GSE3912, N=3 and GSE18497, N=14). RMA-normalized values for the GSE18497 dataset and the MAS5- Signal intensity values for the GSE3912 dataset were log10 transformed and mean-centered to the average value for the diagnosis samples for each gene transcript in each study. Heat map depicts up and down regulated transcripts ranging from red to green respectively for expression values and clustered according to average distance metric (A). T-tests (2 sample) compared B- versus T-lineage ALL subsets for IK target gene expression levels in leukemic cells from initial diagnosis specimens of patients who subsequently experienced a relapse (B). There was a significant increase in the multi-variate mean for the 45 transcripts representing IKZF1 gene (3 transcripts) and 29 IK target genes (42 transcripts) in B-lineage ALL compared with T-lineage ALL (MANOVA, F1,74 = 9.32, P=0.0032). Figure S4, Ikaros Target Gene Transcript Levels in Primary Leukemia Cells from BCR-ABL+ B-precursor ALL (BPL) Patients Compared to Normal Bone Marrow Cells. We compiled the archived “Microarray Innovations in Leukemia” (MILE) study gene expression profiling (GEP) data on primary leukemic cells (GSE13159) from 122 pediatric BCR-ABL+ B-precursor ALL (BPL) patients with t(9;22) translocation and normal hematopoietic cells from 74 non-leukemic bone marrow specimens. Differential expression levels of IKZF1 gene (3 transcripts) and 29 IK target genes (42 transcripts) were compared in T-tests utilizing the DQN3 values (2-sample, Unequal variance correction, p-values<0.05 deemed significant). Gene expression values were transformed into standard deviation units calculated from the mean and standard deviation expression values for all the samples in each study and effect sizes were reported using differences standard deviation units between comparison groups. Heat map depicts up and down regulated transcripts ranging from red to green respectively for expression values and clustered according to average distance metric (A). Statistical differences were assessed utilizing MANOVA that showed no effect in the multivariate mean for the 45 transcripts in BCR-ABL+ BPL cells (F1,94 = 0.768, P = 0.382) and differences in the individual transcripts were reported using T-tests assuming unequal variances (B). Thirteen (7 genes) and 18 transcripts (15 genes) were up regulated and down regulated respectively (P<0.05). Figure S5, Lymphoid Priming Gene Transcript Levels in Primary Leukemia Cells from Pediatric BCR-ABL- and BCR-ABL+ BPL Patients. Expression levels of lymphoid priming gene transcripts were compared for primary leukemic cells from 155 pediatric BCR-ABL- BPL patients vs. 20 BCR-ABL+ BPL patients on the Mullighan study (GSE12995). Transcript signal values were obtained from hybridization onto the Affymetrix Human Genome U133A gene chip arrays. Heat map depicts up and down regulated transcripts ranging from red to green respectively for mean centered log10 transformed expression values and clustered according to average distance metric. Figure S6, Lymphoid Priming Gene Transcript Levels in Primary Leukemia Cells from BCR-ABL- and BCR-ABL+ Pediatric BPL Patients from the MILE Study. Expression levels of lymphoid priming transcripts were compared in primary leukemic cells from BCR-ABL- B-precursor ALL patients vs. BCR-ABL+ B-precursor ALL patients on MILE study (GSE13159). Transcript signal values were obtained from hybridization onto the Affymetrix Human Genome U133A genechip arrays. Heat map depicts up and down regulated transcripts ranging from red to green respectively for standardized expression values and clustered according to average distance metric. Multivariate means between these two groups was not significant (MANOVA, F1,357 = 1.664, P = 0.198). Figure S7, Correlation Cluster of Gene Expression Profiles of Primary Leukemic Cells from Pediatric BPL Patients Forms Distinct Modules of Expression for IKZF1 vs. Iacobucci Signature Genes for Adult Ph+ BPL with IKZF1 Deletions. [A] Pairwise correlations were performed using the adult gene signature for IKZF1 deletions (158 probe sets down regulated in adult Ph+ BPL patients with IKZF1 deletions and the 5 IKZF1 probe sets for Exons 1-4 for pediatric BPL cases using the RMA normalized dataset (N=123 Ph+ cases