File S1 - Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

Figure S1, Expression Levels of Lymphoid-priming Genes in Primary Lymphocyte Precursors from B-lineage ALL Patients in Relationship to BTK Transcript Levels. [A] Gene Pattern (http://www.broadinstitute.org/cancer/software/genepattern/) was used to extract expression values for the 20 Lymphoid-priming genes in the combined data set from 5 studies with a total of 1104 primary leukemia samples of human lymphocyte precursors for further analysis (18 genes/ 27 transcripts were represented on the Human gene chips). Expression values expressed as Standard Deviation units calculated from 1104 samples were compiled for the 5 studies and rank ordered according to the mean expression of 3 highly correlated the BTK transcripts. For each study, the standard deviation values were calculated from the study mean for all the patients. We focused our analysis on human lymphocyte precursors from 884 B-lineage ALL patients. The datasets were combined to test for consistent differences in the Z-scores for high BTK (>0.5 SD units; N = 369 samples) and low BTK (<−0.5 SD units; N = 125 samples) groups. These samples were also rank ordered according to IKZF1 expression level (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)). A one-way agglomerative hierarchical clustering technique was used to organize expression patterns using the average distance linkage method such that genes (rows) having similar expression across patients were grouped together (average distance metric). The heat map depicts expression values represented by standard deviation units above (red) and below the mean (green). Dendrograms were drawn to illustrate similar gene-expression profiles from joining pairs of closely related gene expression profiles, whereby genes joined by short branch lengths showed most similarity in expression profile across patients. [B] Standardized expression values (SD units) for BTK probeset (205504_at) compiled from the 5 studies comprising of 1104 primary leukemia specimens from pediatric acute lymphoblastic leukemia (ALL) patients (GSE3912, N = 113; GSE18497, N = 82; GSE4698, N = 60; GSE7440, N = 99; GSE13159, N = 750) were examined for correlation with the expression of 3 Ikaros/IKZF1 probesets common in all 5 studies (205038_at, 205039_s_at, 216901_s_at). Highly significant correlations were observed for IKZF1 probesets plotted against the BTK probeset: 205038_at (B.1; Correlation Coefficient  = 0.27, T-value  = 9.24, P<0.0001), 205039_s_at (B.2; Correlation Coefficient  = 0.19, T-value  = 6.47, P<0.0001) and 216901_s_at (B.3; Correlation Coefficient  = 0.19, T-value  = 6.37, P<0.0001). Line of best fit and the associated 95% confidence intervals for the fit are shown in the shaded area. [C] 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 15 transcripts representing 12 lymphoid priming genes significantly upregulated in specimens with both high BTK and high IKZF1 expression. Figure S2, Expression Levels of Validated Ikaros Target Genes in Primary Lymphocyte Precursors from B-lineage ALL Patients in Relationship to BTK Transcript Levels. Gene Pattern (http://www.broadinstitute.org/cancer/software/genepattern/) was used for further analysis of expression values for previously published and validated IK target genes [13] in the combined data set from 5 studies with a total of 884 primary leukemia samples of human lymphocyte precursors from B-lineage ALL patients. For each study, the standard deviation values were calculated from the study mean for all the patients. A one-way agglomerative hierarchical clustering technique was used to organize gene expression patterns using the average distance linkage method such that genes (rows) having similar expression across patients were grouped together (average distance metric). The heat map depicts expression values represented by standard deviation units above (red) and below the mean (green). Dendrograms were drawn to illustrate similar gene-expression profiles by joining pairs of closely related gene expression profiles, whereby genes joined by