PARP1-MGMT complex underpins pathway crosstalk in O6-methylguanine repair.

DNA lesions induced by alkylating agents are repaired by two canonical mechanisms, base excision repair dependent on poly(ADP) ribose polymerase 1 (PARP1) and the other mediated by O6-methylguanine (O6meG)-DNA methyltransferase (MGMT) in a single-step catalysis of alkyl-group removal. O6meG is the most cytotoxic and mutagenic lesion among the methyl adducts induced by alkylating agents. Although it can accomplish the dealkylation reaction all by itself as a single protein without associating with other repair proteins, evidence is accumulating that MGMT can form complexes with repair proteins and is highly regulated by a variety of post-translational modifications, such as phosphorylation, ubiquitination, and others. Here, we show that PARP1 and MGMT proteins interact directly in a non-catalytic manner, that MGMT is subject to PARylation by PARP1 after DNA damage, and that the O6meG repair is enhanced upon MGMT PARylation. We provide the first evidence for the direct DNA-independent PARP1-MGMT interaction. Further, PARP1 and MGMT proteins also interact via PARylation of MGMT leading to formation of a novel DNA damage inducible PARP1-MGMT protein complex. This catalytic interaction activates O6meG repair underpinning the functional crosstalk between base excision and MGMT-mediated DNA repair mechanisms. Furthermore, clinically relevant 'chronic' temozolomide exposure induced PARylation of MGMT and increased binding of PARP1 and MGMT to chromatin in cells. Thus, we provide the first mechanistic description of physical interaction between PARP1 and MGMT and their functional cooperation through PARylation for activation of O6meG repair. Hence, the PARP1-MGMT protein complex could be targeted for the development of advanced and more effective cancer therapeutics, particularly for cancers sensitive to PARP1 and MGMT inhibition.

Mitochondrial TrxR2 regulates metabolism and protects from metabolic disease through enhanced TCA and ETC function.

Mitochondrial dysfunction is a key driver of diabetes and other metabolic diseases. Mitochondrial redox state is highly impactful to metabolic function but the mechanism driving this is unclear. We generated a transgenic mouse which overexpressed the redox enzyme Thioredoxin Reductase 2 (TrxR2), the rate limiting enzyme in the mitochondrial thioredoxin system. We found augmentation of TrxR2 to enhance metabolism in mice under a normal diet and to increase resistance to high-fat diet induced metabolic dysfunction by both increasing glucose tolerance and decreasing fat deposition. We show this to be caused by increased mitochondrial function which is driven at least in part by enhancements to the tricarboxylic acid cycle and electron transport chain function. Our findings demonstrate a role for TrxR2 and mitochondrial thioredoxin as metabolic regulators and show a critical role for redox enzymes in controlling functionality of key mitochondrial metabolic systems.

Nanoformulation of Talazoparib Increases Maximum Tolerated Doses in Combination With Temozolomide for Treatment of Ewing Sarcoma.

The Pediatric Preclinical Testing Program previously identified the PARP inhibitor talazoparib (TLZ) as a means to potentiate temozolomide (TMZ) activity for the treatment of Ewing sarcoma. However, the combination of TLZ and TMZ has been toxic in both preclinical and clinical testing, necessitating TMZ dose reduction to ~15% of the single agent maximum tolerated dose. We have synthesized a nanoparticle formulation of talazoparib (NanoTLZ) to be administered intravenously in an effort to modulate the toxicity profile of this combination treatment. Results in Ewing sarcoma xenograft models are presented to demonstrate the utility of this delivery method both alone and in combination with TMZ. NanoTLZ reduced gross toxicity and had a higher maximum tolerated dose than oral TLZ. The dose of TMZ did not have to be reduced when combined with NanoTLZ as was required when combined with oral TLZ. This indicated the NanoTLZ delivery system may be advantageous in decreasing the systemic toxicity associated with the combination of oral TLZ and TMZ.

Dual targeting of androgen receptor and mTORC1 by salinomycin in prostate cancer.

Androgen receptor (AR) and PI3K/AKT/mTORC1 are major survival signals that drive prostate cancer to a lethal disease. Reciprocal activation of these oncogenic pathways from negative cross talks contributes to low/limited success of pathway-selective inhibitors in curbing prostate cancer progression. We report that the antibiotic salinomycin, a cancer stem cell blocker, is a dual-acting AR and mTORC1 inhibitor, inhibiting PTEN-deficient castration-sensitive and castration-resistant prostate cancer in culture and xenograft tumors. AR expression, its transcriptional activity, and androgen biosynthesis regulating enzymes CYP17A1, HSD3ß1 were reduced by sub-micro molar salinomycin. Estrogen receptor-α expression was unchanged. Loss of phosphorylated AR at serine-81, which is an index for nuclear AR activity, preceded total AR reduction. Rapamycin enhanced the AR protein level without altering phosphoAR-Ser81 and CYP17A1. Inactivation of mTORC1, evident from reduced phosphorylation of mTOR and downstream effectors, as well as AMPK activation led to robust autophagy induction. Apoptosis increased modestly, albeit significantly, by sub-micro molar salinomycin. Enhanced stimulatory TSC2 phosphorylation at Ser-1387 by AMPK, and reduced inhibitory TSC2 phosphorylation at Ser-939/Thr-1462 catalyzed by AKT augmented TSC2/TSC1 activity, which led to mTORC1 inhibition. AMPK-mediated raptor phosphorylation further reduced mTOR's kinase function and mTORC1 activity. Our novel finding on dual inhibition of AR and mTORC1 suggests that salinomycin is potentially active as monotherapy against advanced prostate cancer.

Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions.

Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug's impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and microfluidic organs-on-chips, which mimic the physiology of a multicellular environment, will likely replace the current cell-based workflow.

Developmental expression of Toll-like receptors-2 and -4 in preterm baboon lung.

Preterm babies are susceptible to respiratory infection due to immature lung and immune system. Immune cells express Toll-like receptors (TLRs), which may be important in local host defense of preterm infants. We studied the expression of TLR2 and TLR4 in lung tissues of fetal baboons delivered at 125, 140, and 175 days of gestation (dGA; term=185+/-2 days) and preterm baboons that became naturally infected with bacterial/fungal pathogens. The TLR-mRNA and protein were quantified by Northern and Western blotting, respectively. The expression of both TLRs was significantly low at 125 and 140dGA. At 175dGA, the levels reached equivalent to those in adult baboons. However, in naturally infected baboons, the TLR4-mRNA was reduced (p<0.05); TLR2-mRNA expression remained unaltered. The protein expression of both TLRs was found increased in naturally infected baboons. Our results suggest that the lung TLR expression is developmentally regulated and altered during respiratory infection in preterm babies.

Immunophenotype and functions of fetal baboon bone-marrow derived dendritic cells.

Dendritic cells (DCs) are unique antigen-presenting cells that can take up pathogens, pathogens-derived and stress-antigens and stimulate antigen-specific immune response. Here we investigated the immunobiology of fetal DCs and compared their phenotype and activation status against infectious stimuli with those of young and adult baboons. The DCs were obtained from femoral bone-marrow (BMDCs) of fetus (140 and 175 days of gestation), young (4-5 years old) and mature adult (10-35 years old) baboons. The cells were cultured in the presence of GM-CSF and IL-4. To study phagocytic ability of BMDCs, the cells were harvested on 6th day and incubated with fluorescent-labeled Escherichia coli bioparticles. The BMDCs were also treated with E. coli O111:B4 lipopolysaccharide (LPS) for 24h and changes in expression of cell-surface markers and IL-12 were studied using distinct immunoassays. We found that the phenotype and morphology of BMDCs from fetal, young and adult baboons were similar and showed increased expression of HLA-DP, DQ, DR and T cell co-stimulatory molecules upon LPS treatment. However, significant differences were observed in phagocytic activity and IL-12 secretion among BMDCs from these sources. The ability of fetal baboon BMDCs to phagocytose E. coli bioparticles was significantly lower and they secreted lower level of LPS-stimulated IL-12 as compared to the BMDCs from adult baboon. These results suggest that compared to adult BMDCs, fetal baboon BMDCs are less efficient in mounting immune response against Gram-negative bacterial stimuli.

Temporal regulation of agonist efficacy at 5-hydroxytryptamine (5-HT)1A and 5-HT 1B receptors.

Coactivation of purinergic (P 2Y) receptors reduces agonist efficacy at serotonin 1B (5-HT 1B), but not 5-HT 1A receptors. Herein, we report that pretreatment for 5 min with the P 2Y receptor agonist ATP reduced agonist responsiveness at the 5-HT 1A, but not at the 5-HT 1B, receptor. The effect of ATP pretreatment on the 5-HT 1A receptor response rapidly reversed within a 10 min time frame between P 2Y receptor and 5-HT 1A receptor activation. ATP pretreatment effects on 5-HT 1A agonist responsiveness were blocked by the protein kinase inhibitors staurosporine and bisindolylmaleimide, suggesting that the ATP-mediated temporal regulation involves activation of protein kinase C (PKC). Moreover, the temporal effect of ATP was blocked by incubation with 1% ethanol, suggesting that consequences of phospholipase D (PLD) activation play a role. ATP pretreatment blocked the inhibitory effect produced by 5-HT 2C receptor activation on the 5-HT 1A, but not the 5-HT 1B, receptor response, suggesting that the 5-HT 1A receptor itself was the target for PLD/PKC action. Finally, ethanol did not block the reduction in responsiveness of the 5-HT 1A receptor system produced by activation of PKC with phorbol ester treatment, suggesting that PKC activation lies downstream of PLD. Taken together, these data suggest that activation of P 2Y receptors can reduce responsiveness of the 5-HT 1A receptor system via a PLD/PKC-dependent mechanism that is highly dependent upon the temporal pattern of receptor activation. Moreover, this work underscores the importance of time as a variable in receptor signaling cross talk and serves to further illustrate differences between the 5-HT 1A and 5-HT 1B receptor systems.