A Green Light for the Ultimate Cure for Cancer
To develop an unbreachable defense against cancer, SENS Foundation is pursuing the WILT (Wholebody Interdiction of Lengthening of Telomeres, or OncoSENS) strategy of preemptively 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. Without a mechanism for extending its telomeres, the replicative potential of a cancer cell faces an absolute barrier to achieving clinically significant growth, and what is today a death sentence (or at best a chronic or potentially relapsing disease) is transformed into a benign lesion, of no more clinical significance than a cyst or plantar hyperkeratosis.(12-14)
At its core, then, WILT entails the ablation of some gene encoding an element of the telomerase holoenzyme. The strongest challenge to this approach, granting the periodic replenishment of somatic stem-cell pools with autologous but OncoSENS-ready stem cells, has been the possible existence of functions of TERT (telomerase reverse transcriptase -- the catalytic subunit of telomerase), other than the lengthening of telomeres itself. In recent years, several reports(1-10) have emerged claiming to have uncovered such functions, but generally in vitro and in nearly all cases under unphysiologically high and/or persistent forced expression of the telomerase gene. While it is not necessarily the case that TERT itself be knocked out -- ablation of the gene encoding the RNA template for TERT's reverse transcriptase is the other main candidate -- WILT is a sufficiently, complex, sweeping, and essential as the sole strategy currently proposed to render the human body ultimately immune to malignant disease that having one of our main options for implementation taken off of the table because of gene pleiotropy would be an unwelcome restriction on therapeutic options.
A direct in vivo test for deleterious effects of the deletion of TERT that do not involve the critical shortening of telomeres per se is somewhat challenging. Even first-generation TERT-/- mice do exhibit some mild shortening of the normal lifespan,(15) but such animals suffer some loss of tissue renewal across their lives. But a careful test of the development of TERT-/- mice would be expected to provide strong evidence on the subject one way or the other, provided that telomere lengths did not shorten excessively.
Nobel laureate Dr. Carol W. Greider, whose career in telomerase research and path to the Nobel prize began when she first identified the enzyme in 1984, has finally carried out such a test, and she and her collaborators have generated results that strongly support the safety of this element of WILT.
Seeking -- and Not Finding
To rigorously test for any non-TMM effects of TERT requires confidence that any phenotypic abnormalities are not related to telomere shortening per se. Greider's group built two strong safeguards against this major confounder. The first was the comparison of mice with TERT knockout and haploinsufficiency, to mice haploinsufficient and deficient in mTR, the murine TERC. Such mice have a fully functional catalytic telomerase subunits, but suffer progressive telomere shortening. They also made a novel selection for their background strain. In previous studies, TERT-/- mice have been generated on either a C57BL/6J or a 129/C57BL/6J mixed genetic background strain, both of which have "very heterogeneous and unusually long telomeres," again raising the possibility that any developmental abnormalities might be due to subpopulations of stem cells with critically-short telomeres. To avoid this problem, they instead performed their experiments in the CAST/EiJ mouse, a strain with telomere lengths similar to humans and homogeneous telomere length distributions.(11)
Telomere shortening and progressive impairments of tissue renewal in mTERT–/– and mTERT+/– mice on this background was similar to that in mTR–/– and mTR+/– mice, respectively. CAST/EiJ mTR–/– mice exhibit significant deficits in tissue renewal during adulthood, even in the first generation, and progressive worsening of the phenotype with successive generations, similar to what is seen in other background strains with these mutations and in the genetic anticipation observed in successive generations of families with autosomal dominant forms of the human genetic disorder of telomerase components, dyskeratosis congenita. Importantly, however, "mTERT⁻/⁻ mice, from heterozygous mTERT⁺/⁻ mouse crosses, were born at the expected Mendelian ratio (26.5%; n = 1,080 pups), indicating no embryonic lethality of this genotype ... [and] show no additional phenotypes not seen in mTR–/– mice"(11) -- and even more importantly, "mTERT–/– mice show no additional phenotypes not seen in mTR–/– mice."(11)
