NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases

The transcriptional regulation of the murine carbonic anhydrase II gene in mouse erythroleukemia cells

Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide, producing bicarbonate and hydrogen ions. Two primary CA isozymes are expressed in murine erythrocytes, of which form II accounts for about 90% of CA hydration activity. Mouse erythroleukemia (MEL) cells can be induced to undergo erythroid differentiation in culture by treatment with dimethyl sulfoxide (DMSO). Overall CA activity and the level of CAII protein are moderate in uninduced MEL cells, but rise 2- to 3-fold upon induction. This change in expression is regulated at several points, including the rate of transcription of the gene. CAII transcription changes coordinately with that of other erythroid genes, including CAI and $\beta$-globin, peaking after two days at about six times the uninduced level. In order to locate sequences involved in transcriptional regulation, the gene was screened for DNase I hypersensitive sites (HSS). Six promoter and intronic HSS were discovered and partially mapped, three of which intensified upon induction. Plasmids containing luciferase reporter genes driven by the CAII promoter, along with various combinations of the other HSS, were transfected into MEL cells and assayed for luciferase activity. It was necessary to include an exogenous enhancer to obtain readable transient expression. Expression of the basic construct decreased $\sim$50% upon induction, showing that the promoter itself was not inducible. Insertion of a 3\sp\prime region containing at least one of the inducible HSS resulted in a reproducible 2-fold increase in promoter inducibility. Inclusion or elimination of the other intronic sites produced no detectable effect. Using gel-shift and footprinting assays, a protein complex was found to bind to the promoter and was localized to a triple repeat sequence. Complex binding was affected only by mutagenesis of the middle repeat (R2), but transient luciferase expression was increased by mutation of R1 or R2, and decreased by mutation of R3. None of the mutations affected induction. Thus, a region near the 3\sp\prime end of the CAII gene contributes to its transcriptional control, but sequences or combinations of regions in addition to those tested are required for its full expression and appropriate regulation during erythropoiesis

Absence of an Essential Role for Thymic Stromal Lymphopoietin Receptor in Murine B-Cell Development

The murine cytokine thymic stromal lymphopoietin (TSLP) supports the development of B220(+) IgM(+) immature B cells and induces thymocyte proliferation in vitro. Human TSLP, by contrast, activates CD11c(+) dendritic cells, but not B or T cells. Recent studies have demonstrated that the receptor for TSLP consists of a heterodimer of the interleukin 7 (IL-7) α chain and a novel protein that resembles the hematopoietic cytokine receptor common γ chain. We examined signal transduction by the γ-like chains using chimeric receptor proteins. The cytoplasmic domain of the human, but not of the murine, γ-like chain, activates Jak2 and Stat5 and supports the proliferation of hematopoietic cell lines. In order to assess the role of the murine γ-like chain in vivo, we generated γ-like chain-deficient mice. Receptor-deficient mice are unresponsive to TSLP but exhibit no obvious phenotypic defects. In particular, hematopoietic cell development appeared normal. B-cell development, including the IgM(+) compartment, was unaffected by loss of the TSLP pathway, as were T lymphopoiesis and lymphocyte proliferation in vitro. Cytokine receptors that utilize the common γ chain signal through the lymphocyte-specific kinase Jak3. Mice deficient in Jak3 exhibit a SCID phenotype but harbor a residual B220(+) splenic lymphocyte population. We demonstrate here that this residual lymphocyte population is lost in mice lacking both the γ-like chain and Jak3