Ikaros regulates transcription of genes that control the PI3K pathway. (A-B) qChIP analysis of Ikaros occupancy of target genes that control the PI3K pathway identified by ChIP-Seq in (A) Nalm6 B-ALL cells and (B) primary B-ALL cells (representative data from 1 of 4 patients without IKZF1 deletion is shown). (C) Expression of Ikaros target genes that control the PI3K pathway in Nalm6 cells transduced with vector containing Ikaros compared with controls (measured by qRT-PCR). (D) Promoter activity of Ikaros target genes that control the PI3K pathway as measured by luciferase assay following transfection with Ikaros or control vector. (E) Effect of Ikaros knockdown on the gene expression of Ikaros targets that control the PI3K pathway. Gene expression was determined by qRT-PCR using total RNA isolated from the Nalm6 cells transfected with scramble shRNA or Ikaros shRNA and cultured for 2 days. (F) Loss of phosphorylation at serine 473 of AKT kinase in cells transduced with vector containing Ikaros compared with control. The loss of phosphorylation of serine 473 of AKT kinase following imatinib treatment was used as positive control (right). (G) Model of Ikaros-mediated regulation of the PI3K pathway: Ikaros represses transcription of genes that promote the PI3K pathway, but induces transcription of a gene that inhibits the PI3K pathway (INPP5D), leading to negative regulation of the PI3K pathway and cellular proliferation.