Additional file 1 of Ikaros sets the threshold for negative B-cell selection by regulation of the signaling strength of the AKT pathway
Creators: name: Ehm, Patrick A. H.
name: Horn, Stefan
name: Hoffer, Konstantin
name: Kriegs, Malte
name: Horn, Michael
name: Giehler, Susanne
name: Nalaskowski, Marcus
name: Rehbach, Christoph
name: Horstmann, Martin A.
name: Jücker, Manfred
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1. Differential INPP5D expression in B-ALL subtypes. (A) Analysis of INPP5D expression across leukemia subtypes from the gene expression microarray data GSE13204 [41]. INPP5D mRNA expression was compared between different subtypes and to healthy and non-malignant bone marrow controls. (B) Occurrence of INPP5D gene fusions in B-other subtypes. INPP5D gene fusions were listed on the right side. Data were taken from the publicly available dataset of [10]. (C) Establishment of SHIP1-specific knockdown in B-ALL cell lines Reh and SupB15. Two different SHIP1-shRNAs were tested for their knockdown efficiency. Figure S2. Effects of SHIP1-knockdown on the activity profile of cellular kinases in SupB15 cells. (A) Schematic representation of functional kinome profiling on whole cell extracts. (B) Analysis of the influence of SHIP1-knockdown on the activity profile of tyrosine and serine/threonine kinases in BCR-ABL-positive SupB15 cells by kinome tree visualization. The influence on the family of tyrosine kinases (TK) and calcium/calmodulin-dependent protein kinases (CAMK) was shown enlarged. The Tec-kinase BTK was particularly recognizable here. Branch and node color showed the kinase statistic of each kinase after knockdown of SHIP1. Blue indicated a negative regulation and red indicated a positive regulation after SHIP1-knockdown. The node size visualized the specificity score of each kinase after knockdown of SHIP1. (C-E) showed the highest impact of the SHIP1-knockdown (“top list”) on the activity profile of tyrosine and serine/threonine kinases involved in overall (C), receptor tyrosine kinase (RTK, D) and PI3K/AKT signaling. The length of the individual bars corresponded to the strength of the change in phosphorylation. (F) Volcano plot visualization of the effect of SHIP1-knockdown on the activity profile of tyrosine and serine/threonine kinases in SupB15 cells. Significantly regulated kinases were shown in red (dots). Figure S3. Gene set enrichment analysis for INPP5D-high and -low expression in Ph-positive and ETV6-RUNX1 B-ALL subtypes. The strength of INPP5D gene expression was divided into high and low and a subsequent gene set enrichment analysis was carried out in patient samples with Ph- (A) and ETV6-RUNX1-positive (B) B-ALL subtypes. Gene sets with a FDR q-val ≤ 0.25 were considered significant and presented in a bar chart with the Normalized Enrichment Score (NES) value. Identical genes were marked with an asterisk. (C) Gene expression analysis of kinome-specific genes according to the classification of INPP5D with low, intermediate or high gene expression by transcriptome data from B-ALL patient samples with a Ph-positive subtype. In the heatmap analysis, high and low expressed genes were shown in red and blue, respectively. Genes that showed a gradual change in their gene expression pattern from low to high together with INPP5D were marked with an asterisk. (D) Venn diagram of significantly enriched genes for INPP5D expression in the Ph- (blue) and ETV6-RUNX1-positive (red) subtype. Both subtypes were compared for similarities and differences in gene sets. Gene set enrichment analysis of high versus low INPP5D expressing samples was carried out. Gene ontology (GO) analysis of genes correlated with INPP5D in the Ph-positive and ETV6-RUNX1 subtype was performed. Identical genes were marked with an asterisk. Shared genes were further subdivided according to their function as driver, oncogene or tumor suppressor. The genes were further subdivided into groups of transcription factors (E) and surface markers (F). Common genes were listed below in grey rectangle. Gene expression data were taken from the publicly available dataset of [ http://www.stjuderesearch.org/data ]. Figure S4. Representative flow cytometry gating strategy for analysis of SHIP1-positive cell populations. The intact lymphocyte gate was analyzed depending on distinguished FSC vs. SSC properties. The cell doublets were excluded. The relative proportion of human SHIP1-expressing cells in spleen and liver was analysed after staining with an FITC-conjugated anti-hCD45 antibody and an APC-conjugated anti-hSHIP1 antibody for the scrambled control (above) and the SHIP1-knockdown cohort (below). Figure S5: Representative flow cytometry gating strategy for analysis of apoptosis of genetically modified Ph-positive SupB15 cells. (A)The lymphocyte gate was analyzed depending on distinguished FSC vs. SSC properties. The cell doublets were excluded. Apoptotic cells were identified by Annexin V staining. Figure S6. Kinase activity profiles after reconstitution of Ikaros-WT in SupB15 cells. (A) Kinome tree visualization of the differential kinase activity profiles after reconstitution of Ikaros-WT expression. The impact on the family of tyrosine kinases (TK) and calcium/calmodulin-dependent protein kinases (CAMK) was shown enlarged. The NTRK and Src-kinase subfamilies were particularly recognizable. Branch and node color showed the statistic of each kinase after reconstitution of Ikaros-WT. Blue indicated a negative and red indicated a positive change after Ikaros-WT reconstitution. The node size visualized the specificity score. (B–D) showed the highest impact (“top list”) on the activity profile of tyrosine and serine/threonine kinases involved in overall (B), RTK (C) and PI3K/AKT signaling (D) after reconstitution of Ikaros-WT. The length of the individual bars corresponded to the strength of the change in phosphorylation. The length of the individual bars corresponded to the strength of the change in phosphorylation. (E) Volcano plot visualization of the kinome activity profiling data. Significantly regulated kinases were shown in red (dots). (F) Gene expression analysis of kinome-specific genes after categorizing IKZF1 expression into low, intermediate or low expressing groups. Data were taken from transcriptome analysis of Ph-positive B-ALL patient samples [ http://www.stjuderesearch.org/data ]. High and low expressed genes were shown in the heatmap analysis in red and blue, respectively. Genes displaying a gradual change in their expression from low to high together with IKZF1 were marked with an asterisk. Figure S7: SHIP1 expression was upregulated by Ikaros in the Ph-positive CML cell line BV173 cells. (A) Expression of SHIP1 and AKT in human Ph-positive cell lines. Two ALL (TOM-1 and SupB15) and two CML cell lines in blast crisis (Nalm-1 and BV173) were analysed. (B) To investigate the influence of SHIP1 and Ikaros-WT on the signal transduction of Ph-positive CML cells, BV173 control and BV173-SHIP1-knockdown cells were transduced with lentiviral particles containing the empty vector and the Ikaros-WT vector, respectively. The different cells were analysed at the protein level and the SHIP1 expression was quantified relative to the double transduced cells with the scrambled shRNA and with the empty vector control (right panel). (C) Effects of SHIP1-knockdown and ectopic expression of Ikaros-WT on cell proliferation. Representative images were shown (left). The confluence was determined at day five after seeding. Figure S8. Venn diagram of AKT isoform-associated gene expression profiles in Ph-positive B-ALL patients. The strength of the gene expression of AKT1, 2 and 3 was categorized separately into high and low, followed by a gene set enrichment analysis. AKT isoform-specific and overlapping genes were identified and assigned to the three isoforms. Data were extracted from the publicly available dataset of [ http://www.stjuderesearch.org/data ].