W I L T an ambitious but truly un-escapable anti-cancer therapy Aubrey D.N.J. de Grey

1. W I L T an ambitious but truly un-escapable anti-cancer therapy Aubrey D.N.J. de Grey Department of Genetics, University of Cambridge

2. Structure of this talk Why a big cancer session in an aging meeting? Outline of a really, I mean really, crazy idea Analysis (i): Is it crazy enough to work? Analysis (ii): Is it too crazy to work?

3. Cancer: the hardest aspect of aging to combat??? Reason why it might be: every cancer is a moving target the smarter we get, the smarter it gets Treating an “inert” type of damage can be done periodically, and the same treatment works just as well every time :-) Treating a neoplastic type of damage selects for mutants that resist the treatment. 6Gb of DNA is a lot to play with :-(

4. Is such pessimism really justified? My take -- yes, shown by: the huge variety of innovative approaches to treating cancer that show promise but only modest efficacy the inherent subtlety of the cell-biological differences between cancer and non-cancer cells, which limits any (?) treatment’s therapeutic index Must we stick with the “cocktail” approach, or is there a “clean” (even if very ambitious) solution?

5. Tackling genomic instability head-on Selection can only do so much: some events are just too unlikely, so no cells in a cancer will experience them Problem: Gene expression changes are not unlikely enough What about gene existence changes?

6. Better acronym, anyone? • Whole-body • Interdiction of • Lengthening of • Telomeres

7. The proposed therapy, in a nutshell Engineer (in vitro) a patient’s cells to: a) be stem cells of each rapidly-renewing tissue b) be deleted for telomerase and ALT genes c) have (initially) natural-length telomeres d) have genetic resistance to some chemotherapies 2) Introduce these cells prior to chemotherapy Repeat (2) every decade or so, forever Delete telomerase/ALT genes in situ in satellite cells etc.

8. The four options 1) Without any cancer treatment: 2) With current or foreseeable cancer treatment:

9. 3) With uncompensated telomere maintenance deficiency: 4) With compensated telomere maintenance deficiency (WILT):

10. Crazy enough? Too crazy? SENS III, December 2nd 2002, Cambridge, UK Would lack of telomerase and ALT be totally protective? Steve Artandi, Nicola Royle Can cells be genetically engineered to be chemoresistant? Leslie Fairbairn Can all relevant stem cell types be transplanted? Gerry Graham, Colin Jahoda, Charles Campbell Can quiescent precursors (e.g. satellite cells) have their telomerase/ALT genes deleted efficiently by gene therapy? Andrew Porter Can we remain healthy for a decade with no telomerase? Inderjeet Dokal

11. Analysis (i): is WILT crazy enough to work?

12. Cancer protection by lack of telomerase/ALT: current status Fallacy #1: there aren’t enough cell divisions. Log2(1012) is indeed too few, but (a) huge rate of cell death, (b) multi-event nature of cancer development mean that at least a few hundred divisions precede clinical relevance. Fallacy #2: telomere shortening is dangerous because it causes genomic instability (which promotes cancer). It indeed promotes cancer initiation, but it totally prevents cancer progression once the telomeres are gone.

13. ALT: as clear-cut as telomerase? • The bad news: 1) it works by hijacking recombination and possibly constitutive DNA repair systems • 2) there may be many such systems that it can use • The good news: • 1) some DNA repair and recombination systems are dispensable (e.g., those involved in meiosis) • 2) all ALT cancers/lines studied thus far have many phenotypic characteristics in common

14. Engineering stem cell chemoresistance: current status Already being pursued as an anti-cancer strategy: - Alkylating agents: Fairbairn LJ (various), etc - Methotrexate: e.g. Patel et al., Blood 95:2356 - Cisplatin: e.g. Pradat et al., Human Gene Therapy 12:2237 - 5-FU: e.g. Yoshisue et al., Canc Chemo Pharm 46:51

17. Analysis (ii): is WILT too crazy to work?

18. Transplantation of engineered stem cells: current status Blood: bone marrow transplant is routine. Immune system: see next slide Gut: seems very easy by surgery in mice (Tait 1994); may be doable using endoscopy technology in humans. Lung: being actively explored as a cystic fibrosis therapy. Skin: the epidermis renews rapidly, but the (negligibly-dividing) dermis directs its behaviour. Burns research has exploited this (including in tissue engineering).

20. Immune senescence: WILT’s Achilles heel? Doubt #1: who needs memory cells anyway? Elderly people (with few/sluggish naïve cells)! Doubt #2: Maybe memory cells are sufficiently oligoclonal that we can use the same method to relengthen their telomeres in vitro? Doubt #3: Maybe CMV (etc)-induced senescence can also be avoided this way?

21. Mesenchymal cancers, gene targeting: current status Needed because we can’t dilute away the progenitor cells when they only divide very rarely (on demand) A promising approach:changing a gene by triggering the cells’ homologous recombination machinery A big plus, compared to viral (etc.) gene therapy, is that multiple hits to the same cell are harmless (if targeted!) Single-bp changes: many approaches (ssDNA, RNA/DNA hybrids, triplex-forming oligonucleotides) Big changes, e.g. deletions: target flanking sequences

23. Harmful effects of telomere shortening: current status In mice: none at all, unless engineered to have telomeres at birth much shorter than they normally are at death In humans: dyskeratosis congenita (DC) -- age of onset 7-8 years on average (big variance). Symptoms: as you might guess (bone marrow failure, skin disorders, malignancy.Mostly caused by mutations in TERC or dyskerin (a key telomere-maintenance protein) Stem cell therapy (bone marrow transplantation) has long been used against DC -- despite immune problems, which would not be relevant for WILT

25. Promoting stem cell longevity: current status Key idea: inhibited stem cell differentiation  increased stem cell number  slower necessary stem cell division rate  extended time before stem cell telomeres run out Key regulatory genes are being discovered: Blood: MIP-1 (Graham GJ, others) Skin: 14-3-3 (Dellambra et al., J Cell Biol 149:1117) What about the gut?????

26. The gut paradox Literature consensus: human blood and epidermal stem cells divide only every few months, but gut stem cells divide once a week. Thus, other tissues might survive a decade without telomerase but surely the gut would not. However.... if so, why don’t DC sufferers or TERT-/- mice get gut problems far sooner than anything else? A curiosity: crypts are usually monoclonal. Why?

27. A possible explanation: a third form of stem cell population dynamics Option 1: all stem cells divide all the time (but slowly) Option 2: “clonal selection”: one stem cell does all the work until it fails, then another takes over. Much data contradicts this Option 3: most stem cells divide all the time, but a few “ultra-stems” divide only when the “stemness” of their neighbours falls (e.g. a stem neighbour dies), and then usually produce an ultra-stem and a normal stem cell

28. stem (50%) progenitor (50%) few slow stem stem (rarely) progenitor (rarely) committed (usually) cell abun- dance cell div. rate progenitor committed fast differentiated (all) many nil differentiated

29. ultrastem (50%) stem (50%) very very few slow ultrastem stem (50%) progenitor (50%) few slow stem stem (rarely) progenitor (rarely) committed (usually) cell div. rate abun- dance progenitor committed fast differentiated (all) many nil differentiated

30. Conclusion: crazy or not? • Is it crazy enough? • Probably; the only big question is the genetics of ALT • Is it too crazy? • Maybe not: many daunting problems to solve, but all are already at a promising stage • Is it worth pursuing now? • The more successful the other work discussed at this meeting is, the more people will die of cancer in the future if cancer therapy doesn’t keep up. You decide....