Bradley A. Stohr

Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

The Role of Telomere Biology in Cancer

Telomere biology plays a critical and complex role in the initiation and progression of cancer. Although telomere dysfunction resulting from replicative attrition constrains tumor growth by engaging DNA-damage signaling pathways, it can also promote tumorigenesis by causing oncogenic chromosomal rearrangements. Expression of the telomerase enzyme enables telomere-length homeostasis and allows tumor cells to escape the antiproliferative barrier posed by short telomeres. Telomeres and telomerase also function independently of one another. Recent work has suggested that telomerase promotes cell growth through pathways unrelated to telomere maintenance, and a subset of tumors elongate telomeres through telomerase-independent mechanisms. In an effort to exploit the integral link between telomere biology and cancer growth, investigators have developed several telomerase-based therapeutic strategies, which are currently in clinical trials. Here, we broadly review the state of the field with a particular focus on recent developments of interest.

The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.

ATM Mediates Cytotoxicity of a Mutant Telomerase RNA in Human Cancer Cells

Telomeres are elongated by the enzyme telomerase, which contains a template-bearing RNA (TER or TERC) and a protein reverse transcriptase. Overexpression of a particular mutant human TER with a mutated template sequence (MT-hTer-47A) in telomerase-positive cancer cells causes incorporation of mutant telomeric sequences, telomere uncapping, and initiation of a DNA damage response, ultimately resulting in cell growth inhibition and apoptosis. The DNA damage pathways underlying these cellular effects are not well understood. Here, we show that the ataxia-telangiectasia mutated (ATM) protein is activated and forms telomeric foci in response to MT-hTer-47A expression. Depletion of ATM from two cancer cell lines, including the p53-mutant UM-UC-3 bladder cancer line, rendered the cells largely unresponsive to MT-hTer-47A. Relative to ATM-competent controls, ATM-depleted cells showed increased proliferation and clonogenic survival and reduced cell death following MT-hTer-47A treatment. In contrast, ATM depletion sensitized the cancer cells to treatment with camptothecin, a topoisomerase inhibitor that induces DNA double-strand breaks. We show that the effects of ATM depletion on the MT-hTer-47A response were not due to decreased expression of MT-hTer-47A or reduced activity of telomerase at the telomere. Instead, ATM depletion allowed robust cancer cell growth despite the continued presence of dysfunctional telomeres containing mutant sequence. Notably, the number of end-to-end telomere fusions induced by MT-hTer-47A treatment was markedly reduced in ATM-depleted cells. Our results identify ATM as a key mediator of the MT-hTer-47A dysfunctional telomere response, even in cells lacking wild-type p53, and provide evidence that telomere fusions contribute to MT-hTer-47A cytotoxicity.

Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity

Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with liposarcomatous components. EGFR amplification was heterogeneous and present only in the non-heterologous component of one tumor with liposarcomatous differentiation. The results identify novel pathways involved in the pathogenesis of malignant phyllodes tumors, which significantly increase our understanding of tumor biology and have potential clinical impact.

The Shelterin TIN2 Subunit Mediates Recruitment of Telomerase to Telomeres

Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.

In situ visualization of telomere elongation patterns in human cells

The telomerase enzyme plays a critical role in human aging and cancer biology by maintaining telomere length and extending the proliferative lifespan of most stem cells and cancer cells. Despite the importance of this enzyme, our understanding of the mechanisms that regulate its activity and establish telomere length homeostasis in mammalian cells is incomplete, in part because the perfect repetitive nature of telomeric sequence hampers in situ detection of telomere elongation patterns. Here, we describe a novel assay using a mutant telomerase that adds a well-tolerated variant telomeric repeat sequence to telomere ends. By specifically detecting the addition of these variant repeats, we can directly visualize telomere elongation events in human cells. We validate this approach by in situ mapping of telomere elongation patterns within individual nuclei and across a population of cells.

