Inhibition of MLL-Menin interaction by small peptide molecules: a new investigation way and a possible therapeutic tool

 
	
   10	
  
	
  
1.1.HEMATOPOIESIS	
  
Hematopoiesis	
  is	
   an	
  highly	
  orchestrated	
   process	
  by	
  which	
  immature	
  precursor	
  cells 	
  
develop	
  into	
  new	
  mature	
   blood	
  cells,	
  which	
  includes	
  red	
  blood	
  cells	
  ( erythrocytes),	
  
white	
  blood	
  cells	
  ( leukocytes),	
  and	
  platelets	
  (Orkin	
  et	
  al.	
  1995,	
  1996).	
  It	
  begins	
  early	
  in 	
  
embryonic	
  development	
  and	
  continues	
  throughout	
  the	
  lifetime	
  of	
  an	
  organism. 	
  
In	
  developing	
  embryos,	
  blood 	
   formation	
  occurs	
  in	
  aggregates	
  of	
  blood	
  cells	
  in	
  the	
  yolk 	
  
sac.	
  When	
  bone	
  marrow	
  develops,	
  it,	
  with	
  its	
  intersinusoidal	
  spaces,	
  assumes	
  the	
  task	
  of 	
  
forming	
  most	
  of	
  the	
  blood	
  cells	
  for	
  the	
  entire	
  organism.	
  However,	
  maturation, 	
  
activation,	
  and	
  some	
  prolifera tion	
  of	
  lymphoid	
  cells	
  occurs	
  in	
  secondary	
  lymphoid 	
  
organs	
  (spleen,	
  thymus,	
  and	
  lymph	
  nodes).	
  In	
  children,	
  haematopoiesis	
  occurs	
  in	
  the 	
  
marrow	
  of	
  the	
  long	
  bones,	
  in	
  adults,	
  it	
  occurs	
  mainly	
  in	
  the	
  pelvis,	
  cranium,	
  vertebrae, 	
  
and	
  sternum. 	
  
The	
  sinusoids	
   (venous	
  channels)	
  feed	
  into	
  the	
  marrow	
  venous	
  drainage	
  system,	
  they	
  are 	
  
lined	
  with	
  specialized	
  fenestrated	
  endothelial	
  cells.	
  These	
  cells	
  produce	
  growth	
  factors 	
  
and	
  cytokines,	
  which	
  influence	
  proliferation	
  and	
  differentiation	
  of	
  hematopoietic	
  cells 	
  
and	
  thu s	
  play	
  an	
  important	
  regulatory	
  role.	
  Mature	
  blood	
  cells	
  enter	
  the	
  blood	
  stream	
  
by	
   passing	
  through	
  the	
  sinusoidal	
  wall	
  to	
  get	
  into	
  the	
  sinuses.	
  The	
  bone	
  marrow	
  stroma 	
  
contains	
  many	
  different	
  cell	
  types,	
  including	
  macrophages,	
  fibroblasts,	
  endothelial	
  cells, 	
  
smooth	
  muscle	
  cells,	
  T -­‐lymphocytes,	
  monocytes.	
  These	
  cells,	
  in	
  combination	
  with 	
  
components	
  of	
  the	
  extracellular	
  matrix	
  and	
  basement	
  membranes	
  as	
  well	
  as	
  a	
  plethora 	
  
of	
  soluble	
  and	
  membrane -­‐bound	
  cytokines	
  and	
  growth	
  factor,	
  form	
  the	
  so -­‐called	
  
Hematopoietic	
  inductive	
  microenvironment	
   (HIM),	
  which	
  maintains	
  the	
  functional 	
  
integrity	
  of	
  this	
  complex	
  system	
  of	
  resident	
  and	
  circulating	
  cells.	
  (Trentin,	
  JJ.	
  1971) 	
  
All	
  different	
  types	
  of	
  blood	
  cells	
  are	
  derived	
  from	
  a	
  small	
  common	
  pool	
  of	
  totipotent 	
  
cells,	
  called	
  hematopoietic	
  stem	
  cells	
  (HSCs).	
  These	
  cells	
  have	
  the	
  unique	
  properties	
  to 	
  
give	
  rise	
  to	
  new	
  hematopoietic	
  stem	
  cells	
  (self -­‐renewal)	
  and	
  generate	
  primitive 	
  
progenitors	
  that	
  are	
  programmed	
  to	
  differentiate.	
  This	
  process	
  is	
  called	
   Steady-­‐ state	
  
hematopoiesis	
   or	
  Constitutive	
  hematopoiesis .	
  
