ADP-ribose Units Research Articles

Distinct roles for PARP-1 and PARP-2 in c-Myc–driven B-cell lymphoma in mice

AbstractDysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc–driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc–driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the Eμ-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic Eμ-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc–driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc–driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc–driven tumors.

Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.

Why structure and chain length matter: on the biological significance underlying the structural heterogeneity of poly(ADP-ribose).

Poly(ADP-ribosyl)ation (PARylation) is a multifaceted post-translational modification, carried out by poly(ADP-ribosyl)transferases (poly-ARTs, PARPs), which play essential roles in (patho-) physiology, as well as cancer therapy. Using NAD+ as a substrate, acceptors, such as proteins and nucleic acids, can be modified with either single ADP-ribose units or polymers, varying considerably in length and branching. Recently, the importance of PAR structural heterogeneity with regards to chain length and branching came into focus. Here, we provide a concise overview on the current knowledge of the biochemical and physiological significance of such differently structured PAR. There is increasing evidence revealing that PAR’s structural diversity influences the binding characteristics of its readers, PAR catabolism, and the dynamics of biomolecular condensates. Thereby, it shapes various cellular processes, such as DNA damage response and cell cycle regulation. Contrary to the knowledge on the consequences of PAR’s structural diversity, insight into its determinants is just emerging, pointing to specific roles of different PARP members and accessory factors. In the future, it will be interesting to study the interplay with other post-translational modifications, the contribution of natural PARP variants, and the regulatory role of accessory molecules. This has the exciting potential for new therapeutic approaches, with the targeted modulation and tuning of PARPs’ enzymatic functions, rather than their complete inhibition, as a central premise.

Synthesis of ADP-Ribosylated Histones Reveals Site-Specific Impacts on Chromatin Structure and Function.

ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.

Abstract 48: RBN-2397: A potent and selective small molecule inhibitor of PARP7 that induces tumor-derived antitumor immunity dependent on CD8 T cells

Abstract Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. In contrast to PARP1, PARP7 (TIPARP) is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates (MARylation) to change their function and plays a role in suppressing the Type I IFN response. RBN-2397 selectively inhibits PARP7 compared to the approved PARP1 inhibitors and demonstrates > 50-fold selectivity for inhibition of PARP7 over all PARP family members as measured by biochemical assays. The inhibition of PARP1-mediated ADP-ribosylation has been well-characterized for several PARP1 inhibitors using a cellular hydrogen peroxide-induced PARylation assay. Here, we show that RBN-2397 inhibits PARP7-dependent MARylation with an IC50 of 2 nM exhibiting a 300-fold window over PARP1-driven PARylation. Using the mouse CT26 cell line, we showed that RBN-2397, but not the PARP1 inhibitor olaparib, induced Type I IFN signaling demonstrated by STAT1 phosphorylation. The effect on pSTAT1 was phenocopied by PARP7 knockout (KO). To further demonstrate specificity, we show that simultaneous KO of PARP7 prevented any additional increase of STAT1 phosphorylation by RBN-2397; however, KO of PARP1 had no effect on the induction of Type I IFN signaling by RBN-2397. We had previously reported that RBN-2397 dosing of CT26 tumor bearing immune competent BALB/c mice led to complete and durable tumor regressions which could be reversed by interfering with tumor-derived IFN signaling (1). Here we show that in contrast, RBN-2397 showed modest activity with no tumor regressions in CT26-tumor bearing immunodeficient NOG mice. To assess which immune cell populations are involved in the antitumor effects of RBN-2397, CT26 tumor-bearing BALB/c mice were depleted of CD4 T, CD8 T or NK cells. Depletion of CD4 T or NK cells had no effect on RBN-2397 antitumor activity; however, depletion of CD8 T cells significantly reversed the effects of RBN-2397, suggesting that CD8 T cells are responsible for much of the antitumor immunity induced by RBN-2397. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in cancer cells and that this is an on-target effect of inhibiting the catalytic activity of PARP7 and not PARP1. We further show that the adaptive immune response was required for the antitumor effects of RBN-2397. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability, and a Phase I clinical trial is underway (NCT04053673). (1) AACR Jun 22-24, 2020: Cancer Res 2020;80 (16 Suppl): Abstract nr 3405. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Jennifer R. Molina, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Ellen Bamberg, Jan-Rung Mo, David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A potent and selective small molecule inhibitor of PARP7 that induces tumor-derived antitumor immunity dependent on CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 48.