The Terminal Telomeric DNA Sequence Determines the Mechanism of Dysfunctional Telomere Fusion

Mammalian telomeres consist of tandem DNA repeats that bind protective protein factors collectively termed shelterins. Telomere disruption typically results in genome instability induced by telomere fusions. The mechanism of telomere fusion varies depending on the means of telomere disruption. Here, we investigate telomere fusions caused by overexpression of mutant telomerases that add mutated telomeric repeats, thereby compromising shelterin binding to telomeric termini. While all mutant telomeric sequences tested induced heterodicentric chromosome fusions in ATM-competent cells, only those mutant repeat sequences with significant self complementarity induced ATM-independent sister chromatid and isodicentric chromosome fusions. Thus, once a telomere becomes dysfunctional, the terminal telomeric sequence itself determines the fate of that telomere. These results suggest that annealing of self-complementary DNA sequence engages an alternative telomere fusion pathway in human cells, and provide one explanation for the conspicuous lack of self complementarity in the majority of known naturally occurring eukaryotic telomeric sequences.

Autophagy-Independent Senescence and Genome Instability Driven by Targeted Telomere Dysfunction

Telomere dysfunction plays a complex role in tumorigenesis. While dysfunctional telomeres can block the proliferation of incipient cancer clones by inducing replicative senescence, fusion of dysfunctional telomeres can drive genome instability and oncogenic genomic rearrangements. Therefore, it is important to define the regulatory pathways that guide these opposing effects. Recent work has shown that the autophagy pathway regulates both senescence and genome instability in various contexts. Here, we apply models of acute telomere dysfunction to determine whether autophagy modulates the resulting genome instability and senescence responses. While telomere dysfunction rapidly induces autophagic flux in human fibroblast cell lines, inhibition of the autophagy pathway does not have a significant impact upon the transition to senescence, in contrast to what has previously been reported for oncogene-induced senescence. Our results suggest that this difference may be explained by disparities in the development of the senescence-associated secretory phenotype. We also show that chromosome fusions induced by telomere dysfunction are comparable in autophagy-proficient and autophagy-deficient cells. Altogether, our results highlight the complexity of the senescence-autophagy interface and indicate that autophagy induction is unlikely to play a significant role in telomere dysfunction-driven senescence and chromosome fusions.

Application of a Prognostic Gleason Grade Grouping System to Assess Distant Prostate Cancer Outcomes

BACKGROUND There is growing enthusiasm for the adoption of a novel grade grouping system to better represent Gleason scores. OBJECTIVE To evaluate the ability of prognostic Gleason grade groups to predict prostate cancer (PCa)-specific mortality (PCSM) and bone metastatic progression. DESIGN, SETTING, AND PARTICIPANTS We identified patients with PCa enrolled in the Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) registry across treatment strategies, including conservative and nondefinitive therapy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS We examined the prognostic ability of Gleason grade groups to predict risk of PCSM and bone metastasis using the Kaplan-Meier method and unadjusted and adjusted Cox proportional hazards models. RESULTS AND LIMITATIONS We identified 10529 men with PCa followed for a median of 81 mo (interquartile range 40-127), including 64% in group I (< 3 + 4); 17% in group II (3+4); 9% in group III (4+3); 6% in group IV (4+4); and 4% in group V (≥ 4 + 5). Relative to grade group I, the unadjusted risks of PCSM and bone metastasis were significantly associated with prognostic grade groupings for both biopsy and prostatectomy samples (all p<0.01). Pairwise comparisons within Gleason sums collapsed within grade group V were not significant; however, this analysis was limited by a small representation of men with Gleason pattern ≥ 4 + 5. CONCLUSIONS The prognostic grade grouping system is associated with risk of PCSM and metastasis across management strategies, including definitive therapy, conservative management, and primary androgen deprivation. PATIENT SUMMARY A five-level reporting system for prostate cancer pathology is associated with the risk of late prostate cancer endpoints.