Specific and shared biological functions of PARP2 - is PARP2 really a lil' brother of PARP1?

PARP2, that belongs to the family of ADP-ribosyl transferase enzymes (ART), is a discovery of the millennium, as it was identified in 1999. Although PARP2 was described initially as a DNA repair factor, it is now evident that PARP2 partakes in the regulation or execution of multiple biological processes as inflammation, carcinogenesis and cancer progression, metabolism or oxidative stress-related diseases. Hereby, we review the involvement of PARP2 in these processes with the aim of understanding which processes are specific for PARP2, but not for other members of the ART family. A better understanding of the specific functions of PARP2 in all of these biological processes is crucial for the development of new PARP-centred selective therapies.

PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

Leveraging shape screening and molecular dynamics simulations to optimize PARP1-Specific chemo/radio-potentiators for antitumor drug design.

PARP1 plays a pivotal role in DNA repair within the base excision pathway, making it a promising therapeutic target for cancers involving BRCA mutations. Current study is focused on the discovery of PARP inhibitors with enhanced selectivity for PARP1. Concurrent inhibition of PARP1 with PARP2 and PARP3 affects cellular functions, potentially causing DNA damage accumulation and disrupting immune responses. In step 1, a virtual library of 593 million compounds has been screened using a shape-based screening approach to narrow down the promising scaffolds. In step 2, hierarchical docking approach embedded in Schrödinger suite was employed to select compounds with good dock score, drug-likeness and MMGBSA score. Analysis supplemented with decomposition energy, molecular dynamics (MD) simulations and hydrogen bond frequency analysis, pinpointed that active site residues; H862, G863, R878, M890, Y896 and F897 are crucial for specific binding of ZINC001258189808 and ZINC000092332196 with PARP1 as compared to PARP2 and PARP3. The binding of ZINC000656130962, ZINC000762230673, ZINC001332491123, and ZINC000579446675 also revealed interaction involving two additional active site residues of PARP1, namely N767 and E988. Weaker or no interaction was observed for these residues with PARP2 and PARP3. This approach advances our understanding of PARP-1 specific inhibitors and their mechanisms of action, facilitating the development of targeted therapeutics.

Transcription-replication conflicts underlie sensitivity to PARP inhibitors.

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1-6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

Cooperative nucleic acid binding by Poly ADP-ribose polymerase 1.

Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1's normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20 and U20 oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20 and U20 oligonucleotides show that PARP's binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1's ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

Network integration of thermal proteome profiling with multi-omics data decodes PARP inhibition.

Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on disease mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that can serve as proxies of functional protein changes. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a multi-omics integration framework, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover consequences of Poly (ADP-ribose) polymerase (PARP) inhibition in ovarian cancer cells on cell cycle and DNA damage response as well as interferon and hippo signaling. We found that TPP offers a complementary perspective to other omics data modalities, and that its integration allowed us to obtain a more complete molecular overview of PARP inhibition. We anticipate that this strategy can be used to integrate functional proteomics with other omics to study molecular processes.

CRABP2 reduces the sensitivity of Olaparib in ovarian cancer by downregulating Caspase-8 and decreasing the production of reactive oxygen species.

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, such as Olaparib, have been pivotal in treating BRCA-deficient ovarian cancer. However, their efficacy is limited in over 40% of BRCA-deficient patients, with acquired resistance posing new clinical challenges. To address this, we employed bioinformatics methods to identify key genes impacting Olaparib sensitivity in ovarian cancer. Through comprehensive analysis of public databases including GEO, CPTAC, Kaplan Meier Plotter, and CCLE, we identified CRABP2 as significantly upregulated at both mRNA and protein levels in ovarian cancer, correlating with poor prognosis and decreased Olaparib sensitivity. Using colony formation and CCK-8 assays, we confirmed that CRABP2 knockdown in OVCAR3 and TOV112D cells enhanced sensitivity to Olaparib. Additionally, 4D label-free quantitative proteomics analysis, GSEA, and GO/KEGG analysis revealed CRABP2's involvement in regulating oxidation signals. Flow cytometry, colony formation assays, and western blotting demonstrated that CRABP2 knockdown promoted ROS production by activating Caspase-8, thereby augmenting Olaparib sensitivity and inhibiting ovarian cancer cell proliferation. Moreover, in xenograft models, CRABP2 knockdown significantly suppressed tumorigenesis and enhanced Olaparib sensitivity, with the effect being reversed upon Caspase-8 knockdown. These findings suggest that CRABP2 may modulate Olaparib sensitivity in ovarian cancer through the Caspase-8/ROS axis, highlighting its potential as a target for Olaparib sensitization.