Abstract 381: Elevated PARP7 expression in select cancers identifies a target population for RBN-2397 therapy

Abstract PARP7 (TIPARP) is a monoPARP which catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Its expression is upregulated during cellular stress, including viral infection or through the activation of the aryl hydrocarbon receptor after exposure to cigarette smoke. We and others have shown that PARP7 activity suppresses the Type I interferon (IFN) response following activation by cytosolic nucleic acid sensing pathways. RBN-2397 is a first-in class PARP7 inhibitor, which induces cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Here we describe the presence of PARP7 genomic amplification with corresponding increased mRNA expression across select cancers. Elevated PARP7 expression or amplification may identify cancer patients that could derive benefit from treatment with RBN-2397. In characterizing PARP7 copy number and mRNA expression from The Cancer Genome Atlas (TCGA) database, we found the presence of PARP7 copy number amplification in a subset of tumor types, particularly those of squamous histology, as well as ovarian cancer that corresponded to higher mRNA expression levels. High PARP7 expression correlated with poor survival in squamous cancers, while it had no effect on survival in ovarian cancer. Interestingly, tumor types with high PARP7 expression also expressed higher levels of baseline interferon stimulated genes (ISGs). This parallels our findings that cancer cell lines with higher ISGs at baseline are more responsive to PARP7 inhibition. To explore PARP7 copy number variations (CNVs) in advanced cancer patients, we queried the FoundationCore® (Foundation Medicine, Inc) database. Similar to TCGA, squamous cancers as well as ovarian, breast, and pancreatic ductal adenocarcinoma (PDAC) were among the tumor types with PARP7 amplifications. Moreover, PARP7 was found to be amplified both on the background of chromosome 3q arm-level amplifications as well as focally. Congruent to our analysis of PARP7 amplifications, we evaluated PARP7 mRNA expression in both squamous and non-squamous non-small cell lung carcinoma (NSCLC), breast cancer, and PDAC primary tumor samples. Using a validated RNAscope ISH probe set, we analyzed over 1,000 patient samples and found that PARP7 was more highly expressed in tumor cells relative to corresponding normal tissues. Overall, there were varying levels of PARP7 expression across samples with higher expression levels of PARP7 in tumor cells, compared to stroma, across all cancers examined. Our analyses describing the presence of PARP7 amplifications as well as high expression levels in several tumor types including NSCLC, breast, and PDAC provides evidence for the therapeutic relevance of PARP7 inhibition and highlights potential patient selection strategies to identify those patients more likely to benefit from RBN-2397 treatment. Citation Format: Jodie Wong, Kristy Kuplast-Barr, Ryan P. Abo, Anupriya S. Kulkarni, Linda T. Nieman, Azfar Neyaz, Katherine H. Xu, Radwa Sharaf, Lee A. Albacker, David T. Ting, Heike Keilhack, Kristen A. McEachern. Elevated PARP7 expression in select cancers identifies a target population for RBN-2397 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 381.

