How to Disable a Cellular Bomb: Findings and Tools on the Machinery of ALT
To develop an unbreachable defense against cancer, SENS Foundation is pursuing the WILT (Wholebody Interdiction of Lengthening of Telomeres) strategy (otherwise OncoSENS) of systematically deleting genes essential to the cellular telomere-maintenance mechanisms (TMM) from all somatic cells, while ensuring ongoing tissue repair and maintenance through periodic re-seeding of somatic stem-cell pools with autologous TMM-deficient cells whose telomeres have been lengthened ex vivo. In addition to the deletion of one or more genes coding for essential element(s) of the telomerase holoenzyme, success will also require the deletion of some essential element of the machinery [responsible] for the Alternative Lengthening of Telomeres (ALT) phenomenon, observed in a minority of cancer cells.
One of the characteristic phenotypic features of cells utilizing the ALT mechanism is the presence of telomeric DNA and the telomere binding proteins TERF1 and TERF2 in close association with a subset of the cell's promyelocytic leukaemia protein (PML) nuclear bodies. PML nuclear bodies are spherical nuclear structures which, in normal cells, are involved in DNA repair, senescence, apoptosis, and other functions. But their association with telomeric chromatin is rare, except in ALT cells, where they are instead hallmarks of the phenomenon; this has led to such assemblies being designated "ALT-associated PML bodies" (APBs). APBs not only contain recombination proteins demonstrated to be essential to telomere maintenance in ALT cells, but appear themselves to be involved in telomere recombination(1) and to coincide spatially and temporally with telomeric DNA synthesis.(2,3) Therefore, some have hypothesized that APBs may be the nexus of the “Alternative Lengthening of Telomeres” in such cells.
To test whether APBs might indeed be causally involved in the TMM of ALT cells, a team of German researchers devised the novel approach of generating APBs artificially, by recruiting APB subcomponents to telomeric or pericentric DNA, and observing the effects on assembly of APB-like structures and on telomere elongation.(4) To do this, the investigators generated fusion proteins of several APB constituents with the bacterial LacI repressor protein and fluorescent binding proteins as markers, and then exploited LacI's high-affinity binding with the operator region of lac operons to tether these proteins to the repeats of lac operator sequences that are stably integrated into the genome of U2OS osteosarcoma cell lines. To reveal the effects of chromosomal localization on such proteins' ability to form APBs and on the abilities of such experimentlaly-induced entities, the researchers tested the effects of APB constituent protein tethering in two different U2OS cell lines: one (F6B2) with such operator sequences integrated adjacent to telomeric DNA, and another (F42B8) whose sequences were localized pericentrically. Thus, transfection of one or the other cell line with LacI-APB component fusion constructs resulted in the tethering of the various APB component proteins at locations proximate to, or remote from, telomeric DNA.(4)
Bring a Friend
When tethered to telomeric DNA, LacI fusion constructs containing either of the two proteins (PML protein or Sp100) present in PML nuclear bodies, induced recruited the complementary protein in turn; tethering of "empty" fluorescent-labeled LacI constructs yielded no such results.(4) The bringing-together of these proteins at telomeric DNA induced the formation of apparent APBs, as suggested by the observations that (a) the PML protein assumed the same cap-like structures surrounding telomere repeats observed in APBs; (b) the structures appeared almost entirely localized with single telomere structures; and (c) the location of the recruited endogenous proteins appeared enriched with several SUMO isoforms, consistent with the presence of SUMO modifications of these proteins in endogenous PML nuclear bodies.(5)
Sussing Out SUMO
Indeed, modification of at least some APB constituents by SUMO proteins is known to be essential to the formation of APBs: PML protein contains a SUMO-interacting motif (SIM) that is not involved in SUMOylation but that that is required for PML nuclear body formation.(5) Again mirroring observations in native APBs, telomeric recruitment of LacI constructs with several SUMO isoforms led to colocalization of native PML, Sp100, and Rad17 (all present in endogenous APBs), again consistent with APB formation. A series of tests using constructs containing SUMO1 mutants either incapable of the noncovalent interactions with SIMs observed in the APBs in endogenous ALT cells, or incapable of covalent attachments, demonstrated that the role of this SUMO protein in the formation of the de novo APBs involves the former kind of interactions,(4) consistent with the need for noncovalent SIM binding in the formation of native PML nuclear bodies.
On the other hand, several telomere-associated proteins present in APBs in ALT (including TRF1, TRF2, and Rap1) do require SUMOylation, by the SUMO E3 ligase MMS21, and here again the investigators were able to use their tethering system to recapitulate observations of native APBs. Tethering of MMS21 to telomereic or pericentric DNA was "highly efficient in promoting APB assembly," recruiting substantial increases in colocalized PML protein. But importantly, only those MMS21-tethered telomeres with colocalized PML also colocalized with the APB component Rad9. This was hightly similar to what they observed in native APBs: "The vast majority (98%) of endogenous APBs (defined as colocalization of PML and TRF2) contained Rad9, and only 0.9% of the telomeres marked by TRF2 had Rad9 but no PML."(4) This is especially noteworthy, since there is evidence(5) that MMS21 plays a role in DNA repair, and the ALT TMM itself exploits (telomeric) DNA repair.
Back of the Line, Please
Inducing TRF1, TRF2, Rad9, NBS1, and MMS21 recruitment to telomeric DNA also increased formation of APBs.(4) On the other hand, tethering of two other APB constituents (Rad51 or Rad17) to telomeric DNA led to little (Rad51) or no (Rad17) recruitment of PML.(4) Despite this, endogenous Rad17 -- like NBS1 and Rad9 -- was enriched in the de novo APBs formed following PML tethering, consistent with native APBs. The authors note that a previous report (5) had found that knockdown or Rad51 with siRNA in U2OS cells had no effect on APB formation; combined, these results suggest that these repair factors may only be recruited to APBs after the initial assembly of other constituent proteins.
