W I L T

1
W I L T an ambitious but truly un-escapable
anti-cancer therapy (which, conveniently,
involves stem cells) Aubrey D.N.J. de
Grey Department of Genetics, University of
Cambridge
2

• Structure of this talk
• 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
Context the foreseeable control of everything
else age-related - All non-neoplastic aspects of
aging may be curable in mice within a decade.
(Details on request...) - Translation to humans
might take only another decade or two if were
lucky - Doing this but not curing cancer will
give us only 20 years more life, and well all
die horribly This is an unsatisfactory scenario
4

• Cancer the hardest aspect of aging to combat
• 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 -(
• Must we stick with the cocktail approach, or is
  there a clean solution?
• Context it is OK for such a solution to be very
  ambitious, not worth doing until we cure aging

5
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 952356 - Cisplatin e.g. Pradat et al.,
Human Gene Therapy 122237 - 5-FU e.g. Yoshisue
et al., Canc Chemo Pharm 4651
6
(No Transcript)
7
(No Transcript)
8

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

9
Better acronym, anyone? Whole-body
Interdiction of Lengthening of Telomeres
10

• The proposed therapy, in a nutshell
• Engineer (in vitro) a patients 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 quiescent
  cells

11
The four options
1) Without any cancer treatment
2) With current or foreseeable cancer treatment
12
3) With uncompensated telomere maintenance
deficiency
4) With compensated telomere maintenance
deficiency (WILT)
13

• Crazy enough? Too crazy?
• SENS III, December 2nd 2002, Cambridge, UK
• de Grey et al. (2004) Annals NY Acad Sci 1019, in
  press
• 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

14
Analysis (i) is WILT crazy enough to work?
15
Cancer protection by lack of telomerase/ALT
current status

• Fallacy 1 there arent 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.

16
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
• 3) ALT research is very fashionable well know
  soon

17
Analysis (ii) is WILT too crazy to work?
18

• Transplantation of engineered stem cells current
  status
• Blood bone marrow transplant is routine.
• Gut seems easy by surgery in mice (Tait 1994)
  should 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).

19
(No Transcript)
20

• Mesenchymal cancers, gene targeting current
  status
• Needed because we cant 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
• Single-bp changes many techniques (ssDNA,
  RNA/DNA hybrids, triplex-forming
  oligonucleotides)
• Big changes, e.g. deletions target flanking
  sequences

21
(No Transcript)
22
Major limitation of in vivo gene targeting
random integration is very common Potential
solution phage integrases (see esp. work of
Michele Calos, Stanford)
23
Circular dsDNA random integration v.
rare attP ? attB control of integration vs
excision
24

• And better yet
• specificity is just weak enough that there are a
  few natural sites in mammalian genomes introns
  preferred (open chromatin)
• target site can be varied by in vitro evolution,
  and maybe soon by design

25
Could WILT stem cells last 10 years? Literature
consensus in humans, bone marrow 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. But.... if so, why
dont DC sufferers or TERT-/- mice get gut
problems far sooner than anything else?
26

• 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

27
stem (50) progenitor (50)
stem
slow
few
stem (rarely) progenitor (rarely) committed
(usually)
cell div. rate
cell abun- dance
progenitor
committed
fast
differentiated (all)
many
nil
differentiated
28
ultrastem (50) stem (50)
very very few slow
ultrastem
stem (50) progenitor (50)
stem
slow
few
stem (rarely) progenitor (rarely) committed
(usually)
cell div. rate
cell abun- dance
progenitor
committed
fast
differentiated (all)
many
nil
differentiated
29
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 other work on
age-related decline is, the more people will die
of cancer in the future if cancer therapy doesnt
keep up. You decide....
30
(No Transcript)
31

• 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
  rejection problems, which would not be relevant
  for WILT

32
(No Transcript)
33

• 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
  1491117) What about the gut?????