and 327 Ph- cases). Correlation coefficients (r) were determined between all gene pairs and cluster analysis was applied to the matrix of correlation coefficients for both rows and columns of probe set identifications whereby red indicated positive correlations between probe set pairs and blue indicated negative correlation between probe set pairs. The majority of correlations between the 158 probe sets were significantly positive in direction (92% with P<0.05 (11456/12403 pairs) (Median correlation coefficient = 0.43; Interquartile Range = 0.28 to 0.57), whereas the majority of correlations for each of the IKZF1 probe sets with each of the 158 probe sets for the adult IKZF1 deletion signature exhibited either a positive correlation (42% with P<0.05 [329/790 pairs]) or no correlation (54% with P>0.05 [428/790 pairs], Median correlation coefficient = -0.07, Interquartile Range = -0.16 to -0.01) in pediatric BPL cases. [B] Probeset correlations were clustered for a subset of 29 IK target genes that harbored IK binding sites in mice identified by cross-referencing this gene set with the archived CHiPseq data (GSM803110 archived in GSE32311 [3] and IKZF1 probe sets specific for Exons 1-4 in pediatric BPL samples. Figure S8, Ikaros target gene transcript levels in primary leukemia cells from newly diagnosed pediatric BPL patients who subsequently experience an early vs. late relapse. Expression levels of IK target genes were examined for primary leukemic cells from matched-pair diagnosis vs. relapse specimens of BPL patients using archived GEP datasets from 2 independent studies (GSE 3912, GSE18497). IK target gene expression levels were compared for leukemic cells from initial diagnostic specimens of patients who subsequently experienced an early relapse (N=40; <36 months) versus a late relapse (N=19; >36months) (2-sample T-test). Heat map depicts up and down regulated transcripts ranging from red to green respectively for log10 transformed mean centered expression values and clustered according to average distance metric (A). T-tests (2 sample) compared for Early versus Late relapse subsets for IK target gene expression levels in leukemic cells from initial diagnosis specimens of these patients. (B) There was a significant increase in the multivariate mean for 45 transcripts (3 transcripts for IKZF1 and 42 transcripts for 29 IK target genes) comparing Early to Late Relapse subsets (Average fold increase = 1.3; MANOVA, F1,57 = 7.11, P=0.01). Figure S9, Primary Leukemia Cells from Pediatric BPL Patients in Relapse are not Characterized by Ikaros Deficiency. [A] Matched pair gene expression values for leukemia cells obtained from 59 BPL patients at diagnosis and then at relapse (combined from GSE3912, N=32 and GSE18497, N=27). RMA-normalized values for the GSE18497 dataset and the MAS5- Signal intensity values for the GSE3912 dataset were log10 transformed and mean-centered to the average value for the diagnosis samples for each gene transcript in each study. To determine the differential expression of each gene, paired T-tests were performed for the combined mean-centered values from GSE3910 and GSE18497 datasets (Unequal variance correction, P<0.05 deemed significant). Heat map depicts up and down regulated transcripts ranging from red to green respectively for standardized expression values and clustered according to average distance metric. [B] Exon-specific genomic PCR with DNA sequencing on purified genomic DNA samples from 9 patients with BPL in first bone marrow relapse that occurred within 12 months of the completion of primary therapy. IKZF1 Exons E4-7 and their intron-exon junctions were PCR amplified, as described in Materials and Methods using the PCR primers listed in Table S3. Normal size PCR products were obtained in each of the 9 patients, providing strong evidence against the existence of homozygous deletions of the entire IKZF1 locus or within IKZF1 exons 4-7. [C] IK Western blot analysis on whole cell lysates was performed as described in Materials and Methods. See text for discussion. Figure S10, BPL Xenograft Cells are not Ikaros-Deficient. [A] IK Western blot analysis was performed on ALL xenograft cells (and RAJI cell line that was included as a positive control). Xenograft cells were CD10+CD19+CD34+HLA-DR+ and caused overt leukemia in NOD/SCID mice after reinjection [2]. [B] IK expression in BPL xenograft cells was also detected by intracellular flow cytometry. B1 = Xeno-1 in A; B2 = Xeno-2 in A; B3 = Xeno-3 in A; B4 = Xeno-12.2 in A. [C] Confocal fluorescence microscopy showed punctate nuclear and perinuclear localization of IK in xenograft cells from two separate cases (C1-C3 = Xeno-1 in A and C4-C6 = Xeno-5 in A). System Magnification: 630 (Magnification[objective]: 63 x Magnification[Eyepiece]: 10). [D] Shown in D1 are the mean Ct values for Exon 4 and Exon 7 amplicons obtained by qPCR of genomic DNA samples from 3 BPL xenograft cases (Xeno-1, Xeno-2, and Xeno-5 in A) and normal hematopoietic cells from 2 non-leukemic normal bone marrow specimens in two independent experiments. Depicted in D2 are the bar graphs of Ct data from [D1] for Exon 4-specific qPCR after normalization by referencing the raw data to the Ct values from Exon 7-specific qPCR. Depicted in D3 and D4 the are representative cycle number vs. Log (∆Rn) (fluorescence signal) amplification plots obtained using the IKZF1 Exon 4-specific test primers and IKZF1 Exon 7-specific reference primers respectively for the normal hematopoietic cells and the 3 BPL xenograft cases. Table S1, Expression Levels of Ikaros Target Genes in Ph(BCR-ABL) + vs. Ph(BCR-ABL)- BPL Patients – Mullighan Study. Expression levels of IK target genes were compared in primary leukemic cells from 155 pediatric BCR-ABL- BPL patients and 20 BCR-ABL+ BPL patients on the Mullighan study (GSE12995). Trimmed mean target intensity of each array was globally scaled to 500 (MAS5 values) as the normalization method. T-tests were performed using log10 transformed MAS5 signal values (2-sample, Unequal variance correction, p-values<0.05 deemed significant) to identify differentially regulated transcripts for IK target genes. Table S2, Expression Levels of Ikaros Target Genes in BCR-ABL+ vs. BCR-ABL- BPL Patients – MILE Study. Expression levels of IK target genes were compared in primary leukemic cells from 122 BCR-ABL+ and 237 BCR-ABL- pediatric BPL patients (MILE Study, GSE13159). Transcript signal values obtained from hybridization onto the Affymetrix Human Genome U133 Plus 2.0 Arrays were calculated using non-central trimmed mean of differences between perfect match and mismatch intensities with quantile normalization (DQN3, signal normalized with quartiles of the beta distribution with parameters p=1.2 and q=3. Of the 42 IK target transcripts representing 29 genes, only 7 transcripts representing 5 genes were significantly down regulated in BCR-ABL+ patients. 25 transcripts representing 16 genes were expressed at significantly higher levels in leukemia cells from BCR-ABL+ patients. Hierarchical cluster analysis identified MDFIC (0.69 SD units, P = 8.8 x 10-12), DHRS3 (0.6 SD units, P = 3.7 x 10-11), GSN (0.49 SD units, P = 3.4 x 10-10), ITGA4 (0.53 SD units, P = 2.6 x 10-9) and TSPAN13 (0.2 SD units, P = 1.2 x 10-7) as the most significantly up-regulated genes in the 122 BCR-ABL+ patient samples. Table S3, Primer Sets used for Amplifying and Sequencing Ikaros/IKZF1 Exons 4, 5, 6 and 7 and their Exon-Intron Junctions. Table S4, Expression Levels of IK Target Genes in Primary Leukemia Cells from Matched-Pair Relapse vs. Diagnosis Specimens of Pediatric BPL Patients. Matched pair gene expression values of IK target genes for leukemia cells obtained from 59 BPL patients at diagnosis and then at relapse (combined from GSE3912, N=32 and GSE18497, N=27). RMA-normalized values for the GSE18497 dataset and the MAS5- Signal intensity values for the GSE3912 dataset were log10 transformed and mean-centered to the average value for the diagnosis samples for each gene transcript in each study. To determine the differential expression of each gene, paired T-tests were performed for the combined mean-centered values from GSE3910 and GSE18497 datasets (Unequal variance correction, P<0.05 deemed significant). We also compared the IK target gene expression levels in leukemic cells from initial diagnosis specimens of patients who subsequently experienced an early relapse (N=40; <36 months) versus a late relapse (N=19; >36months) (2-sample T-test).
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