Abstract 2043: Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies

Abstract Background:The enzyme Poly(ADP-ribose) glycohydrolase (PARG) plays an important role during the DNA damage repair process through hydrolysis of poly(ADP-ribose)(PAR) chains and accounts for approximately 90% of dePARylation activity within the cell. PAR chain synthesis is achieved by poly(ADP-ribose) polymerases (PARPs), which use donor NAD+ molecules to link repeating ADP-ribose units. The resulting PAR chains serve as a platform to recruit DNA repair proteins that are critical for single-strand break (SSB) repair. Following DNA repair PARG hydrolyzes the linkages to break down PAR chains, completing the cycle. Recent studies also found that dePARylation is necessary for the translocation of DNA repair proteins such as XRCC1 to SSB suggesting a more direct role for PARG in DNA damage repair. PARP inhibitors (PARPi), demonstrate synthetic lethality in cells with impaired homologous recombination (HR)-mediated DNA repair. Clinical studies have shown that responses to PARPi are often accompanied by high rates of resistance, generating a need for additional therapies for patients with HR-deficient tumors. The ability of PARG inhibition (PARGi) to exacerbate replication deficiencies makes it a promising therapeutic target for a broad range of cancer types with genomic instability. We therefore sought out to evaluate the effects of PARGi in HR-deficient ovarian and breast cancer models. Methods: To understand the effects of PARGi in cell growth and survival, a panel of HR-deficient cell lines and cell line derived xenografts (CDX) were exposed to PARGi alone or in combination with DNA damage response inhibitors (DDRi). In these studies, PAR chain accumulation was assessed as a marker of target engagement and PARPi was used to assess on target effects in vitro. Results: Using cellular proliferation assays and xenograft models, we find that PARGi increases the cellular levels of PAR and significantly decreases the viability of HR-deficient cancer cell lines. Furthermore, inhibition of cell proliferation by PARGi is antagonized by PARPi, which is consistent with an on-target cellular mechanism of action (MOA). In conclusion, PARGi induces significant accumulation of PAR chains and decreases cell proliferation both in vitro and in vivo in HR-deficient tumor cells. Ongoing exploratory studies will further delineate the molecular underpinning of the relationship between HR-deficiency and PARGi. Citation Format: Monah Abed, Diana Munoz, Firoz Jaipuri, Nandini Ravindran, Vidya Seshadri, Marya Liimatta, Claire Neilan, Xin Linghu, An Nguyen, Marie-Claire Wagle, Zineb Mounir. Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2043.

The PARP Enzyme Family and the Hallmarks of Cancer Part 2: Hallmarks Related to Cancer Host Interactions.

Poly (ADP-ribose) polymerases (PARPs) modify target proteins with a single ADP-ribose unit or with a poly (ADP-ribose) (PAR) polymer. PARP inhibitors (PARPis) recently became clinically available for the treatment of BRCA1/2 deficient tumors via the synthetic lethality paradigm. This personalized treatment primarily targets DNA damage-responsive PARPs (PARP1-3). However, the biological roles of PARP family member enzymes are broad; therefore, the effects of PARPis should be viewed in a much wider context, which includes complex effects on all known hallmarks of cancer. In the companion paper (part 1) to this review, we presented the fundamental roles of PARPs in intrinsic cancer cell hallmarks, such as uncontrolled proliferation, evasion of growth suppressors, cell death resistance, genome instability, replicative immortality, and reprogrammed metabolism. In the second part of this review, we present evidence linking PARPs to cancer-associated inflammation, anti-cancer immune response, invasion, and metastasis. A comprehensive overview of the roles of PARPs can facilitate the identification of novel cancer treatment opportunities and barriers limiting the efficacy of PARPi compounds.

The PARP Way to Epigenetic Changes.

ADP-ribosylation, is a reversible post-translational modification implicated in major biological functions. Poly ADP-ribose polymerases (PARP) are specialized enzymes that catalyze the addition of ADP ribose units from “nicotinamide adenine dinucleotide-donor molecules” to their target substrates. This reaction known as PARylation modulates essential cellular processes including DNA damage response, chromatin remodeling, DNA methylation and gene expression. Herein, we discuss emerging roles of PARP1 in chromatin remodeling and epigenetic regulation, focusing on its therapeutic implications for cancer treatment and beyond.

Kaposi's sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication.

Kaposi’s sarcoma–associated herpesvirus (KSHV), which belongs to the gammaherpesvirus subfamily, is associated with the pathogenesis of various tumors. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the polymerization of ADP-ribose units on target proteins. In KSHV-infected cells, PARP1 inhibits replication and transcription activator (RTA), a molecular switch that initiates lytic replication, through direct interaction. Thus, for efficient replication, KSHV has to overcome the molecular barrier in the form of PARP1. Previously, we have demonstrated that KSHV downregulates the expression of PARP1 through PF-8, a viral processivity factor. PF-8 induces ubiquitin–proteasome system–mediated degradation of PARP1 via direct physical association and enhances RTA transactivation activity. Here, we showed that dimerization domains of PF-8 are crucial not only for PARP1 interaction and degradation but also for enhancement of the RTA transactivation activity. PF-8 recruited CHFR for the PARP1 degradation. A knockdown of CHFR attenuated the PF-8–induced PARP1 degradation and enhancement of the RTA transactivation activity, leading to reduced KSHV lytic replication. These findings reveal a mechanism by which KSHV PF-8 recruits a cellular E3 ligase to curtail the inhibitory effect of PARP1 on KSHV lytic replication.