The Road to Perdition -- and a Bridge to Nowhere
In addition to observations at telomeric DNA, APB component proteins were also recruited when the researchers tethered PML to the pericentric lac operator sequences in the F442B8 U2OS line; in fact, these components were recruited "to a similar or even higher degree than at the telomeric sites." Again in general agreement with observations using telomerically-tethered proteins, recruiting MMS21 to pericentric DNA also led to elevated colocalization of APB proteins, whereas tethering of Rad51 at this locus did not.(4)
But the purpose of the investigation was not to determine if APBs could induced to form at these loci, but to determine the function of these structures: whether APBs play a causal role in telomere maintenance in ALT cells, or are merely phenotypic hallmarks of the ALT phenomenon. The investigators therefore tested the ability of de novo APBs to lengthen telomeres in a manner consistent with the ALT TMM.
When PML was tethered to telomeric chromosomal loci, the resulting de novo APBs were associated with elevated phosphorylated γ-H2AX histone. Phosphorylated γ-H2AX is a component of APBs, and indicator of double-strand break (DSB) repair, which is involved in the ALT TMM.(4) Similarly, the generation of de novo APBs at telomeric chromosomes was associated with increased non-replicative DNA synthesis, as detected by elevated pulsed 5-bromo-2′-deoxyuridine (BrdU) incorporation at the limited number of foci where it occurs during non-replicative phases of the cell cycle. Such increases in DNA synthesis, arising in association with the formation of APBs at telomeric DNA, are consistent with telomere lengthening. Again, a fluorescence in situ hybridization (FISH) probe was used to detect the percentage chromosomal ends too short to register on the probe, and thus primed for telomere extension by the ALT TMM. Tethering PML to telomeric DNA reduced the fraction of chromosomes with indetectably-short telomeres from ~43% to ~19%, suggesting the extension of critically-short telomeres; by contrast, tethering "empty" fluorescent-labeled LacI constructs to telomeric DNA led to no such increase.(4)
And importantly, none of these phenomena -- neither increased phosphorylated γ-H2AX, nor increased non-replicative BrdU incorporation, nor a reduction in indetectably-short telomeres -- appeared subsequent to APB formation at pericentric lac operator sequences. Thus, these phenomena -- each consistent with the lengthening of telomeres by an ALT-like mechanism -- were specific to the presence of APBs at telomeric DNA.(4)
Pause del Silenzio
It is worth here quoting the authors' own conclusions at length:
[Our findings] establish APBs as functional intermediates of the ALT pathway [my emphasis]. ... Accordingly, we conclude that the formation of bona fide APBs promotes the extension of the telomere repeat sequence by a DNA- repair-coupled synthesis process. Our study does not provide information on the nature of the telomere repeat template used for synthesis ... Furthermore, given the large number of partially contradicting results in the literature, it is well conceivable that different telomerase-independent mechanisms for telomere repeat extension exist. ... Thus, it will be important to further dissect the exact combination of protein factors that are sufficient to trigger telomere extension in APBs and to investigate the effects of their presence or absence. We anticipate that the experimental approach introduced here ... will allow us to precisely identify all protein components that are sufficient to form a telomeric PML-NB subcompartment structure, as well as the additional factors needed to induce telomere extension at these sites. This will serve to select protein targets for inhibiting telomere extension, and thus cell proliferation, in tumors that make use of the ALT pathway.(4)
All of the known constituents of APBs play a role in normal cellular metabolism. But full implementation of the WILT strategy will require not only that we "select [APB] protein targets for inhibiting telomere extension," but that one or more element of the ALT TMM be irreversibly disabled, in all of the cells of the body (and as a major intermediate goal, in those tissues in which ALT cancers most commonly arise). Thus, success with WILT will require a detailed elaboration of all the structures responsible for the lengthening of telomeres in ALT cells -- those discovered and undiscovered, constitutive of APBs and not -- and an understanding of their role in telomere lengthening in disease, as well as physiological functions in health. By providing significant support for the role of APBs in telomere extension, the beginnings of further understanding of the process of APB assembly and the roles of some constituent proteins in the APB TMM, and new tools for dissecting APB structure and function, Chung et al have taken us a significant step toward this benchmark, and thus toward a decisive cure for malignant disease.
1: Draskovic I, Arnoult N, Steiner V, Bacchetti S, Lomonte P, Londoño-Vallejo A. Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination. Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15726-31. Epub 2009 Aug 26. PubMed PMID: 19717459; PubMed Central PMCID: PMC2747187.
2: Nabetani A, Yokoyama O, Ishikawa F. Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. J Biol Chem. 2004 Jun 11;279(24):25849-57. Epub 2004 Apr 9. PubMed PMID: 15075340.
3:Wu G, Lee WH, Chen PL. NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres. J Biol Chem. 2000 Sep 29;275(39):30618-22. PubMed PMID: 10913111.
4: Chung I, Leonhardt H, Rippe K. De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation. J Cell Sci. 2011 Nov 1;124(Pt 21):3603-18. Epub 2011 Nov 1. PubMed PMID: 22045732.
5: Shen TH, Lin HK, Scaglioni PP, Yung TM, Pandolfi PP. The mechanisms of PML-nuclear body formation. Mol Cell. 2006 Nov 3;24(3):331-9. PubMed PMID: 17081985; PubMed Central PMCID: PMC1978182
6: Potts PR, Yu H. Human MMS21/NSE2 is a SUMO ligase required for DNA repair. Mol Cell Biol. 2005 Aug;25(16):7021-32. PubMed PMID: 16055714; PubMed Central PMCID: PMC1190242.