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.

Targeting cyclooxygenase by indomethacin decelerates progression of acute lymphoblastic leukemia in a xenograft model.

Acute lymphoblastic leukemia (ALL) develops in the bone marrow in the vicinity of stromal cells known to promote tumor development and treatment resistance. We previously showed that the cyclooxygenase (COX) inhibitor indomethacin prevents the ability of stromal cells to diminish p53-mediated killing of cocultured ALL cells in vitro, possibly by blocking the production of prostaglandin E2 (PGE2). Here, we propose that PGE2 released by bone marrow stromal cells might be a target for improved treatment of pediatric ALL. We used a xenograft model of human primary ALL cells in nonobese diabetic-scid IL2rγnull mice to show that indomethacin delivered in the drinking water delayed the progression of ALL in vivo. The progression was monitored by noninvasive in vivo imaging of the engrafted leukemic cells, as well as by analyses of CD19+CD10+ leukemic blasts present in spleen or bone marrow at the termination of the experiments. The indomethacin treatment increased the level of p53 in the leukemic cells, implying that COX inhibition might reduce progression of ALL by attenuating protective paracrine PGE2 signaling from bone marrow stroma to leukemic cells.

cAMP-mediated autophagy inhibits DNA damage-induced death of leukemia cells independent of p53.

Autophagy is important in regulating the balance between cell death and survival, with the tumor suppressor p53 as one of the key components in this interplay. We have previously utilized an in vitro model of the most common form of childhood cancer, B cell precursor acute lymphoblastic leukemia (BCP-ALL), to show that activation of the cAMP signaling pathway inhibits p53-mediated apoptosis in response to DNA damage in both cell lines and primary leukemic cells. The present study reveals that cAMP-mediated survival of BCP-ALL cells exposed to DNA damaging agents, involves a critical and p53-independent enhancement of autophagy. Although autophagy generally is regarded as a survival mechanism, DNA damage-induced apoptosis has been linked both to enhanced and reduced levels of autophagy. Here we show that exposure of BCP-ALL cells to irradiation or cytotoxic drugs triggers autophagy and cell death in a p53-dependent manner. Stimulation of the cAMP signaling pathway further augments autophagy and inhibits the DNA damage-induced cell death concomitant with reduced nuclear levels of p53. Knocking-down the levels of p53 reduced the irradiation-induced autophagy and cell death, but had no effect on the cAMP-mediated autophagy. Moreover, prevention of autophagy by bafilomycin A1 or by the ULK-inhibitor MRT68921, diminished the protecting effect of cAMP signaling on DNA damage-induced cell death. Having previously proposed a role of the cAMP signaling pathway in development and treatment of BCP-ALLs, we here suggest that inhibitors of autophagy may improve current DNA damage-based therapy of BCP-ALL - independent of p53.

AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint.

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.