Amongst the studies claiming to have found evidence of a TMM-independent function of TERT, two(9.10) have drawn a putative connection to Wnt signaling. But in investigator-blinded comparisons of tissue sections from each group of mice, no evidence of defects related to loss of Wnt signaling were found in embryonic or adult pulmonary, renal, cerebral, or skeletal tissues. In particular, (9) had reported that some of their mTERT–/– mice were missing ribs, which was interpreted as a developmental defect of impaired Wnt signaling; comparison of wild-type and mTERT–/– CAST/EiJ and C57BL/6J mice showed normal numbers and morphology of ribs in all animals.(11) To test for possible developmental compensation for more subtle Wnt defects, they examined haploinsufficient crosses of CAST/EiJ mTERT+/– mice; none of the multiple defects observed in mice with knockouts of multiple different Wnt subtypes; again, no such phenotypes were observed.(11) To more directly test for defects in Wnt signaling, CAST/EiJ and C57BL/6J WT and mTERT–/– embryonic fibroblasts were transfected with a luciferase reporter plasmid with a Wnt3a ligand; luciferase expression was indistinguishable between WT and mTERT–/– cells.(11)
No Hairy Deal, No Silent Partner
Similarly, another group had reported excessive hair growth in mice with genetically augmented TERT activity under an unphysiologic promoter,(5) suggesting a possible role of TERT in follicular (and possibly stem) cell growth. But mTERT-/- mice exhibited no defects in hair loss.(11) Yet another group had suggested, based on unphysiologic cell models, that TERT might be involved in production of some small interfering RNAs as part of a ribonucleoprotein complex with RNA component of mitochondrial RNA processing endoribonuclease (RMRP);(8) again, no phenotypes suggestive of RMRP deficiency were observed in mTERT–/– mice.(11)
Opening the Lanes for A Long Drive Home
This little-heralded, meticulous investigation into the effects of ablation of the telomerase catalytic subunit in mice with human-like telomeres provides us with strong reassurance that, should it prove to be the preferred approach for implementing the OncoSENS strategy, the effects of knocking out TERT would be limited to those dictated by the loss of telomere-lengthening per se, and would not lead to an unintentional loss of some essential but hitherto-unknown phsyiological function. In light of the importance of WILT as an element of a comprehensive suite of rejuvenation biotechnologies,this news is welcome -- but insufficient, despite its rigor. SENS Foundation is funding additional research to further open up the path toward its realization. Amongst these are a research project in the lab of Dr. Jan Vijg, Chair of the Department of Genetics at Albert Einstein College of Medicine, who with research associate Dr. Silvia Gravina is performing the first single-cell analysis of the rate of incidence of epimutations in the aging mouse brain, to test for any unexpected involvement of non-cancerous mutations in the degenerative aging process, whose existence might render WILT an insufficient strategy to address nuclear genomic epimutations during the course of the "normal" life expectancies of today. Another is to monitor the effects of transplanting telomerase-deficient but ex vivo telomere-extended bone marrow into late-generation, TMM-disabled mice, so as to be certain that the niche of such animals (or, by implication, aging humans) will support the homing, engraftment, and initial development and differentiation of such cells; the necessary research is underway now thanks to a SENS Foundation grant to Dr. Zhenyu Ju of the Institute of Laboratory Animal Sciences and Max-Planck-Partner-Group on Stem Cell Aging in the Chinese Academy of Medical Sciences, and research partner of prominent telomere biologist Dr. K. Lenhard Rudolph.
WILT is the most ambitious plank in the SENS platform, with the longest and most logistically intricate path to development. The results of this and additional SENS Foundation-funded studies provide researchers with a confident basis to proceed toward an end to the scourge of cancer.
1. Ju Z, Jiang H, Jaworski M, Rathinam C, Gompf A, Klein C, Trumpp A, Rudolph KL. Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment. Nat Med. 2007 Jun;13(6):742-7. Epub 2007 May 7. PubMed PMID: 17486088.