During	
  or	
  after	
  cell	
  division	
  the	
  two	
  daughter	
  cells	
  of	
  a	
  HSC	
  have	
  to	
  decide	
  their	
  fate. 	
  
 
	
   11	
  
They	
  can	
  either	
  choose	
  the	
  same	
  (symmetric	
  division)	
  or	
  different	
  (asymmetric 	
  
division)	
  fate.	
  Thus,	
  they	
  can	
  either	
  choose	
  to	
   remain	
  as	
  HSCs,	
  commit	
  to 	
  
differentiation,	
  to	
  die	
  by	
  apoptosis	
  and	
  also	
  to	
  stay	
  in	
  the	
  bone	
  marrow	
  or	
  migrate	
  to 	
  
periphery.	
  (Domen,J	
  and	
  Weissman,	
  IL.,	
  1999).	
  The	
  HSC	
  cell -­‐fate	
  decision	
   involves	
  a 	
  
complex	
  interplay	
  between	
  intrinsic	
  genetic	
  processes	
  of	
  bl ood	
  cells	
  and	
  their 	
  
environment.	
   (Brown	
  G.,et	
  al,	
  1985;	
  Orkin	
  SH.,	
  1995;	
  Singh,	
  H.,	
  1996) 	
  
When	
  the	
  totipotent	
  hematopoietic	
  stem	
  cells	
  choose	
  to	
  divide	
  through	
  asymmetric	
  cell 	
  
division,	
  advance	
  in	
  their	
  differentiative	
  program	
  gradually	
  producing	
  specific	
   receptors	
  
and/or	
  molecular	
  markers,	
  which	
  reflect	
  both	
  the	
  degree	
  of	
  differentiation	
  achieved	
  and 	
  
their	
  ability	
  to	
  recognize	
  certain	
  differentiation	
  factors	
  (cytokines).In	
  this	
  way,	
  gradually 	
  
are	
  characterized	
  different	
  progenitor	
  cells	
  that	
  are	
  still	
  imma ture	
  but	
  "committed", 	
  
directed	
  towards	
  one	
  or	
  other	
  of	
  mature	
  cell	
  lines.	
  By	
  defining	
  their	
  differentiation 	
  
programs,	
  progenitor	
  cells	
  lost	
  their	
  ability	
  to	
  self -­‐renewal	
  and	
  moving	
  toward	
  the 	
  
desired	
  phenotype	
  to	
  become,	
  at	
  the	
  same	
  time,	
  more	
  mitotically	
   active.	
  
HSCs	
  can	
  be	
  divided	
  into	
  a	
  long -­‐term	
  subset	
  (LT -­‐HSC),	
  capable	
  of	
  indefinite 	
  
selfrenewal,	
  and	
  a	
  short -­‐term	
  subset	
  (ST -­‐HSC)	
  that	
  self -­‐renews	
  for	
  a	
  defined	
  interval. 	
  
LT-­‐HSCs	
  divide	
  into	
  ST -­‐HSCs	
  that	
  give	
  rise	
  to	
  the	
  briefly	
  self -­‐renewing	
  multipotent 	
  
progenitors	
  (MPPs)	
  which	
  then	
  differentiate	
  into	
  oligolineage -­‐restricted	
  progenitors 	
  
through	
  functionally	
  irreversibile	
  maturation	
  steps.(Fig.1)	
  (Morrison,	
  SJ.	
  et	
  al.,	
  1997) 	
  
