Recent findings have thrust poly(ADP-ribose) polymerases (PARPs) into the limelight as potential chemotherapeutic targets. To provide a framework for understanding these recent observations, we review what is known about the structures and functions of the family of PARP enzymes, and then outline a series of questions that should be addressed to guide the rational development of PARP inhibitors as anticancer agents.Current efforts to develop poly(ADP-ribose) polymerase (PARP) inhibitors as anticancer drugs represent the culmination of over 40 years of research. After Paul Mandel's research group first described a nuclear enzymatic activity that synthesizes an adenine-containing RNA-like polymer 1 , independent studies by French and Japanese teams demonstrated that this polymer, designated poly(ADP-ribose) (pADPr), is composed of two ribose moieties and two phosphates per unit polymer [2][3][4][5] . The purification of an enzyme that could generate large amounts of pADPr, PARP1 (REFS 6,7 ), led to the discovery that PARP1 is activated by DNA strand breaks [8][9][10] . Seminal work by Sydney Shall's group showed that PARP1 is involved in DNA repair and also suggested the potential use of PARP inhibitors to enhance the cytotoxic effects Correspondence to G.G.P. guy.poirier@crchul.ulaval.ca. Competing interests statementThe authors declare no competing financial interests. DATABASES NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of alkylating agents 10 . Examination of knockout mouse models 11 strengthened the hypothesis that PARP1 participates in DNA repair and simultaneously provided the first evidence for the existence of PARP2 (REF. 12 ). A parallel set of experiments demonstrated that PARP1 hyperactivation leads to nicotinamide adenine dinucleotide (NAD + ) and ATP depletion after various types of DNA damage 13,14 (BOX 1), potentially contributing to a unique form of metabolic cell death, which is now termed parthanatos 15 . PARP was thrust into the limelight by the discovery that PARP inhibition is particularly toxic in cancer cell lines 16,17 and human tumours 18 that lack BRCA1 or BRCA2. Despite this progress, there is still much that we do not understand about the biology of the PARP family and pADPr, as detailed below. Box 1 PARP1 hyperactivation and cell deathNicotinamide adenine dinucleotide (NAD + ) is the source of ADP-ribose used by poly(ADPribose) polymerases (PARPs) to produce poly(ADP-ribose) (pADPr). Because hyperactivation of PARP1 consumes the cytosolic and nuclear pools of NAD + to generate pADPr, pADPr synthesis translates DNA damage intensity into changes in cellular energy. Low to moderate DNA damage triggers pADPr-dependent DNA repair. In the context of excessive DNA damage, however, PARP1 hyperactivation leads to extended pADPr synthesis and extensive NAD + consumption 8,13,14 . Depending on the cellular context, this intense pADPr synthesis can induce cell death through several mechanisms. Long and branched pADPr (60mers and longer) can directly trigg...

Poly(ADP-ribose) polymerase (PARP) inhibitors are strikingly toxic to cells with defects in homologous recombination (HR). The mechanistic basis for these findings is incompletely understood. Here, we show that PARP inhibitor treatment induces phosphorylation of DNA-dependent protein kinase substrates and stimulates error-prone nonhomologous end joining (NHEJ) selectively in HRdeficient cells. Notably, inhibiting DNA-dependent protein kinase activity reverses the genomic instability previously reported in these cells after PARP inhibition. Moreover, disabling NHEJ by using genetic or pharmacologic approaches rescues the lethality of PARP inhibition or down-regulation in cell lines lacking BRCA2, BRCA1, or ATM. Collectively, our results not only implicate PARP1 catalytic activity in the regulation of NHEJ in HR-deficient cells, but also indicate that deregulated NHEJ plays a major role in generating the genomic instability and cytotoxicity in HR-deficient cells treated with PARP inhibitors.chemotherapy | DNA repair | synthetic lethality | double-strand break repair P oly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that synthesizes poly(ADP-ribose) polymer when activated by DNA nicks or breaks. Activation of PARP1 has important effects on a variety of cellular processes, including base excision repair (BER), transcription, and cellular bioenergetics (1). The role of PARP1 in the DNA damage response sparked interest in the development of PARP inhibitors as potential chemosensitizers for the treatment of cancer (1, 2). The more recent observation that PARP inhibition is particularly lethal to cells deficient in homologous recombination (HR) proteins (3-8) has generated additional excitement in the cancer chemotherapy community. The current explanation for this hypersensitivity focuses on a mechanism (Fig. 1A) in which loss of PARP1 activity is thought to result in accumulation of DNA single-strand breaks (SSBs), which are subsequently converted to DNA double-strand breaks (DSBs) by the cellular replication and/or transcription machinery. These DSBs, which are repaired by HR in BRCApositive cells, are presumed to accumulate in BRCA1-or BRCA2-deficient cells, leading to subsequent cell death. Heightened sensitivity to PARP inhibition has also been observed in cells with other genetic lesions that affect HR, including phosphatase and tensin homolog (PTEN) deficiency (5), ataxia telangiectasia mutated (ATM) deficiency (7,8), and Aurora A overexpression (6).Although the preceding studies underscore the importance of PARP1 and HR in maintaining genomic stability, they do not address the role of nonhomologous end joining (NHEJ), an alternate DSB repair modality that directly joins broken ends of DNA with little or no regard for sequence homology (9). NHEJ is initiated when free DNA ends are bound by Ku70 and Ku80, which recruit the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The resulting complex, known as the DNAdependent protein kinase (DNA-PK) complex, phosphorylates downstream targe...