2. Flores I, Cayuela ML, Blasco MA. Effects of telomerase and telomere length on epidermal stem cell behavior. Science. 2005 Aug 19;309(5738):1253-6. Epub 2005 Jul 21. PMID: 16037417 [PubMed - indexed for MEDLINE]
3. Liu L, DiGirolamo CM, Navarro PA, Blasco MA, Keefe DL. Telomerase deficiency impairs differentiation of mesenchymal stem cells. Exp Cell Res. 2004 Mar 10;294(1):1-8. PMID: 14980495 [PubMed - indexed for MEDLINE]
4. Passos JF, Saretzki G, von Zglinicki T. DNA damage in telomeres and mitochondria during cellular senescence: is there a connection? Nucleic Acids Res. 2007;35(22):7505-13. PMID: 17986462
5. Sarin KY, Cheung P, Gilison D, Lee E, Tennen RI, Wang E, Artandi MK, Oro AE, Artandi SE. Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature. 2005 Aug 18;436(7053):1048-52. PMID: 16107853 [PubMed - indexed for MEDLINE]
6: Masutomi K, Possemato R, Wong JM, Currier JL, Tothova Z, Manola JB, Ganesan S, Lansdorp PM, Collins K, Hahn WC. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8222-7. Epub 2005 May 31. PubMed PMID: 15928077; PubMed Central PMCID: PMC1149439.
7: Geserick C, Tejera A, González-Suárez E, Klatt P, Blasco MA. Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-beta signaling. Oncogene. 2006 Jul 20;25(31):4310-9. Epub 2006 Feb 27. PubMed PMID: 16501597.
8: Maida Y, Yasukawa M, Furuuchi M, Lassmann T, Possemato R, Okamoto N, Kasim V, Hayashizaki Y, Hahn WC, Masutomi K. An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA. Nature. 2009 Sep 10;461(7261):230-5. Epub 2009 Aug 23. PubMed PMID: 19701182; PubMed Central PMCID: PMC2755635.
9: Park JI, Venteicher AS, Hong JY, Choi J, Jun S, Shkreli M, Chang W, Meng Z, Cheung P, Ji H, McLaughlin M, Veenstra TD, Nusse R, McCrea PD, Artandi SE. Telomerase modulates Wnt signalling by association with target gene chromatin. Nature. 2009 Jul 2;460(7251):66-72. PubMed PMID: 19571879.
10: Choi J, Southworth LK, Sarin KY, Venteicher AS, Ma W, Chang W, Cheung P, Jun S, Artandi MK, Shah N, Kim SK, Artandi SE. TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program. PLoS Genet. 2008 Jan;4(1):e10. Epub 2007 Dec 13. PubMed PMID: 18208333; PubMed Central PMCID: PMC2211538.
11: Strong MA, Vidal-Cardenas SL, Karim B, Yu H, Guo N, Greider CW. Phenotypes in mTERT⁺/⁻ and mTERT⁻/⁻ mice are due to short telomeres, not telomere-independent functions of telomerase reverse transcriptase. Mol Cell Biol. 2011 Jun;31(12):2369-79. Epub 2011 Apr 4. PubMed PMID: 21464209; PubMed Central PMCID: PMC3133422.
12: de Grey AD, Campbell FC, Dokal I, Fairbairn LJ, Graham GJ, Jahoda CAB, Porter ACG. Total deletion of in vivo telomere elongation capacity: an ambitious but possibly ultimate cure for all age-related human cancers. Ann N Y Acad Sci. 2004 Jun;1019:147-70. PubMed: 15247008.
13: de Grey AD. Whole-body interdiction of lengthening of telomeres: a proposal for cancer prevention. Front Biosci 2005;10:2420-2429. PubMed: 15970505.
14: de Grey AD. WILT: Necessity, feasibility, affordability. In: Fahy GM, West M, Coles LS, Harris SB (eds) The Future of Aging: Pathways to Human Life Extension. 2010; Springer, 667-684.
15: García-Cao I, García-Cao M, Tomás-Loba A, Martín-Caballero J, Flores JM, Klatt P, Blasco MA, Serrano M. Increased p53 activity does not accelerate telomere-driven ageing. EMBO Rep. 2006 May;7(5):546-52. Epub 2006 Mar 31. PubMed PMID: 16582880; PubMed Central PMCID: PMC1479549.