Disruption of double-negative feedback loop between PI3Kδ and GR enhances dex cytotoxicity. (A) Schematic feedback loop based on combined data from the shRNA screen and microarray gene expression data. Dex-induced repression of PIK3CD (blue blocking arrow, PI3Kδ) and activation of PIK3IP1 (red arrow) gene expression. shRNA knockdown of PTEN and PIK3R2 was protective (purple), whereas knockdown specifically of PIK3CD sensitized cells to dex (green). Thus, interruption of PIK3δ inhibition of GR is expected to synergistically induce cell death. (B-D) Results of the shRNA screen. Bar graphs show the log10(P) of the hits from the shRNA screen. Sensitizing hits have been depicted as negative (green), protective as positive (purple). (E) Effect of dex on gene expression. Fold change of gene expression across sensitive B-ALL samples as measured by microarray (left) and GR occupancy as measured by ChIP-seq (right) post-dex treatment. Primary and PDX samples are marked red; cell lines, black. ChIP-seq data are shown for sensitive (B1) and resistant (HM3101) samples. The presence of GR-binding sites in sensitive cells for both PIK3IP1 and PIK3CD indicates potential direct regulation by dex. (F) The combination index of dex and CAL-101 in sensitive (NALM-6, SUP-B15) and resistant (RCH-ACV) cell lines, a resistant patient sample (HM3101), and a multiply relapsed refractory patient-derived mouse xenograft (ALL121) (super additive <1; CalcuSyn). Numbers reflect isobolograms depicted in supplemental Figure 9. (G) Quantification of westerns against phospho-S203 of GR in the absence and presence of PI3Kδ inhibition (error bars represent standard error of the mean [SEM] across 4 time points). CAL-101 treatment reduces GR S203 phosphorylation, likely increasing GR activity. (H) Spleens of mice (n = 5 mice/cohort) engrafted with relapsed B-ALL cells (ALL121) and treated with vehicle, dex (7.5 mg/kg), idela (50 mg/kg), or both for 2 weeks. Enlarged spleens indicate the accumulation of lymphoblasts. Treatment with either dex or idela alone failed to significantly reduce spleen size compared with untreated control; however, treatment with both dex and idela significantly reduced spleen size, indicating a synergistic effect between the 2 drugs. (I) Total number of human ALL cells (y-axis) in spleens of mice in panel H as measured by quantitative flow cytometry.