Targeting PARP14 with lomitapide suppresses drug resistance through the activation of DRP1-induced mitophagy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that remains incurable, primarily due to the high likelihood of relapse or development of resistance to current treatments. To explore and discover new medications capable of overcoming drug resistance in MM, we conducted cell viability inhibition screens of 1504 FDA-approved drugs. Lomitapide, a cholesterol-lowering agent, was found to exhibit effective inhibition on bortezomib-resistant MM cells in vitro and in vivo. Our data also indicated that lomitapide decreases the permeability of the mitochondrial outer membrane and induces mitochondrial dysfunction in MM cells. Next, lomitapide treatment upregulated DRP1 and PINK1 expression levels, coupled with the mitochondrial translocation of Parkin, leading to MM cell mitophagy. Excessive mitophagy caused mitochondrial damage and dysfunction induced by lomitapide. Meanwhile, PARP14 was identified as a direct target of lomitapide by SPR-HPLC-MS, and we showed that DRP1-induced mitophagy was crucial in the anti-MM activity mediated by PARP14. Furthermore, PARP14 is overexpressed in MM patients, implying that it is a novel therapeutic target in MM. Collectively, our results demonstrate that DRP1-mediated mitophagy induced by PARP14 may be the cause for mitochondrial dysfunction and damage in response to lomitapide treatment.

A Novel Inhibitor of Poly(ADP-Ribose) Polymerase-1 Inhibits Proliferation of a BRCA-Deficient Breast Cancer Cell Line via the DNA Damage-Activated cGAS-STING Pathway.

Loss-of-function mutations in the Breast Cancer Susceptibility Gene (BRCA1 and BRCA2) are often detected in patients with breast cancer. Poly(ADP-ribose) polymerase-1 (PARP1) plays a key role in the repair of DNA strand breaks, and PARP inhibitors have been shown to induce highly selective killing of BRCA1/2-deficient tumor cells, a mechanism termed synthetic lethality. In our previous study, a novel PARP1 inhibitor─(E)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(4-fluorophenyl) hydrazine-1-carbothioamide (4F-DDC)─was synthesized, which significantly inhibited PARP1 activity with an IC50 value of 82 ± 9 nM. The current study aimed to explore the mechanism(s) underlying the antitumor activity of 4F-DDC under in vivo and in vitro conditions. 4F-DDC was found to selectively inhibit the proliferation of BRCA mutant cells, with highly potent effects on HCC-1937 (BRCA1-/-) cells. Furthermore, 4F-DDC was found to induce apoptosis and G2/M cell cycle arrest in HCC-1937 cells. Interestingly, immunofluorescence and Western blot results showed that 4F-DDC induced DNA double strand breaks and further activated the cGAS-STING pathway in HCC-1937 cells. In vivo analysis results revealed that 4F-DDC inhibited the growth of HCC-1937-derived tumor xenografts, possibly via the induction of DNA damage and activation of the cGAS-STING pathway. In summary, the current study provides a new perspective on the antitumor mechanism of PARP inhibitors and showcases the therapeutic potential of 4F-DDC in the treatment of breast cancer.

Cannabinoids induce cell death in leukaemic cells through Parthanatos and PARP-related metabolic disruptions.

BACKGROUND: Several studies have described a potential anti-tumour effect of cannabinoids (CNB). CNB receptor 2 (CB2) is mostly present in hematopoietic stem cells (HSC). The present study evaluates the anti-leukaemic effect of CNB. METHODS: Cell lines and primary cells from acute myeloid leukaemia (AML) patients were used and the effect of the CNB derivative WIN-55 was evaluated in vitro, ex vivo and in vivo. RESULTS: We demonstrate a potent antileukemic effect of WIN-55 which is abolished with CB antagonists. WIN-treated mice, xenografted with AML cells, had better survival as compared to vehicle or cytarabine. DNA damage-related genes were affected upon exposure to WIN. Co-incubation with the PARP inhibitor Olaparib prevented WIN-induced cell death, suggesting PARP-mediated apoptosis which was further confirmed with the translocation of AIF to the nucleus observed in WIN-treated cells. Nicotinamide prevented WIN-related apoptosis, indicating NAD+ depletion. Finally, WIN altered glycolytic enzymes levels as well as the activity of G6PDH. These effects are reversed through PARP1 inhibition. CONCLUSIONS: WIN-55 exerts an antileukemic effect through Parthanatos, leading to translocation of AIF to the nucleus and depletion of NAD+, which are reversed through PARP1 inhibition. It also induces metabolic disruptions. These effects are not observed in normal HSC.

Excavatolide C/cisplatin combination induces antiproliferation and drives apoptosis and DNA damage in bladder cancer cells.

