N-acetyl cysteine turns EPAC activators into potent killers of acute lymphoblastic leukemia cells.

Today, the majority of patients with pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell-permeable cAMP analog 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.

cAMP-Mediated Autophagy Promotes Cell Survival via ROS-Induced Activation of PARP1: Implications for Treatment of Acute Lymphoblastic Leukemia.

DNA-damaging therapy is the basis for treatment of most cancers, including B-cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL). We have previously shown that cAMP-activating factors present in the bone marrow render ALL cells less sensitive to DNA damage-induced apoptosis, by enhancing autophagy and suppressing p53. To sensitize ALL cells to DNA-damaging therapy, we have searched for novel targets that may counteract the effects induced by cAMP signaling. In the current study, we have identified PARP1 as a potential target. We show that the PARP1 inhibitors olaparib or PJ34 inhibit cAMP-mediated autophagy and thereby potentiate the DNA-damaging treatment. Furthermore, we reveal that cAMP-mediated PARP1 activation is preceded by induction of reactive oxygen species (ROS) and results in depletion of nicotinamide adenine dinucleotide (NAD), both of which are autophagy-promoting events. Accordingly, we demonstrate that scavenging ROS by N-acetylcysteine and repleting NAD independently reduce DNA damage-induced autophagy. In addition, olaparib augmented the effect of DNA-damaging treatment in a human xenograft model of ALL in NOD-scidIL2Rgammanull mice. On the basis of the current findings, we suggest that PARP1 inhibitors may enhance the efficiency of conventional genotoxic therapies and thereby provide a novel treatment strategy for pediatric patients with ALL. IMPLICATIONS: PARP1 inhibitors augment the DNA damage-induced killing of ALL cells by limiting the opposing effects of cAMP-mediated autophagy, which involves ROS-induced PARP1 activation and depletion of cellular NAD levels.

Nuclear IL-33 restrains the early conversion of fibroblasts to an extracellular matrix-secreting phenotype.

Interleukin (IL)-33 is a cytokine that appears to mediate fibrosis by signaling via its receptor ST2 (IL-33R/IL1RL1). It is also, however, a protein that after synthesis is sorted to the cell nucleus, where it appears to affect chromatin folding. Here we describe a novel role for nuclear IL-33 in regulating the fibroblast phenotype in murine kidney fibrosis driven by unilateral ureteral obstruction. Transcriptional profiling of IL-33-deficient kidneys 24 h after ligation revealed enhanced expression of fibrogenic genes and enrichment of gene sets involved in extracellular matrix formation and remodeling. These changes relied on intracellular effects of IL-33, because they were not reproduced by treatment with a neutralizing antibody to IL-33 that prevents IL-33R/ST2L receptor signaling nor were they observed in IL-33R/ST2-deficient kidneys. To further explore the intracellular function of IL-33, we established transcription profiles of human fibroblasts, observing that knockdown of IL-33 skewed the transcription profile from an inflammatory towards a myofibroblast phenotype, reflected in higher levels of COL3A1, COL5A1 and transgelin protein, as well as lower expression levels of IL6, CXCL8, CLL7 and CCL8. In conclusion, our findings suggest that nuclear IL-33 in fibroblasts dampens the initial profibrotic response until persistent stimuli, as enforced by UUO, can override this protective mechanism.

Lack of interleukin-33 and its receptor does not prevent calcipotriol-induced atopic dermatitis-like inflammation in mice.

Current studies addressing the influence of interleukin-33 or its receptor (IL-33R/ST2) on development of atopic dermatitis-like inflammation in mice have reported conflicting results. We compared the response in single- and double-deficient IL-33-/-/ST2-/- C57BL/6J BomTac mice in the well-established calcipotriol-induced model of atopic dermatitis. All genotypes (groups of up to 14 mice) developed atopic dermatitis-like inflammation yet we observed no biologically relevant difference between groups in gross anatomy or ear thickness. Moreover, histological examination of skin revealed no differences in mononuclear leukocyte and granulocyte infiltration nor Th2 cytokine levels (IL-4 and IL-13). Finally, skin CD45+ cells and CD3+ cells were found at similar densities across all groups. Our findings indicate that lack of interleukin-33 and its receptor ST2 does not prevent the development of AD-like skin inflammation.

Hypo-osmotic Stress Drives IL-33 Production in Human Keratinocytes-An Epidermal Homeostatic Response.

Although inflammation has traditionally been considered a response to either exogenous pathogen-associated signals or endogenous signals of cell damage, other perturbations of homeostasis, generally referred to as stress, may also induce inflammation. The relationship between stress and inflammation is, however, not well defined. Here, we describe a mechanism of IL-33 induction driven by hypo-osmotic stress in human keratinocytes and also report interesting differences when comparing the responsiveness of other inflammatory mediators. The induction of IL-33 was completely dependent on EGFR and calcium signaling, and inhibition of calcium signaling not only abolished IL-33 induction but also dramatically changed the transcriptional pattern of other cytokines upon hypo-osmotic stress. IL-33 was not secreted but instead showed nuclear sequestration, conceivably acting as a failsafe mechanism whereby it is induced by potential danger but released only upon more extreme homeostatic perturbations that result in cell death. Finally, stress-induced IL-33 was also confirmed in an ex vivo human skin model, translating this mechanism to a potential tissue-relevant signal in the human epidermis. In conclusion, we describe hypo-osmotic stress as an inducer of IL-33 expression, linking cellular stress to nuclear accumulation of a strong proinflammatory cytokine.

Epidermal Expression and Regulation of Interleukin-33 during Homeostasis and Inflammation: Strong Species Differences.

IL-33 is a novel IL-1 family member with a putative role in inflammatory skin disorders and a complex biology. Therefore, recent conflicting data regarding its function in experimental models justify a close assessment of its tissue expression and regulation. Indeed, we report here that there are strong species differences in the expression and regulation of epidermal IL-33. In murine epidermis, IL-33 behaved similar to an alarmin, being constitutively expressed in keratinocyte nuclei and rapidly lost during acute inflammation. By contrast, human and porcine IL-33 were weakly expressed or absent in keratinocytes of noninflamed skin but induced during acute inflammation. To this end, we observed that expression of IL-33 in human keratinocytes but not murine keratinocytes was strongly induced by IFN-γ, and this upregulation completely depended on the presence of EGFR ligands. Accordingly, IFN-γ increased the expression of IL-33 in the basal layers of the epidermis in human ex vivo skin cultures only, despite good evidence of IFN-γ activity in cultures from both species. Together these findings demonstrate that a full understanding of IL-33 function in clinical settings must take species-specific differences into account.