PARP-1 activation after oxidative insult promotes energy stress-dependent phosphorylation of YAP1 and reduces cell viability.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyze the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using WRL68 (HeLa derivate) cells, we previously showed that PARP-1 activation induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promoted cell death. In this work, LC-MS/MS-based phosphoproteomics in WRL68 cells showed that the oxidative damage induced by H2O2 increased the phosphorylation of YAP1, a transcriptional co-activator involved in cell survival, and modified the phosphorylation of other proteins involved in transcription. Genetic or pharmacological inhibition of PARP-1 in H2O2-treated cells reduced YAP1 phosphorylation and degradation and increased cell viability. YAP1 silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells exposed to oxidative damage. Supplementation of NAD+ also reduced YAP1 phosphorylation, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of the metabolic sensor AMPK. Since NAD+ supplementation reduced the phosphorylation of some AMPK substrates, we hypothesized that the loss of cellular NAD+ after PARP-1 activation may induce an energy stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in which PARP-1 activation reduced cell viability by promoting the phosphorylation and degradation of YAP1 through a mechanism that involves the depletion of NAD+.

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5′-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5′-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5′-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR–DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR–DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1′ of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

Tankyrase inhibitors: emerging and promising therapeutics for cancer treatment

Cancer is a disease portrayed by the uncontrolled growth of irregular cells. Tankyrase, a member of poly(ADP-ribose)polymerase family, mediates Wnt signal transduction and has emerged as a new molecular target for the therapy of different kinds of cancer. Wnt/β-catenin signaling functions significantly in a wide scope of biological events, such as the upkeep of genomic stability, transcriptional control, energy metabolism, and apoptosis. ADP-ribosylation is a reversible posttranslational modification process that regulates several cellular signaling pathways in which transferase enzymes such as mono (ADP-ribosyl) and poly(ADP-ribosyl) transferases move a unit of ADP-ribose moiety from the NAD+ co-substrate to specific amino acid side chains and/or potentially ADP-ribose units on target proteins. Recently, inhibition of tankyrase has risen as an appealing strategy for the discovery of novel anticancer drugs. The current review offers an understanding of the ongoing improvements on new lead structures as inhibitors of tankyrase and their activities. A special spotlight is set on the structure-activity relationship, molecular docking, polypharmacology profile, and binding mode at the active center.

Magnetic bead-based electrochemical and colorimetric methods for the detection of poly(ADP-ribose) polymerase-1 with boronic acid derivatives as the signal probes

Poly(ADP-ribose) polymerase-1 (PARP-1), as a highly conserved nuclear enzyme, can catalyze the transfer of multiple ADP-ribose units from β-nicotinamide adenine dinucleotide (NAD+) to substrate proteins, including PARP-1 itself. In this work, two boronic acid derivatives, ferroceneboronic acid (FcBA) and 4-mercaptophenylboronic acid (MPBA) were used as the signal probes for electrochemical and colorimetric detection of PARP-1, respectively. The detection platform was constructed by modification of double-stranded DNA (dsDNA) on the surface of magnetic beads (MBs). Once PARP-1 was captured by the MB-dsDNA, auto-PARylation was initiated and the PAR products consisting of abundant ribose units were covalently bound to FcBA or MPBA through the formation of boronate ester covalent bonds. Sequestration of FcBA or MPBA by the PAR-covered MBs led to the decrease in the electrochemical signal of FcBA or preventing the MPBA-triggered aggregation of gold nanoparticle indicators. The change of oxidation current or adsorption intensity (color) was dependent upon the concentration and activity of PARP-1. A wide linear range of 0.1 ∼ 50 U was obtained for the electrochemical or colorimetric assays. The methods have been used to evaluate the inhibition efficiency and monitor the PARP-1 activity in living and apoptotic cancer cells.

