Alternative Lengthening of Telomeres: Building Bridges To Connect Chromosome Ends

Abstract

Alternative lengthening of telomeres (ALT) is a mechanism of telomere maintenance that is observed in many of the most recalcitrant cancer subtypes. Telomeres in ALT cancer cells exhibit a distinctive nucleoprotein architecture shaped by the mismanagement of chromatin that fosters cycles of DNA damage and replicative stress that activate homology-directed repair (HDR). Mutations in specific chromatin-remodeling factors appear to be key determinants of the emergence and survival of ALT cancer cells. However, these may represent vulnerabilities for the targeted elimination of ALT cancer cells that infiltrate tissues and organs to become devastating tumors. In this review we examine recent findings that provide new insights into the factors and mechanisms that mediate telomere length maintenance and survival of ALT cancer cells.

Keywords: DNA damage; cancer; homology-directed repair; replicative stress; telomere.

FIGURE 1.. TELOMERE MAINTENANCE DURING CANCER EVOLUTION

In normal somatic cells, telomeres shorten overtime after each cell division due to incomplete replication of terminal DNA sequences. Progressive and irreversible loss of telomeres eventually reaches a critical point when cellular senescence halts proliferation to prevent genomic instability. (T1) To bypass this barrier, cells exhibit deregulation of p53 and retinoblastoma (Rb) proteins to gain proliferative activity. (T2) However, due to telomere dysfunction-induced chromothripsis and polyploidization, cells undergo crisis and are eliminated by autophagic death. Thus, cancer cells adopt telomere maintenance mechanisms to achieve replicative immortality. (T3) The majority of cancers reactivate the specialized reverse transcriptase telomerase to synthesize de novo telomeric DNA. Telomerase re-expression arises from hTERT (catalytic subunit of telomerase) promoter mutations and upstream genomic rearrangements. (T4) 10-15% of cancers utilize the recombination-mediated pathway termed Alternative Lengthening of Telomeres (ALT). ALT telomeres display a permissive chromatin state, stochastic DNA damage and replication stress, as well as inactivation of type I interferon response – all of which contribute to a favorable environment for homology-directed repair (HDR) pathways that mediate telomere maintenance. (T5) The inhibition of ALT based telomere maintenance has been shown and often elicits strong cytotoxic effects without telomere shortening. Yet, there have been instances where telomere shortening correlates with proliferative arrest and even the activation of cytoprotective autophagy.

FIGURE 2.. THE BIOGENESIS AND FUNCTION OF APBs

SUMOylation of Shelterin components, TRF1 and TRF2, recruits PML/SP100 via SUMO-SIM interactions. SUMOylation-induced APB condensates lead to clustering of telomeres and DNA repair factors within the PML/SP-100 shell. A positive SUMOylation feedback loop enhances APB formation. APBs act as a platform to concentrate repair and recombination factors to perhaps facilitate telomeric DNA synthesis, particularly of extra-chromosomal C-circles. APBs are subsequently disassembled as a result of deSUMOylation events.

FIGURE 3.. HOMOLOGY DIRECTED REPAIR MECHANISM OF ALT

Telomeres pose as a challenge for the replication machinery, leading to replication fork stalling. The stalled replication fork can undergo fork reversal and restart by SMARCAL1 and FANCM. Here, replication stress is relieved at telomeres and ALT activity is restrained. However, unresolved replication stress leads to fork collapse, which provides direct DSB substrates for ALT-mediated recombination and telomere synthesis by RAD51-dependent (A) and RAD52-dependent (B and C) mechanisms. (A) Rad51-dependent HR: HR components, such as ATR, MRN, RAD51AP1, HOP2, BRCA1, and SMC5/6, facilitate the loading of RAD51 onto a telomeric end for subsequent long-range homology search. This results in semi-conservative replication, in which both recipient and donor telomeric strands contain the original template and newly synthesized telomeric DNA. (B) RAD51-dependent HDR: BLM-DNA2 resect the telomeric end to initiate PCNA-RFC-Polδ-mediated DNA synthesis. The outcome is conservative replication, which leads to newly synthesized DNA on both strands of the recipient telomere. In (A) and (B), the BTR complex (BLM-TOP3A-RMI1/2) initiates telomere dissolution for long-tract ALT-mediated telomere synthesis. (C) RAD52/SLX4-dependent Mitotic DNA synthesis (MiDaS): SLX4IP can antagonize the BTR complex to favor SMX (SLX1-SLX4, MUS81-EME1)-dependent resolution. The SMX complex promotes resolution of telomeric recombination intermediates in the absence of telomere extension.

FIGURE 4.. THE FATE OF ALT-INHIBITED CANCER CELLS.

ALT cancer cells must maintain a balance between pro- and anti-recombinogenic signals. A shift from this equilibrium poses a therapeutic vulnerability that can be exploited for selective killing of ALT cancers. Synthetic lethality with ATRX/DAXX is a promising target since ATRX/DAXX loss of function mutations are prevalent in ALT cancers. Similarly, ALT-associated PML bodies (APBs) is an attractive target for therapy since APBs are unique to ALT cancers and are implicated in telomere metabolism of ALT cells. Disruption of APBs with infection of Herpes Simplex Virus-1 (HSV-1) or depletion of TSPYL5 have been shown to elicit cytotoxicity. In addition, several lines of evidence report that inhibition of HDR factors perturbs ALT activity and provokes to cell death. Intriguingly, the absence of STING in ALT cancers enables activation of the pro-survival autophagic pathway. This reliance on autophagy may present a unique opportunity in ALT cancers to tip the balance in favor of apoptotic cell death.