Two	
  kinds	
  of	
  oligolineage -­‐restricted	
  progenitors	
  have	
  been	
  identified:	
  the	
  common 	
  
lymphoid	
  progenitors	
  (CLPs),	
  which	
  at	
  clonal	
  level	
  are	
  restricted	
  to	
  give	
  rise	
  to	
  T 	
  
lymphocites,	
  B	
  lymphocites	
  and	
  natural	
  killer	
  cells	
  (Kondo,	
  M.	
  et	
  al.,1997),	
  and	
  the 	
  
common	
  myeloid	
  progenitors	
  (CMPs)	
  which	
  are	
  progenitors	
  for	
  the	
  myelo -­‐erythroid	
  
lineages	
   (Akashi,	
  K.	
  et	
  al.,	
  2000).	
  CMPs	
  give	
  rise	
  to	
  myelomonocytic	
  progenitors 	
  
(GMPs),	
  which	
  in	
  turn	
  produce	
  monocyte/macrophages	
  and	
  granulocytes,	
  and	
  to 	
  
megacaryotic/erithroid	
  progenitors,	
  which	
  differentiate	
  into	
  megakaryocytes/platelets 	
  
and	
  erythrocyte. 	
  
 
	
   12	
  
	
  
Fig.1:	
   Hematopoietic	
  stem	
  cell	
  differentiation	
  in	
  the	
  bone	
  marrow	
  microenviroment 	
  
	
  
Lymphopoiesis	
  and	
  myelopoiesis	
  differentiation	
  are	
  stepwise	
  processes	
  characterized	
  by 	
  
the	
   alternate	
   expression	
   of	
   growth	
   factors,	
   their	
   receptors	
   and	
   transcriptional	
  
regulators.	
  
Transcription	
  factors	
  present	
  in	
  a	
  particular	
  cell	
  are	
  characteristic	
  of	
  the	
  hematopoietic 	
  
line	
  to	
  which	
  it	
  belongs	
  and	
  its	
  stage	
  of	
  development	
  and	
  maturation.	
  So	
  the	
  complex 	
  
interaction	
  between	
  transcription	
  factors,	
  co -­‐regulatory	
  molecules	
  a nd	
  specific 	
  
sequences	
  of	
  DNA	
  binding,	
  determine	
  the	
  line -­‐specific	
  gene	
  expression	
  and	
  subsequent 	
  
cell	
  differentiation.	
  The	
  self -­‐renewal	
  of	
  the	
  stem	
  cell	
  population	
  in	
  the	
  bone	
  marrow,	
  
the	
  
proliferation	
  and	
  differentiation	
  of	
  hematopoietic	
  progenitor	
  cells, 	
   their	
  survival,	
  and 	
  
also	
  all	
  functional	
  activities	
  of	
  the	
  circulating	
  mature	
  forms	
  are	
  subject	
  to	
  regulation	
  by 	
  
a	
  cascade	
  of	
  proteins	
  that	
  are	
  generally	
  known	
  as	
  growth	
  factors	
  or	
  cytokines.	
  These 	
  
factors	
  are	
  products	
  of	
  stromal	
  cells	
  and	
  other	
  cells	
  and	
   they	
   are	
  produced	
  through	
  both 	
  
autocrine	
  and	
  paracrine	
  mechanisms .	
   Cytokines	
  mediate	
  positive	
  and	
  negative	
  effects 	
  
on	
  multiple	
  cellular	
  functions	
  by	
  engaging	
  a	
  specific	
  receptor	
  and	
  activating	
  a	
  variety	
  of 	
  
signaling	
  pathways.	
  (Ogawa.,	
  M.,	
  1993,	
   Zhu	
  J,	
  Emer son	
  SG.,	
  2002 )	
  
They	
  are	
  many	
  and	
  of	
  different	
  origin: 	
  
·∙	
  Stem	
  cell	
  factor	
   (SCF):	
  It	
  binds	
  to	
  the	
  c -­‐Kit	
  receptor	
  and	
  plays	
  a	
  role	
  in	
  the 	
  
 
	
   13	
  
regulation	
  of	
  HSCs	
  in	
  the	
  bone	
  marrow	
  stem	
  cell	
  niche	
  increasing	
  the	
  adhesion 	
  
capacity	
  of	
  HSCs	
  to	
  ECM	
  proteins	
  and	
   stromal	
  cells.	
  (Broudy	
  VC.,	
  1997) 	
  