We report herein thermally responsive elastin-like polypeptides (ELPs) in a linear AB diblock architecture with an N-terminal peptide ligand that self-assemble into spherical micelles when heated slightly above body temperature. A series of 10 ELP block copolymers (ELP BC s) with different molecular weights and hydrophilic-to-hydrophobic block ratios were genetically synthesized by recursive directional ligation. The self-assembly of these polymers from unimers into micelles was investigated by light scattering, fluorescence spectroscopy and cryo-TEM. These ELP BC s undergo two phase transitions as a function of solution temperature: a unimer to spherical micelle transition at an intermediate temperature, and a micelle to bulk aggregate transition at a higher temperature when the hydrophilic-to-hydrophobic block ratio is between 1:2 and 2:1. The critical micelle temperature is controlled by the length of the hydrophobic block and the size of the micelle is controlled by both the total ELP BC length and hydrophilic-to-hydrophobic block ratio. These polypeptide micelles display a critical micelle concentration in the range of 4-8 μM demonstrating high stability of these structures. These studies have also identified a subset of ELP BC s bearing terminal peptide ligands that are capable of forming multivalent spherical micelles that present multiple copies of the ligand on their corona in the clinically relevant temperature range of 37-42 °C and target cancer cells. These ELP BC s may be useful for drug targeting by thermally triggered multivalency. More broadly, the design rules uncovered by this study should be applicable to the design of other thermally reversible nanoparticles for diverse applications in medicine and biology.

umors encompass complex cellular ecosystems of malignant and non-malignant cells, whose diversity and interactions affect cancer progression and drug response and resistance. Recent advances in single-cell genomics, especially single-cell RNA-Seq (scRNA-Seq), have transformed our ability to analyze tumors, revealing cell types, states, genetic diversity and interactions in the complex tumor ecosystem 1-6. Single-cell analysis of tumors is rapidly expanding, including the launch of a Human Tumor Atlas Network (HTAPP) as part of the Cancer Moonshot 7. Successful scRNA-Seq of clinical tumor specimens poses several challenges. First, it requires quick dissociation tailored to the tumor type, and involves enzymatic digestion, which can lead to loss of sensitive cells or changes in gene expression. Moreover, obtaining fresh tissue is time-sensitive and requires tight coordination

AbstractSingle cell genomics is essential to chart the complex tumor ecosystem. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumor tissues, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each strategy requires modifications to fit the unique characteristics of different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We tested eight tumor types of varying tissue and sample characteristics (resection, biopsy, ascites, and orthotopic patient-derived xenograft): lung cancer, metastatic breast cancer, ovarian cancer, melanoma, neuroblastoma, pediatric sarcoma, glioblastoma, pediatric high-grade glioma, and chronic lymphocytic leukemia. Analyzing 212,498 cells and nuclei from 39 clinical samples, we evaluated protocols by cell quality, recovery rate, and cellular composition. We optimized protocols for fresh tissue dissociation for different tumor types using a decision tree to account for the technical and biological variation between clinical samples. We established methods for nucleus isolation from OCT embedded and fresh-frozen tissues, with an optimization matrix varying mechanical force, buffer, and detergent. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types and intrinsic expression profiles, but at different proportions. Our work provides direct guidance across a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.