Excavatolide C (EXCC), a marine coral-derived compound, exhibits an antiproliferation effect on bladder cancer cells. The present study evaluated the improvement in the antiproliferation ability of EXCC by co-treatment with cisplatin in bladder cancer cells. EXCC/cisplatin (12.5 and 1 µg/mL) showed higher antiproliferation effects on bladder cancer cells than single treatments (EXCC or cisplatin alone) in the 48 h ATP assay. EXCC/cisplatin also enhanced the increase in subG1, annexin V-mediated apoptosis, and activation of poly (ADP-ribose) polymerase (PARP) and several caspases (caspases 3, 8, and 9) compared to the single treatments. Cellular and mitochondrial oxidative stress was enhanced with EXCC/cisplatin compared to the single treatments according to analyses of reactive oxygen species (ROS), mitochondrial superoxide, and mitochondrial membrane potential; in addition, cellular antioxidants, such as glutathione (GSH), and the mRNA expressions of antioxidant signaling genes (catalase and NFE2-like bZIP transcription factor 2) were downregulated. EXCC/cisplatin treatment produced more DNA damage than the single treatments, as indicated by γH2AX and 8-hydroxy-2'-deoxyguanosine levels. Moreover, several DNA repair genes for homologous recombination (HR) and non-homologous end joining (NHEJ) were downregulated in EXCC/cisplatin compared to others. The addition of the GSH precursor N-acetylcysteine, which has ROS scavenging activity, attenuated all EXCC/cisplatin-induced changes. Notably, EXCC/cisplatin showed lower antiproliferation, apoptosis, ROS induction, GSH depletion, and γH2AX DNA damage in normal cells than in bladder cancer cells. Therefore, the co-treatment of EXCC/cisplatin reduces the proliferation of bladder cancer cells via oxidative stress-mediated mechanisms with normal cell safety.

Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection.

Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.

Loss of Adaptive DNA Breaks in Alzheimer's Disease Brains.

Background: DNA breaks accumulate in Alzheimer's disease (AD) brains. While their role as true genomic lesions is recognized, DNA breaks also support cognitive function by facilitating the expression of activity-dependent immediate early genes. This process involves TOP2B, a DNA topoisomerase that catalyzes the formation of DNA double-strand breaks. Objective: To characterize how AD impacts adaptive DNA breaks at nervous system genes. Methods: We leveraged the ability of DNA single- and double-strand breaks to activate poly(ADP-ribose) polymerases (PARPs) that conjugate poly(ADP-ribose) (PAR) to adjacent proteins. To characterize the genomic sites harboring DNA breaks in AD brains, nuclei extracted from 3 AD and 3 non-demented autopsy brains (frontal cortex, all male donors, age 78 to 91 years of age) were analyzed through CUT&RUN in which we targeted PAR with subsequent DNA sequencing. Results: Although the AD brains contained 19.9 times more PAR peaks than the non-demented brains, PAR peaks at nervous system genes were profoundly lost in AD brains, and the expression of these genes was downregulated. This result is consistent with our previous CUT&RUN targeting γH2AX, which marks DNA double-strand breaks. In addition, TOP2B expression was significantly decreased in the AD brains. Conclusions: Although AD brains contain a net increase in DNA breaks, adaptive DNA breaks at nervous system genes are lost in AD brains. This could potentially reflect diminished TOP2B expression and contribute to impaired neuron function and cognition in AD patients.

PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.

A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers.

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

Natural pentacyclic triterpenoid from Pristimerin sensitizes p53-deficient tumor to PARP inhibitor by ubiquitination of Chk1.

Inhibition of checkpoint kinase 1 (Chk1) has shown to overcome resistance to poly (ADP-ribose) polymerase (PARP) inhibitors and expand the clinical utility of PARP inhibitors in a broad range of human cancers. Pristimerin, a naturally occurring pentacyclic triterpenoid, has been the focus of intensive studies for its anticancer potential. However, it is not yet known whether low dose of pristimerin can be combined with PARP inhibitors by targeting Chk1 signaling pathway. In this study, we investigated the efficacy, safety and molecular mechanisms of the synergistic effect produced by the combination olaparib and pristimerin in TP53-deficient and BRCA-proficient cell models. As a result, an increased expression of Chk1 was correlated with TP53 mutation, and pristimerin preferentially sensitized p53-defective cells to olaparib. The combination of olaparib and pristimerin resulted in a more pronounced abrogation of DNA synthesis and induction of DNA double-strand breaks (DSBs). Moreover, pristimerin disrupted the constitutional levels of Chk1 and DSB repair activities. Mechanistically, pristimerin promoted K48-linked polyubiquitination and proteasomal degradation of Chk1 while not affecting its kinase domain and activity. Importantly, combinatorial therapy led to a higher rate of tumor growth inhibition without apparent hematological toxicities. In addition, pristimerin suppressed olaparib-induced upregulation of Chk1 and enhanced olaparib-induced DSB marker γΗ2ΑΧ in vivo. Taken together, inhibition of Chk1 by pristimerin has been observed to induce DNA repair deficiency, which may expand the application of olaparib in BRCA-proficient cancers harboring TP53 mutations. Thus, pristimerin can be combined for PARP inhibitor-based therapy.

