PI3Kδ inhibition potentiates glucocorticoids in B-lymphoblastic leukemia by decreasing receptor phosphorylation and enhancing gene regulation.

Glucocorticoids, including dexamethasone and prednisone, are the cornerstone of B-lymphoblastic leukemia (B-ALL) therapy. Because response to glucocorticoids alone predicts overall outcomes for B-ALL, enhancing glucocorticoid potency is a route to improving outcomes. However, systematic toxicities prevent the use of higher dose and more potent glucocorticoids. We therefore took a functional genomic approach to identify targets to enhance glucocorticoid activity specifically in B-ALL cells. Here we show that inhibition of the lymphoid-restricted PI3Kδ, signaling through the RAS/MAPK pathway, enhances both prednisone and dexamethasone activity in almost all ex vivo B-ALL specimens tested. This potentiation is most synergistic at sub-saturating doses of glucocorticoids, approaching the EC50. Potentiation correlates with global enhancement of glucocorticoid-induced gene regulation, including regulation of effector genes that drive B-ALL cell death. Idelalisib reduces phosphorylation of the glucocorticoid receptor (GR) at MAPK1/ERK2 targets S203 and S226, and ablation of these phospho-acceptor sites enhances glucocorticoid potency. We further show that phosphorylation of S226 reduces the affinity of GR for DNA in vitro, which impairs DNA binding. We therefore propose that PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation, increasing DNA binding affinity, and enhancing downstream gene regulation. The overall enhancement of GR function suggests that idelalisib will provide benefit to most patients with B-ALL by improving outcomes for patients whose disease is less responsive to glucocorticoid-based therapy, including high-risk disease, and allowing less toxic glucocorticoid-sparing strategies for patients with standard-risk disease.

PI3Kδ Inhibition Potentiates Glucocorticoids in B-lymphoblastic Leukemia by Decreasing Receptor Phosphorylation and Enhancing Gene Regulation.

Glucocorticoids are the cornerstone of B-lymphoblastic leukemia (B-ALL) therapy. Because response to glucocorticoids alone predicts overall outcomes for B-ALL, enhancing glucocorticoid potency should improve treatment. We previously showed that inhibition of the lymphoid-restricted PI3Kδ with idelalisib enhances glucocorticoid activity in B-ALL cells. Here, we show that idelalisib enhances glucocorticoid potency in 90% of primary B-ALL specimens and is most pronounced at sub-saturating doses of glucocorticoids near the EC50. Potentiation is associated with enhanced regulation of all glucocorticoid-regulated genes, including genes that drive B-ALL cell death. Idelalisib reduces phosphorylation of the glucocorticoid receptor (GR) at PI3Kδ/MAPK1 (ERK2) targets S203 and S226. Ablation of these phospho-acceptor sites enhances sensitivity to glucocorticoids with ablation of S226 in particular reducing synergy. We also show that phosphorylation of S226 reduces the affinity of GR for DNA in vitro. We propose that PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation, increasing DNA binding affinity, and enhancing downstream gene regulation. This mechanism and the response of patient specimens suggest that idelalisib will benefit most patients with B-ALL, but particularly patients with less responsive, including high-risk, disease. This combination is also promising for the development of less toxic glucocorticoid-sparing therapies.

Deacylcortivazol-like pyrazole regioisomers reveal a more accommodating expanded binding pocket for the glucocorticoid receptor.

Glucocorticoids (GCs) are widely used, potent anti-inflammatory and chemotherapeutic drugs. They work by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor, inducing translocation to the nucleus and regulation of genes that influence a variety of cellular activities. Despite being effective for a broad number of conditions, GC use is limited by severe side effects. To identify ligands that are more selective, we synthesized pairs of regioisomers in the pyrazole ring that probe the expanded binding pocket of GR opened by deacylcortivazol (DAC). Using an Ullmann-type reaction, a deacylcortivazol-like (DAC-like) backbone was modified with five pendant groups at the 1'- and 2'-positions of the pyrazole ring, yielding 9 ligands. Most of the compounds were cytotoxic to leukemia cells, and all required GR expression. Both aliphatic and other aromatic groups substituted at the 2'-position produced ligands with GC activity, with phenyl and 4-fluorophenyl substitutions exhibiting high cellular affinity for the receptor and >5× greater potency than dexamethasone, a commonly used strong GC. Surprisingly, phenyl substitution at the 1'-position produced a high-affinity ligand with ∼10× greater potency than dexamethasone, despite little apparent room in the expanded binding pocket to accommodate 1'-modifications. Other 1'-modifications, however, were markedly less potent. The potency of the 2'-substituted and 1'-substituted DAC-like compounds tracked linearly with cellular affinity but had different slopes, suggesting a different mode of interaction with GR. These data provide evidence that the expanded binding pocket opened by deacylcortivazol is more accommodating that expected, allowing development of new, and possibly selective, GCs by substitution within the pyrazole ring.

Relapse-associated AURKB blunts the glucocorticoid sensitivity of B cell acute lymphoblastic leukemia.

Glucocorticoids (GCs) are used in combination chemotherapies as front-line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although effective, many patients relapse and become resistant to chemotherapy and GCs in particular. Why these patients relapse is not clear. We took a comprehensive, functional genomics approach to identify sources of GC resistance. A genome-wide shRNA screen identified the transcriptional coactivators EHMT2, EHMT1, and CBX3 as important contributors to GC-induced cell death. This complex selectively supports GC-induced expression of genes contributing to cell death. A metaanalysis of gene expression data from B-ALL patient specimens revealed that Aurora kinase B (AURKB), which restrains GC signaling by phosphorylating EHMT1-2, is overexpressed in relapsed B-ALL, suggesting it as a potential contributor to relapse. Inhibition of AURKB enhanced GC-induced expression of cell death genes, resulting in potentiation of GC cytotoxicity in cell lines and relapsed B-ALL patient samples. This function for AURKB is distinct from its canonical role in the cell cycle. These results show the utility of functional genomics in understanding mechanisms of resistance and rapidly identifying combination chemotherapeutics.

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

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.

Age-related increase in the frequency of CD4(+) T cells that produce interferon-gamma in response to staphylococcal enterotoxin B during childhood.

BACKGROUND: The susceptibility of infants to infections is well defined clinically, and immunologic abnormalities have been described. Immune maturation is complex, however, and the interval during which changes occur during childhood has not been identified. METHODS: To assess age-related differences in the CD4(+) T cell responses, we evaluated the frequency of CD4(+) T cells that produced interferon (IFN) gamma in response to staphylococcal enterotoxin B (SEB) stimulation in 382 healthy infants and children (2 months to 11 years of age) and 66 adults. Flow cytometry was used to assess SEB-induced CD69 and CD40 ligand (CD40-L) expression and IFN-gamma production by CD4(+) and CD45RO(+)CD4(+) T cells. RESULTS: CD69 and CD40-L expression by CD4(+) and CD45RO(+)CD4(+) T cells were similar to adult levels from infancy, but the frequency of activated T cells that produced IFN-gamma remained lower than adult responses until children were 10 years of age. CONCLUSIONS: These observations indicate that the IFN-gamma response of CD4(+) T cells to SEB remains limited for a much longer interval than was reported elsewhere, extending to the second decade of life. Observed differences in CD45RO(+)CD4(+) T cell function indicate that CD4(+) T cells with the same phenotypes do not possess equivalent functional capabilities.