·∙	
  FLT3	
  ligand	
  ( FLT3-­‐L)	
   of	
  the	
  Flt3	
  receptor.	
  This	
  is	
  a	
  tyrosine	
  kinase	
  receptor	
  that 	
  
was	
  at	
  first	
  noted	
  on	
  stem	
  cells	
  and	
  committed	
  lymphoid	
  precursors.(Matthews	
  W.	
  et 	
  
al.,	
  1991)	
  Its	
  ligand	
  (FL)	
  was	
  shown	
  to	
  be	
  an	
  active	
  p roliferative	
  stimulus	
  for	
  stem 	
  
and	
  developing	
  dendritic	
  cells,	
  particularly	
  when	
  acting	
  in	
  synergy	
  with	
  other 	
  
growth	
  factors.	
  (Vigon	
  I.	
  et	
  al.,	
  1992) 	
  
·∙	
  Multylineage	
  colony	
  stimulating	
  factor	
   (multi-­‐CSF	
  o	
  IL -­‐3):	
  It	
  stimulates	
  the 	
  
differentiation	
  of	
  multipot ent	
  hematopoietic	
  stem	
  cells	
  into	
  myeloid	
  progenitor	
  cells. 	
  
(Metcalf,	
  D.	
  2008) 	
  
·∙	
  Macrophage	
  colony	
  stimulating	
  factor	
   (M-­‐CSF):	
  It’s	
  involved	
  in	
  the	
  proliferation, 	
  
differentiation,	
  and	
  surival	
  of	
  monocytes,	
  macrophages,	
  and	
  bone	
  marrow	
  progenitor 	
  
cells.	
  (St anley	
  ER.,	
  1975) 	
  
·∙	
  Granulocyte-­‐ macrophage	
  colony	
  stimulating	
  factor	
   (GM-­‐CSF):	
  stimulates	
  stem 	
  
cells	
  to	
  produce	
  granulocytes	
  (neutrophils,	
  eosinophils,	
  and	
  basophils)	
  and 	
  
monocytes	
  (Metcalf	
  D.,	
  2008) 	
  
·∙	
  Granulocyte	
  colony	
  stimulating	
  factor	
   (G-­‐CSF):	
  It	
  initi ates	
  proliferation	
  and 	
  
differentiation	
  into	
  mature	
  granulocytes.	
  (Metcalf	
  D.	
  and	
  Nicola	
  NA.,	
  1983) 	
  
·∙	
  EPO:	
  it	
  is	
  the	
  humoral	
  regulator	
  of	
  red	
  cell	
  formation 	
  
·∙	
  IL5:	
   it	
  is	
  the	
  major	
  regulator	
  of	
  eosinophil	
  production 	
  
·∙	
  TPO:	
  regulates	
  platelet	
  production.(Lok, 	
   S.	
  et	
  al.,	
  1994) 	
  
The	
  most	
  important	
  feature	
  of	
  cytokines	
  was	
  their	
  polyfunctionality.They	
  were	
  not 	
  
simply	
  proliferative	
  stimuli	
  but	
  also	
  had	
  actions	
  affecting	
  survival,	
  differentiation 	
  
commitment,	
  induction	
  of	
  maturation,	
  and	
  functional	
  activation	
  of	
  matu re	
  cells,	
  they 	
  
may	
  also	
  facilitate	
  the	
  interactions	
  between	
  stem	
  cells	
  and	
  elements	
  in	
  the 	
  
microenvironment	
  including	
  extracellular	
  matrix	
  (ECM)	
  components.(	
  Kinashi	
  T,	
  and 	
  
Springer	
  TA.,	
  1994)	
  Newly	
  discovered	
  cytokines	
  including	
  Wnt	
  and	
  the	
  notch	
  ligand 	
  
family	
  may	
  also	
  have	
  important	
  effects	
  on	
  stem	
  cell	
  biology.(	
  Milner	
  LA	
  and	
  Bigas	
  A. 	
  