Abstract 1038: A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants

Abstract PARPs (poly-ADP-ribose polymerases) are a family of enzymes that regulate a wide variety of important cellular processes including cellular stress signaling pathways implicated in inflammation and cancer. PARP14 is a member of the monoPARP sub-family of PARPs, which catalyze transfer of a single ADP ribose unit (mono-ADP-ribosylation or MARylation) to their substrates, unlike their polyPARP counterparts which construct chains of ADP ribose units (poly-ADP-ribosylation or PARylation). PARP14 is an interferon-stimulated gene (ISG) that is overexpressed in tumors compared to normal tissues and has been implicated by genetic knockout studies to promote pro-tumor macrophage polarization and suppress antitumor inflammatory response due to its role in modulating IL-4 and IFN-γ signaling pathways. Efforts to further explore and validate the role of PARP14 catalytic activity in these pathways have been hampered by a lack of potent, highly selective PARP14 inhibitors. Here we describe the discovery of the first such chemical probe, RBN012759, which inhibits PARP14 with an IC50 of 0.003 µM and exhibits >300-fold selectivity over all mono- and polyPARP family members. Medicinal chemistry efforts that began with an unselective micromolar screening hit were enabled by a detailed understanding of the PARP14 and broader PARP family binding pockets. X-ray co-crystal structures of RBN012759 and key early analogs bound to PARP14 clearly illustrate the origins of their potency and selectivity. RBN012759 is a cell permeable, soluble probe that demonstrates robust, dose-dependent stabilization of endogenous PARP14 and inhibition of MARylation in primary human macrophages. RBN012759 reverses IL-4 driven (pro-tumor) gene expression in macrophages, confirming published data generated with PARP14 KO and supporting an immune suppressive role of PARP14 in tumors. Moreover, we demonstrate that PARP14 inhibition in primary human tumor explants can induce an inflammatory mRNA signature similar to immune checkpoint therapy. Summary: Structure-based design of the first potent and highly selective inhibitor of PARP14, RBN012759, is described. The discovery of this chemical probe enabled exploration of the role of PARP14 in macrophage polarization and inflammatory pathways. RBN012759 reverses IL-4 driven gene expression in macrophages and induces an inflammatory mRNA signature in human tumor explants, data which support an immune suppressive role of PARP14 in tumors. Citation Format: Laurie Schenkel, Jennifer Molina, Kerren Swinger, Ryan Abo, Danielle Blackwell, Anne Cheung, William Church, Kristy Kuplast-Barr, Alvin Lu, Elena Minissale, Mario Niepel, Melissa Vasbinder, Tim Wigle, Victoria Richon, Heike Keilhack, Kevin Kuntz. A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1038.

Abstract DDT02-01: RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways

Abstract RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function (MARylation). PARP7 expression is increased by cellular stresses, including aromatic hydrocarbons and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 has also been reported to negatively regulate the Type I interferon (IFN) response by interacting with TBK1 during viral infection. As part of our drug discovery efforts to identify inhibitors of PARP7, we utilized structure-based drug design to optimize an unselective monoPARP inhibitor identified by screening Ribon's internal compound collection of PARP inhibitors. Further optimization of potency and physicochemical properties led to the discovery of RBN-2397, a potent and selective small molecule inhibitor of PARP7 catalytic function. A co-crystal structure of RBN-2397 demonstrated binding of the compound in the NAD+-binding pocket. Binding to cellular PARP7 is demonstrated by the ability of RBN-2397 to displace an active site probe in a NanoBRET assay. Functionally, RBN-2397 leads to the inhibition of MARylation of multiple intracellular proteins in PARP7-overexpressing SK-MES-1 cells. We identified a subset of cancers exhibiting dependency on PARP7 for proliferation. Cell lines with higher baseline expression of interferon stimulated genes are more sensitive to RBN-2397 in proliferation assays. We further show that inhibition of PARP7 by RBN-2397 restores Type I IFN signaling as demonstrated by the induction of STAT1 phosphorylation and upregulation of genes enriched for Type I IFN signaling in NCI-H1373 lung cancer cells. Oral dosing of RBN-2397 results in durable, complete tumor regression in a NCI-H1373 lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model that is dependent on tumor-derived Type I IFN signaling. Herein, we describe the discovery of the small molecule PARP7 inhibitor RBN-2397, the first therapeutic agent targeting PARP7 to enter clinical trials, and the first disclosure of the inhibitor. We demonstrate PARP7 is a novel therapeutic target and inhibition of PARP7 by RBN-2397 induces both cancer cell autonomous and immune stimulatory effects via enhanced IFN signaling. Citation Format: Melissa M. Vasbinder, Joseph M. Gozgit, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Zacharenia A. Varsamis, Sunaina P. Nayak, Ellen Bamberg, Jan-Rung Mo, W David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr DDT02-01.

