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Supplementary Data from Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC50 alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3–7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2–5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ. [Mol Cancer Ther 2009;8(8):2232–42]

The DNA damage response (DDR) is a concerted response involving a myriad of pathways that cells elicit in the presence of DNA injuries. Patients bearing mutations in DDR genes have an increased cancer incidence derived from their diminished ability to respond to DNA damage, and the consequent increase in mutations. Intriguingly, mutations in ATM, the chief DDR regulator, can cause ataxia telangiectasia, a neurodegenerative disorder characterized by progressive loss of movement coordination, weak immune system, and increased cancer risk. The relationship between ATM and neural system development and degeneration remains to be fully elucidated and will be discussed in this chapter.

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC50 alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3–7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2–5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ. [Mol Cancer Ther 2009;8(8):2232–42]

The Ku heterodimer, comprised of Ku70 and Ku80 subunits, is a conserved complex involved in nonhomologous end-joining (NHEJ). However, it also functions in maintenance of telomeres, chromosome termini normally resistant to end-joining events. To elucidate the spatial organization of these functions, we rationally guided Ku mutagenesis in yeast with real-valued evolutionary trace (rvET). This revealed two ancestrally related alpha-helices: one on the Ku70 surface that is required in yeast for NHEJ, and a second on the Ku80 surface that is required in yeast for telomeric heterochromatin formation. When bound to a DNA end, the surface containing the NHEJ-specific Ku70 helix is oriented toward the DNA terminus, whereas the surface containing the telomeric function-specific Ku80 helix faces inward, toward telomeric chromatin, when bound to a telomere. We propose a 'two-face' model for Ku and that divergent evolution of these faces allowed Ku's dual role in NHEJ and telomere maintenance.

Motivation: The S/TQ cluster domain (SCD) constitutes a new type of protein domain that is not defined by sequence similarity but by the presence of multiple S/TQ motifs within a variable stretch of amino acids. SCDs are recognized targets for DNA damage response (DDR) kinases like ATM and ATR. Characterizing DDR targets is of significant interest. The aim of this work was to develop a web-based tool to allow for easy identification and visualization of SCDs within specific proteins or in whole proteome sets, a feature not supported by current domain and motif search tools. Results: We have developed an algorithm that (i) generates a list of all proteins in an organism containing at least one user-defined SCD within their sequence, or (ii) identifies and renders a visual representation of all user-defined SCDs present in a single sequence or batch of sequences. Availability and implementation: The application was developed using Pearl and Python, and is available at the following URL: http://ustbioinfo.webfactional.com/scd/. Contact: ribesza@stthom.edu or lariosm@stthom.edu

In order to sustain life, cells must protect their genetic information from the constant threat posed by mutagenic agents such as ultraviolet light, irradiation or reactive oxy‐ gen species, as well as from mistakes introduced during the replication of their ge‐ nomes [1]. To deal with this problem, natural selection has favored a system that repairs the damage caused by these DNA lesions while allowing the highly infrequent but steady production of mutations that constitute the source for adaptive changes dur‐ ing evolution. To repair damaged DNA, cells have developed a myriad of highly speci‐ alized pathways that recognize and repair specific types of injuries produced by specific types of mutagenic events [2, 3]. For instance, while base excision repair detects and re‐ pairs chemically damaged nucleotide bases typically produced by oxygen radicals or al‐ kylating agents, nucleotide excision repair is responsible for the removal of thymine dimers caused by ultraviolet light exposure. Additionally, the mismatch repair pathway specializes in fixing errors introduced during DNA replication. More globally, these pathways are part of the DNA damage response (DDR), a signal transduction cascade coordinated by the ATM/ATR kinases in mammalian cells that halts cell cycle progres‐ sion while DNA is being repaired, and it can trigger apoptosis when the damage is deemed non-repairable [4]. The importance of these pathways is underlined by their high conservation, both in prokaryotes and eukaryotes, and it is emphasized by the role that they play in disease when impaired. Malfunctioning DNA repair pathways are as‐ sociated with several disorders such as Xeroderma pigmentosum or Nijmegen syn‐ drome as well as with increased cancer risk, as they boost the formation of spontaneous mutations that can lead to tumorigenesis [5].