1999;	
  Van	
  Den	
  Berg	
  DJ.	
  et	
  al	
  1998 )	
  
Chemokines	
  are	
  another	
  class	
  of	
  compounds	
  that	
  in	
  hematopoiesis	
  function	
  as	
  positive 	
  
and	
  negative	
  regulators	
  of	
  proliferation,	
  cell	
  traf ficking	
  and	
  homing	
  (	
  Christopherson	
  K 	
  
2nd,	
  Hromas	
  R.	
  2001).	
  Other	
  important	
  environmental	
  regulators	
  of	
  hematopoiesis 	
  
 
	
   14	
  
include	
  the	
  ECM	
  components,	
  hematopoietic	
  and	
  nonhematopoietic	
  cells,	
  nutrients	
  and 	
  
vitamins,	
  and	
  a	
  variety	
  of	
  physiologic	
  processes.	
  ECM	
   components	
  provide	
  a	
  scaffold 	
  
for	
   colocalizing	
   progenitors	
   and	
   HSCs	
   with	
   a	
   wide	
   array	
   of	
   positive	
   and	
   negative	
  
cytokines	
  and	
  other	
  growth	
  regulators.	
  In	
  addition,	
  ECM	
  and	
  stromal	
  components	
  may 	
  
directly	
  mediate	
  signaling	
  to	
  HSCs	
  to	
  activate	
  growth,	
  protect 	
   cells	
  from	
  apoptosis,	
  or 	
  
modulate	
  responses	
  to	
  positive	
  and	
  negative	
  regulatory	
  factors.	
  HSCs	
  and	
  progenitors 	
  
binding	
  to	
  these	
  ECM	
  components	
  is	
  mediated	
  by	
  adhesion	
  molecules,	
  including 	
  
integrins,	
   selectins,	
   and	
   mucins.	
   Hematopoietic	
   and	
   nonhematopoietic 	
  cells	
   that	
  
regulate	
  hematopoiesis	
   are	
   NK	
   cells,T	
   cells,	
   macrophages,	
   fibroblasts,	
   osteoblasts.	
  
(Taichman	
   RS.	
  et	
  al.,	
  2000)	
  These	
  cells	
  may	
  produce	
  important	
  growth	
  factors,	
  facilitate 	
  
engraftment,	
  or	
  induce	
  apoptosis. 	
  
In	
  addition	
  to	
  this	
  wide	
  array	
  of	
  env ironmental	
  factors	
  that	
  regulate	
  hematopoiesis,	
  a 	
  
number	
  of	
  intrinsic	
  genetic	
  events	
  are	
  critical	
  to	
  determine	
  cell	
  fate.	
  The	
  Rb	
  family, 	
  
cyclins,	
  Hox,	
  and	
  other	
  gene	
  families	
  appear	
  to	
  regulate	
  proliferation	
  and	
  self -­‐renewal	
  
of	
  early	
  hematopoietic	
  cells.	
  T he	
  bcl	
  family	
  and	
  Fas	
  receptor	
  with	
  its	
  ligand,	
  caspases, 	
  
regulate	
  apoptosis	
  in	
  hematopoietic	
  cells.	
   Among	
  progenitors,	
  lineage	
  commitment	
  is 	
  
accompanied	
  by	
  loss	
  of	
  expression	
  of	
  genes	
  associated	
  with	
  unrelated	
  lineages	
  (i.g.	
  Hox 	
  
genes).(	
  Park,	
  IK.	
  Et	
  al.	
   2002)	
  This	
  promiscuous	
  gene	
  expression	
  by	
  HSC	
  may	
  provide 	
  
a	
  framework	
  for	
  stochastic	
  fluctuations	
  in	
  expression	
  of	
  signaling	
  or	
  transcriptional 	
  
complexes,	
  which	
  ultimately	
  are	
  amplified	
  or	
  repressed	
  to	
  cement	
  lineage	
  choice.	
  These 	
  
findings	
  support	
  the	
  so	
  c alled	
  stochastic	
  model	
  according	
  to	
  which	
  HSC	
  randomly 	
  
commit	
  to	
  either	
  self -­‐renew	
  or	
  differentiate.	
  Cytokines	
  present	
  in	
  the	
  bone	
  marrow 	
  
milieu	
  do	
  not	
  direct	
  this	
  choice	
  per	
  se,	
  but	
  do	
  allow	
  survival	
  and	
  proliferation	
  of	
  the 	
  
cells	
  that	
  ultimately	
  develop	
   into	
  mature	
  lineages.(Wagers,	
  AJ.	
  et	
  al.,	
  2002)	
  Earlier,	
  the 	
  
instructive	
  model	
  suggested,	
  on	
  the	
  contrary,	
  that	
  differentiation	
  of	
  cells	
  into	
  one	
  of 	
  
several	
  lineages	
  critically	
  depends	
  on	
  the	
  nature	
  of	
  factors	
  acting	
  on	
  these	
  cells	
  at	
  a 	
  
particular	
  time,	
  at 	
   a	
  particular	
  concentration,	
  and/or	
  in	
  a	
  particular	
  sequence.	
  (Trentin 	
  