Abstract 3405: PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition leads to tumor regression

Abstract Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. PARP7 is a member of the monoPARP class of enzymes, which catalyze the transfer of single units of ADP-ribose onto substrates to change their function. PARP7 expression is increased by cellular stress and aromatic hydrocarbons, and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 has also been reported to negatively regulate the Type I IFN response by interacting with TBK1 during viral infection. Herein, we identify PARP7 as a novel negative regulator of cytosolic nucleic acid sensing in tumor cells. RBN-2397, is a potent and selective small molecule inhibitor of PARP7 catalytic function. We identified a subset of cancers exhibiting dependency on PARP7 for proliferation and found that cell lines with higher baseline expression of interferon stimulated genes were more sensitive. We further show that inhibition of PARP7 by RBN-2397 restores Type I IFN signaling as demonstrated by the induction of STAT1 phosphorylation and up-regulation of genes enriched for Type I IFN signaling in NCI-H1373 lung cancer cells. We examined the antitumor effects of once daily orally administered RBN-2397 in SCID mice with subcutaneous NCIH1373 xenograft tumors and observed a dose-dependent effect of RBN-2397 on tumor growth, with regressions at dose levels ≥30 mg/kg. To evaluate the antitumor immune response in vivo, we administered RBN-2397 to CT26 tumor-bearing, immunocompetent BALB/c mice, and observed significant tumor growth inhibition at all dose levels with complete and durable regressions in a subset of mice. All of these tumor-free mice rejected a challenge of injected CT26 cells, but were able to develop 4T1 tumors, demonstrating induction of tumor-specific adaptive immune memory. The antitumor effects of RBN-2397 were further enhanced when combined with an immune checkpoint inhibitor, anti-PD1. Using CRISPR-Cas9 to knockout either TBK1 or IFNAR1 in CT26 cells, we demonstrated that RBN-2397 antitumor immunity is dependent on the effects of tumor-derived Type I interferon on immune cells. Here, we show for the first time that cancer cells use PARP7 to suppress the Type I IFN response to cytosolic nucleic acids. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in the tumor, causes complete tumor regressions and adaptive immunity in murine models. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Zacharenia A. Varsamis, Sunaina P. Nayak, Ellen Bamberg, Jan-Rung Mo, William Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition leads to tumor regression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3405.

In Vitro and Cellular Probes to Study PARP Enzyme Target Engagement

Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in invitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The invitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.

PARG has a robust endo-glycohydrolase activity that releases protein-free poly(ADP-ribose) chains

Poly(ADP-ribosyl)ation (PARylation) regulates DNA damage response, chromatin structure, and cell-fate. Dynamic regulation of cellular PAR levels is crucial for the maintenance of genomic integrity and excessive cellular PAR activates a PAR-dependent cell death pathway. Thus, PAR serves as a cell-death signal; however, it has been debated how the protein-free PAR is generated. Here, we demonstrate that PAR glycohydrolases (PARGs) from mammals to bacteria have a robust endo-glycohydrolase activity, releasing protein-free PAR chains longer than three ADP-ribose units as early reaction products. Released PAR chains are transient and rapidly degraded to monomeric ADP-ribose, which is consistent with a short half-life of PAR during DNA damage responses. Computational simulations using a tri-ADP-ribose further support that PARG can efficiently bind to internal sites of PAR for the endo-glycosidic cleavage. Our collective results suggest PARG as a key player in producing protein-free PAR during DNA damage signaling and establish bacterial PARG as a useful tool to enrich short PAR chains that emerge as important reagents for biomedical research.