P. gingivalis-Induced TLR2 Interactome Analysis Reveals Association with PARP9.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9's role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing.

Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks.

Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.

High expression of BCAT1 sensitizes AML cells to PARP inhibitor by suppressing DNA damage response.

Previous evidence has confirmed that branched-chain aminotransferase-1 (BCAT1), a key enzyme governing branched-chain amino acid (BCAA) metabolism, has a role in cancer aggression partly by restricting αKG levels and inhibiting the activities of the αKG-dependent enzyme family. The oncogenic role of BCAT1, however, was not fully elucidated in acute myeloid leukemia (AML). In this study, we investigated the clinical significance and biological insight of BCAT1 in AML. Using q-PCR, we analyzed BCAT1 mRNAs in bone marrow samples from 332 patients with newly diagnosed AML. High BCAT1 expression independently predicts poor prognosis in patients with AML. We also established BCAT1 knockout (KO)/over-expressing (OE) AML cell lines to explore the underlying mechanisms. We found that BCAT1 affects cell proliferation and modulates cell cycle, cell apoptosis, and DNA damage/repair process. Additionally, we demonstrated that BCAT1 regulates histone methylation by reducing intracellular αKG levels in AML cells. Moreover, high expression of BCAT1 enhances the sensitivity of AML cells to the Poly (ADP-ribose) polymerase (PARP) inhibitor both in vivo and in vitro. Our study has demonstrated that BCAT1 expression can serve as a reliable predictor for AML patients, and PARP inhibitor BMN673 can be used as an effective treatment strategy for patients with high BCAT1 expression. KEY MESSAGES: High expression of BCAT1 is an independent risk factor for poor prognosis in patients with CN-AML. High BCAT1 expression in AML limits intracellular αKG levels, impairs αKG-dependent histone demethylase activity, and upregulates H3K9me3 levels. H3K9me3 inhibits ATM expression and blocks cellular DNA damage repair process. Increased sensitivity of BCAT1 high expression AML to PARP inhibitors may be used as an effective treatment strategy in AML patients.

Poly-(ADP-ribose) polymerases inhibition by olaparib attenuates activities of the NLRP3 inflammasome and of NF-κB in THP-1 monocytes.

Poly-(ADP-ribose) polymerases (PARPs) are a protein family that make ADP-ribose modifications on target genes and proteins. PARP family members contribute to the pathogenesis of chronic inflammatory diseases, including atherosclerosis, in which monocytes/macrophages play important roles. PARP inhibition is protective against atherosclerosis. However, the mechanisms by which PARP inhibition exerts this beneficial effect are not well understood. Here we show that in THP-1 monocytes, inhibition of PARP by olaparib attenuated oxidized low-density lipoprotein (oxLDL)-induced protein expressions of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing-3 (NLRP3) inflammasome components: NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. Consistent with this effect, olaparib decreased oxLDL-enhanced interleukin (IL)-1ß and IL-18 protein expression. Olaparib also decreased the oxLDL-mediated increase in mitochondrial reactive oxygen species. Similar to the effects of the NLRP3 inhibitor, MCC950, olaparib attenuated oxLDL-induced adhesion of monocytes to cultured human umbilical vein endothelial cells and reduced foam cell formation. Furthermore, olaparib attenuated the oxLDL-mediated activation of nuclear factor (NF)-κB through the oxLDL-mediated increase in IκBα phosphorylation and assembly of NF-κB subunits, demonstrated by co-immunoprecipitation of IκBα with RelA/p50 and RelB/p52 subunits. Moreover, PARP inhibition decreased oxLDL-mediated protein expression of a NF-κB target gene, VCAM1, encoding vascular cell adhesion molecule-1. This finding indicates an important role for NF-κB activity in PARP-mediated activation of the NLRP3 inflammasome. Thus, PARP inhibition by olaparib attenuates NF-κB and NLRP3 inflammasome activities, lessening monocyte cell adhesion and macrophage foam cell formation. These inhibitory effects of olaparib on NLRP3 activity potentially protect against atherosclerosis.