JJ.,	
  1971) 	
  
Finally	
  gene -­‐regulatory	
  microRNAs	
  can	
  modulate	
  hematopoietic	
  cell	
  differentiation	
  and 	
  
proliferation	
  and	
  also	
  the	
  activity	
  of	
  hematopoietic	
  cells,	
  in	
  particular	
  those	
  related	
   to	
  
immune	
  function.	
  (Garzon	
  R	
  and	
  Croce,CM.,	
  2008). 	
  
Alterations	
  in	
  the	
  balance	
  between	
  self -­‐renewal	
  and	
  differentiation	
  can	
  lead	
  to	
  the 	
  
 
	
   15	
  
emergence	
  of	
  cells	
  that	
  survive	
  and	
  grow	
  in	
  situations	
  unfavorable	
  for	
  the	
  growth	
  of 	
  
normal	
  cells	
  and	
  hence	
  to	
  the	
  estab lishment	
  of	
  leukemias. 	
  
 
	
   16	
  
1.2.LEUKEMIA	
  
Leukemia	
  is	
  a	
  cancer	
  of	
  blood -­‐forming	
  cells	
  in	
  the	
  bone	
  marrow. 	
  
Cytogenetic	
  analysis	
  of	
  hematologic	
  malignancies	
  reveals	
  a	
  sole	
  abnormality,	
  such	
  as	
  a 	
  
single	
  balanced	
  chromosomal	
  translocation.	
  A	
  chromosome	
  translocatio n	
  is	
  caused	
  by 	
  
rearrangement	
  of	
  parts	
  between	
  nonhomologous	
  chromosomes.	
  Translocations	
  can	
  be 	
  
balanced	
   (Fig.2)	
  or	
   unbalanced,	
  in	
  this	
  case	
  the	
  exchange	
  of	
  chromosome	
  material	
  is 	
  
unequal	
  resulting	
  in	
  extra	
  or	
  missing	
  genes. 2	
  
	
  
Fig.	
  2 :	
  Scheme	
  of	
  balanced	
   chromosomal	
  translocation:	
  pieces	
  of	
  chromosomes	
  are 	
  rearranged	
  
but	
  no	
  genetic	
  material	
  is	
  gained	
  or	
  lost	
  in	
  the	
  cell. 	
  
	
  
Mechanistically	
  chromosomal	
  translocations	
  require	
  the	
  presence	
  of	
  DSBs	
  in	
  DNA	
  at 	
  
two	
  locations.	
  These	
  breaks	
  induce	
  cells	
  to	
  arrest	
  in	
   mitosis	
  or	
  to	
  undergo	
  apoptosis, 	
  
then	
  the	
  appearance	
  of	
  DSBs	
  activates	
  the	
  cellular	
  DNA	
  repair	
  machinery	
  that	
  catalyzes 	
  
the	
  joining	
  of	
  broken	
  chromosome	
  ends	
  (Lieber	
  MR.	
  et	
  al .,	
   2003).	
  A	
  variety	
  of 	
  
rearrangements	
  can	
  result	
  from	
  this	
  joining.	
  For	
  instance, 	
   precise	
  joining	
  of	
  broken	
  ends 	
  
can	
  regenerate	
  a	
  normal	
  chromosome.	
  Deletions,	
  duplications,	
  and	
  inversions	
  can	
  occur 	
  
when	
  joining	
  involves	
  two	
  broken	
  ends	
  on	
  the	
  same	
  chromosome.	
  Furthermore, 	
  
translocations	
  may	
  occur	
  when	
  the	
  broken	
  ends	
  of	
  two	
  nonhomolog ous	
  chromosomes 	
  
are	
  joined	
  together.	
  In	
  this	
  case	
  the	
  chromosomal	
  translocation	
  breaks	
  within	
  the	
  exons 	
  
of	
  the	
  two	
  chromosomes,	
  allowing	
  the	
  transcription	
  product	
  to	
  encompass	
  the	
  linked 	
  
exons,	
  creating	
  a	
  tumor -­‐specific	
  fusion	
  mRNA	
  and	
  in	
  turn	
  fusion	
  prote in.	
  
