TSPYL5 Depletion Induces Specific Death of ALT Cells through USP7-Dependent Proteasomal Degradation of POT1.

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT+ cancer-specific therapeutic target.

Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin.

Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway.

Photo-induced telomeric DNA damage in human cancer cells.

Herein we report on the study of novel dinuclear ruthenium(ii) complexes designed to target and to photo-react with G-quadruplex telomeric DNA. Upon irradiation, complexes efficiently generate guanine radical cation sites as photo-oxidation products. The compounds also display efficient cell penetration with localization to the nucleus and show strong photocytotoxicity toward osteosarcoma cells. Thanks to a microscopic-based telomere dysfunction assay, which allows the direct visualization of DNA damage in cells, we brought the first evidence of forming photo-oxidative damage at telomeres in cellulo. This emphasizes interesting prospects for the development of future cancer phototherapies.

Specific death of ALT cells through TSPYL5 depletion.

Some tumors acquire replicative immortality by activating an ALTernative telomerase-independent telomere maintenance mechanism. ALT offers interesting therapeutic perspectives but lacks any known specific targets. We discovered a crucial role for TSPYL5 (Testis-Specific Y-encoded-Like Protein 5) in keeping ALT cell viability by protecting POT1 (Protection Of Telomeres 1) from proteasomal degradation.

Impaired telomere integrity and rRNA biogenesis in PARN-deficient patients and knock-out models.

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.

Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription.

DNA breaks activate the DNA damage response and, if left unrepaired, trigger cellular senescence. Telomeres are specialized nucleoprotein structures that protect chromosome ends from persistent DNA damage response activation. Whether protection can be enhanced to counteract the age-dependent decline in telomere integrity is a challenging question. Telomeric repeat-containing RNA (TERRA), which is transcribed from telomeres, emerged as important player in telomere integrity. However, how human telomere transcription is regulated is still largely unknown. We identify nuclear respiratory factor 1 and peroxisome proliferator-activated receptor γ coactivator 1α as regulators of human telomere transcription. In agreement with an upstream regulation of these factors by adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), pharmacological activation of AMPK in cancer cell lines or in normal nonproliferating myotubes up-regulated TERRA, thereby linking metabolism to telomere fitness. Cycling endurance exercise, which is associated with AMPK activation, increased TERRA levels in skeletal muscle biopsies obtained from 10 healthy young volunteers. The data support the idea that exercise may protect against aging.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.