Suppression of B-cell development genes is key to glucocorticoid efficacy in treatment of acute lymphoblastic leukemia

Abstract

Glucocorticoids (GCs), including dexamethasone (dex), are a central component of combination chemotherapy for childhood B-cell precursor acute lymphoblastic leukemia (B-ALL). GCs work by activating the GC receptor (GR), a ligand-induced transcription factor, which in turn regulates genes that induce leukemic cell death. Which GR-regulated genes are required for GC cytotoxicity, which pathways affect their regulation, and how resistance arises are not well understood. Here, we systematically integrate the transcriptional response of B-ALL to GCs with a next-generation short hairpin RNA screen to identify GC-regulated "effector" genes that contribute to cell death, as well as genes that affect the sensitivity of B-ALL cells to dex. This analysis reveals a pervasive role for GCs in suppression of B-cell development genes that is linked to therapeutic response. Inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ), a linchpin in the pre-B-cell receptor and interleukin 7 receptor signaling pathways critical to B-cell development (with CAL-101 [idelalisib]), interrupts a double-negative feedback loop, enhancing GC-regulated transcription to synergistically kill even highly resistant B-ALL with diverse genetic backgrounds. This work not only identifies numerous opportunities for enhanced lymphoid-specific combination chemotherapies that have the potential to overcome treatment resistance, but is also a valuable resource for understanding GC biology and the mechanistic details of GR-regulated transcription.

Figure 1.

Dex regulates B-cell development genes in sensitive B-ALL samples. (A) Heatmap clustering genes commonly regulated (KS test, q ≤ 10⁻⁴) by dex across 16 samples. Primary and PDX samples are marked red; cell lines, black. (B) Ingenuity pathway analysis of regulated genes shows enrichment for hematological development genes. (C) Stop or push through model for dex in B-cell development highlighting the roles of dex-repressed ITGA4, IL7R, and BCL6. (D-F) Differential gene expression values across sensitive B-ALL sample across samples measured by microarray (left) and GR occupancy in sensitive (B1) and resistant (HM3101) samples measured by ChIP-seq in response to dex suggest ITGA4, IL7R, and BCL6 are direct targets of GR regulation.

Figure 2.

Next-generation shRNA screen identifies sources of sensitivity and resistance to dex in B-ALL. (A) Venn diagrams showing that 247 of the CRGs are covered by the screen, 63 of which affect dex sensitivity. (B) Volcano plot of the effect of shRNA gene knockdown on dex sensitivity. Each point is a gene with the log significance on the y-axis, with relative effect (phenotype) on dex-induced cell death on the x-axis. GR is the most protective when knocked down, and knockdown of PIK3CD makes NALM-6 cell more sensitive. Top hits (Mann-Whitney, P ≤ .05) are green: sensitizing; purple: protective; gray: P > .05. (C) Zoom of volcano plot showing genes commonly mutated in treatment resistant or relapsed patients with B-ALL have an effect on dex sensitivity when knocked down (supplemental Table 1). (D) Identification of effector genes from among the CRGs. Plot of dex sensitivity phenotype when knocked down (x-axis) vs the average change in expression in response to dex (y-axis) for genes that are significantly regulated by dex and are top hits in the screen. Genes validated as effectors of dex-induced cell death are either: (1) downregulated by dex and cause sensitivity when knocked down (green shaded) or (2) upregulated by dex and are protective when knocked down (purple shaded). Genes involved in B-cell development or previously identified as effectors are in bold.

Figure 3.

Suppression of BCR signaling is detrimental to growth and sensitizes B-ALL to dex. The effects of gene knockdown on growth (A) and dex sensitivity (B) are overlaid on components of the BCR pathway. Genes are present when included in the screen, and shaded when the effect of knockdown is significant (Mann-Whitney, P ≤ .05). Dashed lines indicate repression of PIK3CD and IL7R expression by dex (diagrams based on Ingenuity pathways, and other literature^,).

Figure 4.

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.

Figure 5.

Inhibition of PI3Kδ synergizes with dex in regulating cell-death effector genes. (A) Change in gene expression measured by quantitative polymerase chain reaction (qPCR) in response to 2 concentrations of dex at 24 hours in 3 cell lines. (B) Change in gene expression measured by qPCR in response to 2 concentrations of CAL-101 alone and in combination with 2 concentrations of dex (C) at 24 hours in the same cell lines. Experiments represent at least 3 biological repeats. *P ≤ .05 (see “Methods” for details). Dashed boxes highlight genes whose regulation is restored by CAL-101 (idela).