Sonja Schaetzlein

Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation

Telomere shortening limits the proliferative lifespan of human cells by activation of DNA damage pathways, including upregulation of the cell cycle inhibitor p21 (encoded by Cdkn1a, also known as Cip1 and Waf1)) (refs. 1–5). Telomere shortening in response to mutation of the gene encoding telomerase is associated with impaired organ maintenance and shortened lifespan in humans and in mice. The in vivo function of p21 in the context of telomere dysfunction is unknown. Here we show that deletion of p21 prolongs the lifespan of telomerase-deficient mice with dysfunctional telomeres. p21 deletion improved hematolymphopoiesis and the maintenance of intestinal epithelia without rescuing telomere function. Moreover, deletion of p21 rescued proliferation of intestinal progenitor cells and improved the repopulation capacity and self-renewal of hematopoietic stem cells from mice with dysfunctional telomeres. In these mice, apoptotic responses remained intact, and p21 deletion did not accelerate chromosomal instability or cancer formation. This study provides experimental evidence that telomere dysfunction induces p21-dependent checkpoints in vivo that can limit longevity at the organismal level.

Exonuclease-1 Deletion Impairs DNA Damage Signaling and Prolongs Lifespan of Telomere-Dysfunctional Mice

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.

Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma

Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio‐phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose‐dependent manner in vitro and in vivo. The potency and efficacy of the lipid‐conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere‐free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy. (HEPATOLOGY 2005.)

The cellular level of telomere dysfunction determines induction of senescence or apoptosis in vivo

Telomere dysfunction induces two types of cellular response: cellular senescence and apoptosis. We analysed the extent to which the cellular level of telomere dysfunction and p53 gene status affect these cellular responses in mouse liver using the experimental system of TRF2 inhibition by a dominant‐negative version of the protein (TRF2ΔBΔM). We show that the level of telomere dysfunction correlates with the level of TRF2ΔBΔM protein expression resulting in chromosomal fusions, aberrant mitotic figures and aneuploidy of liver cells. These alterations provoked p53‐independent apoptosis, but a strictly p53‐dependent senescence response in distinct populations of mouse liver cells depending on the cellular level of TRF2ΔBΔM expression. Apoptosis was associated with higher expression of TRF2ΔBΔM, whereas cellular senescence was associated with low levels of TRF2ΔBΔM expression. Our data provide experimental evidence that induction of senescence or apoptosis in vivo depends on the cellular level of telomere dysfunction and differentially on p53 gene function.

Mitogen Stimulation Cooperates with Telomere Shortening To Activate DNA Damage Responses and Senescence Signaling

ABSTRACT Replicative senescence is induced by critical telomere shortening and limits the proliferation of primary cells to a finite number of divisions. To characterize the activity status of the replicative senescence program in the context of cell cycle activity, we analyzed the senescence phenotypes and signaling pathways in quiescent and growth-stimulated primary human fibroblasts in vitro and liver cells in vivo. This study shows that replicative senescence signaling operates at a low level in cells with shortened telomeres but becomes fully activated when cells are stimulated to enter the cell cycle. This study also shows that the dysfunctional telomeres and nontelomeric DNA lesions in senescent cells do not elicit a DNA damage signal unless the cells are induced to enter the cell cycle by mitogen stimulation. The amplification of senescence signaling and DNA damage responses by mitogen stimulation in cells with shortened telomeres is mediated in part through the MEK/mitogen-activated protein kinase pathway. These findings have implications for the further understanding of replicative senescence and analysis of its role in vivo.

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes.

Significance Exonuclease1 (EXO1) is involved in a variety of DNA repair pathways and is implicated in multiple biological processes. To determine the contribution of the enzymatic and structural functions of EXO1 in these processes, we compared mice with catalytically inactive EXO1-knockin and complete EXO1-knockout mutations. We found that the catalytic function of EXO1 is essential for the DNA damage response, double-strand break repair, chromosomal stability, and tumor suppression, whereas EXO1’s structural role alone is critical for mismatch repair, antibody diversification, and meiosis. Our study reveals differential requirements for both EXO1 functions in DNA repair and tumorigenesis in vivo. Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1EK) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1null) mouse. In contrast to Exo1null/null mice, Exo1EK/EK mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1null mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.

Mechanism of DNA resection during intrachromosomal recombination and immunoglobulin class switching

CtBP-interacting protein, exonuclease 1, and RecQ helicases contribute to the processing of DNA ends during double-strand break repairs in primary lymphocytes.

Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells

BackgroundThe evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes.MethodsHigh-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSβ heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSβ (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells.ResultsThis study identified distinct genetic profiles for MutSα and MutSβ, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers.ConclusionOur findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors.

Abstract A13: Identification of evolutionarily conserved genetic interactions in DNA mismatch repair

The DNA mismatch repair (MMR) system is responsible for correcting base substitution and insertion-deletion mutations (IDLs) generated during erroneous replication in bacteria, yeast and mammals. MMR complexes also recognize damaged-base mispairs resulting from environmental exposure to DNA damaging agents. The loss of MMR causes a “mutator” phenotype, resulting in the genome-wide accumulation of mutations, and is the underlying cause for Hereditary Non-polyposis Colorectal Cancer / Lynch Syndrome (HNPCC/LS) and a significant proportion of sporadic colorectal cancer (CRC). MMR-deficient tumors also display resistance to DNA damaging agents and, as a consequence, are resistant to a variety of commonly chemotherapeutic agents. Thus, the development of novel therapeutic strategies that more efficiently target MMR-deficient cancer cells would be highly desirable. A promising new direction to identify potential targets for anticancer treatment is the harnessing of synthetic lethality. Here, we have identified conserved synthetic lethal / sick genetic interactions (GIs) for MMR factors between two distant yeast species, S. pombe and S. cerevisiae. We have confirmed several of these negative GIs in mammalian cells, indicating that they are evolutionarily conserved. Specifically, we have identified a negative genetic relationship between Msh2, a major MMR component mutated in colorectal cancer, and Senp6 protease, which regulates the SUMOylation of a range of genome maintenance proteins. Our findings suggest that Senp6 is a promising new target for the treatment of MMR-deficient CRCs. Citation Format: Elena Tosti, Joseph A. Katakowski, Sonja Schaetzlein, Hyun-Soo Kim, Colm J. Ryan, Michael Shales, Assen Roguev, Nevan J. Krogan, Deborah Palliser, Michael-Christopher Keogh, Winfried Edelmann. Identification of evolutionarily conserved genetic interactions in DNA mismatch repair. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A13.