short branch lengths showed most similarity in expression profile across patients. Samples were assigned to the “high BTK expression” group if their expression level was >0.5 standard deviation unit higher than the mean expression level (N = 369) and to the “low BTK expression” group if their expression level was >0.5 standard deviation unit lower than the mean expression level (N = 125) resulted in identification of 25 IK target genes upregulated in high BTK expression group. T-tests were performed using standardized expression values combined from 5 datasets (2-sample, Unequal variance correction, p-values<0.05 deemed significant) revealing an intersect of 34 transcripts representing 24 genes that were significantly up-regulated in both high BTK and high IKZF1 expression groups (29 genes were up regulated in high IKZF1 samples, of which 5 were not upregulated in high BTK samples versus 24 upregulated in both high IKZF1 and high BTK samples, Fisher's Exact Test, 1-Tailed, P <0.0001). Hierarchical cluster analysis identified a set of genes highly co-regulated with BTK expression: LAMC1 (2 transcripts; 0.68 and 0.97 SD Units, P = 9.2×10−16 and 7.3×10−33 respectively; PTK2 (2 transcripts; 0.42 and 0.46 SD Units, P = 4.5×10−6 and 2.7×10−6 respectively); TSPAN13 (0.63 SD units, P = 2.3×10−17); PREP (2 transcripts; 0.82 and 0.89 SD Units, P = 2.3×10−18 and 7.1×10−22 respectively); and SERPINI1 (0.84 SD units, P = 1.7×10−27) were the most significantly up-regulated 5 genes with the most significant effect sizes in 369 patient samples with high BTK expression. Figure S3, Co-localization and Physical Interactions of Native Ikaros and BTK Proteins in Human Cells. [A] Nuclear co-localization of Native IK and BTK. RAJI and DAUDI cells were fixed and stained with polyclonal rabbit anti-IK1 (primary Ab)/ Alexa Fluor 568 F(ab')2 fragment of goat anti-rabbit IgG (secondary Ab) (red) and mouse anti-BTK MoAb (primary Ab)/ Alexa Fluor 488 goat anti-mouse IgG (secondary Ab) (green) antibodies. Nuclei were stained with blue fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI). MERGE panels depict the merge three-color confocal image showing co-localization of IK1 and BTK in DAPI-stained nucleus as magenta immunofluorescent foci (System magnification: 315×). [B & C] Co-immunoprecipitation of Native IK and BTK. [B] depicts the results of the BTK and IK Western blot analysis of the IK immune complexes immunoprecipitated (IP) from RAJI and DAUDI cells. [C] depicts the results of the IK and BTK Western blot analysis of the BTK immune complexes from the same cells. Figure S4, Effects of BTK Inhibition on CD19-Mediated Activation of IK in Human B-lineage Lymphoid Cells. [A] Anti-phosphoserine (A.1) and Anti-IK (A.2) Western blot analysis of IK immune complexes from BCL1 cells before and after stimulation with an anti-CD19 monoclonal antibody homoconjugate (CD19xCD19) (1 µg/mL) for 15 min and 30 min. A.1: CD19 engagement caused increased serine phosphorylation of IK, which was abrogated by the BTK inhibitor LFM-A13 (50 µM). A.2: LFM-A13 treatment did not affect the IK protein level. [B] EMSA was performed using nuclear extracts (NE) from BCL-1 cells with and without a 2-hour stimulation with an anti-CD19 monoclonal antibody homoconjugate (CD19xCD19) (1 µg/mL) using the radiolabeled IK-BS1 oligonucleotide probe containing a high-affinity IK1 binding site. CD19 engagement caused increased the sequence-specific DNA binding activity of native IK (Lane 7 vs. Lane 2) and this response was blocked by the BTK inhibitor LFM-A13 in a concentration-dependent fashion (Lanes 3–6). The specificity of the binding was confirmed by demonstrating that the mobility shifts caused by binding of native IK in NE to the radiolabeled IK-BS1 probe are abrogated via homologous competition using 60-fold molar excess unlabeled IKBS1 probe (Lane 8). Lanes 9&10: No mobility shifts were observed with the control oligonucleotide probe IK-BS5 that has a single base pair (G>A) substitution at position 3 within the core consensus
and does not bind IK.
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