The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding–defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance., Author Summary The telomeric cap modulates telomere replication and prevents natural chromosome ends from being processed as DNA double-strand breaks (DSBs). In multiple species, including budding yeast, a detailed picture exists of the factors that comprise the telomeric cap and how they associate with telomeric DNA. It is less clear where to place Ku, a conserved heterodimer involved in multiple aspects of telomere biology and DSB repair. Although Ku avidly binds DNA ends, its access to telomeric ends might be restricted by telomere binding proteins and/or higher-order telomere structure. Ku might also be recruited to telomeres via its telomere-associated binding partners. Here, we address whether Ku loads directly onto telomeric ends and whether direct DNA binding is crucial for its telomeric functions. Using structure-guided mutagenesis, we generated end binding–defective yeast Ku heterodimers that retained the ability to associate with Ku's known telomeric binding partners. These end binding–defective heterodimers showed a dramatic reduction in telomere association and were defective for all of Ku's telomeric functions. Our findings indicate that Ku is indeed a component of the telomere cap and that its loading onto telomeric ends is crucial for its telomeric functions and, perhaps, a specific telomere architecture.

Dyskeratosis congenita (DC) is a telomere biology disorder characterized by a mucocutaneous triad, aplastic anemia, and predisposition to cancer. Mutations in a narrow segment of TINF2 exon 6 have been recognized to cause often-severe DC that is either sporadic or autosomal dominant. We describe three children with very early presentations of DC, including one with the severe variant known as Revesz syndrome. Although most TINF2 mutations reported to date are missense changes, each of our patients carried a novel heterozygous nonsense or frameshift mutation, revealing a new 5' boundary to the affected gene segment in patients with DC. Examination of patient-derived lymphoblastoid cell lines revealed stable expression of the predicted truncated TIN2 proteins. In co-immunoprecipitation assays, the ability of a truncation mutant to interact with TRF1 was severely impaired, whereas the ability of the most common DC-associated mutant was much less affected. This suggests that the disruption of TIN2-TRF1 interaction may contribute to the severe clinical phenotype observed in the context of the TIN2 truncation mutation, but is unlikely to be the primary cause of telomere shortening associated with the more prevalent TIN2 missense mutations. Telomere flow-fluorescent in situ hybridization (FISH) analysis of one pedigree showed the dramatic effect a de novo nonsense TINF2 mutation had on telomere length in early development. These cases underscore the severe manifestations of truncating TINF2 mutations.

Background The cellular response to DNA damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. During the DNA damage response (DDR), the sensor kinases Tel1 and Mec1 in Saccharomyces cerevisiae and ATM and ATR in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and DNA repair. A subset of kinase substrates are recognized by the S/T-Q cluster domain (SCD), which contains motifs of serine (S) or threonine (T) followed by a glutamine (Q). However, the full repertoire of proteins and pathways controlled by Tel1 and Mec1 is unknown., Results To identify all putative SCD-containing proteins, we analyzed the distribution of S/T-Q motifs within verified Tel1/Mec1 targets and arrived at a unifying SCD definition of at least 3 S/T-Q within a stretch of 50 residues. This new SCD definition was used in a custom bioinformatics pipeline to generate a census of SCD-containing proteins in both yeast and human. In yeast, 436 proteins were identified, a significantly larger number of hits than were expected by chance. These SCD-containing proteins did not distribute equally across GO-ontology terms, but were significantly enriched for those involved in processes related to the DDR. We also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel Tel1/Mec1 targets in these pathways. In the human proteome, a wide range of similar proteins were identified, including homologs of some SCD-containing proteins found in yeast. This list also included high concentrations of proteins in the Mediator, spindle pole body/centrosome and actin cytoskeleton complexes. Conclusions Using a bioinformatic approach, we have generated a census of SCD-containing proteins that are involved not only in known DDR pathways but several other pathways under Tel1/Mec1 control suggesting new putative targets for t