 
	
   17	
  
This	
  type	
  of	
  event	
  is	
  common	
  in	
  leukemias	
  (Rabbitts	
  TH.,1994;	
  Look	
  AT.,1997). 	
  
The	
  product	
  of	
  this	
  rearrangement	
  is	
  a	
  chimeric	
  gene	
  which	
  alters	
  the	
  normal	
  processes 	
  
of	
  growth,	
  differentiation	
  and	
  survival	
  of	
  haematopoietic	
  cells. 	
  
At	
  present	
  leukemia	
  is 	
   viewed	
  as	
  a	
  newly	
  formed	
  abnormal	
  hematopoietic	
  tissue 	
  
initiated	
  by	
  few	
  leukemic	
  stem	
  cells	
  (LSCs)	
  that	
  undergo	
  an	
  aberrant	
  and	
  poorly 	
  
regulated	
  proliferation.	
  LSCs	
  can	
  either	
  be	
  HSCs	
  ,	
  which	
  have	
  become	
  leukemic	
  as	
  the 	
  
result	
  of	
  accumulated	
  mutation,	
  or	
   more	
  committed	
  progenitors	
  or	
  even	
  differentiated 	
  
mature	
  cells	
  which	
  reacquire	
  the	
  stem	
  cell	
  capability	
  of	
  self -­‐renewal.	
   (Fig.3)	
  (Passegue 	
  
E.	
  et	
  al	
  2003) 	
   	
  
	
  
Fig.3.	
  Origin	
  of	
  the	
  LSC.	
  A	
  given	
  leukemia	
  can	
  be	
  viewed	
  as	
  a	
  newly	
  formed 	
  
abnormal	
   hematopoietic	
  tissue	
  initiated	
  by	
  a	
  few	
  LSCs	
  that	
  undergo	
  an	
  aberrant 	
  
and	
  poorly	
  regulated	
  process	
  of	
  organogenesis	
  analogous	
  to	
  that	
  of	
  normal 	
  
HSCs.	
  LSCs	
  can	
  either	
  be	
  HSCs,	
  which	
  have	
  become	
  leukemic	
  as	
  the	
  result	
  of 	
  
accumulated	
  mutations,	
  or	
  more	
   restricted	
  progenitors,	
  which	
  have	
  reacquired 	
  
the	
  stem	
  cell	
  capability	
  of	
  self	
  renewal .	
  
	
  
In	
  all	
  cases	
  the	
  development	
  of	
  a	
  leukemia	
  is	
  a	
  stepwise	
  process	
  in	
  which	
  genetic 	
  
rearrangements	
  and	
  leukemic -­‐associated	
  fusion	
  genes	
  have	
  a	
  critical	
  function	
  by 	
  
interfering	
  with	
  the	
  hematopoietic	
  differentiation	
  programs,	
  and	
  thereby	
  dictating	
  the 	
  
nature	
  of	
  the	
  leukemia.	
  However	
  they	
  require	
  additional	
  cooperative	
  mutations	
  to 	
  
induce	
  fully	
  malignant	
  diseases.	
  Generally,	
  fusion	
  proteins	
  that	
  give	
  rise	
  from 	
  
chromosomal	
   t ranslocations	
   function	
   as	
   transcriptional	
   regulators,	
   which	
   directly 	
  
 
	
   18	
  
interfere	
  with	
  the	
  hematopoietic	
  differentiation	
  programs	
  (Zhu	
  J	
  and	
  Emerson	
  SG 	
  
2002).	
  These	
  interferences	
  occur	
  through	
  common	
  mechanism	
  including	
  recruitment	
  of 	
  
aberrant	
  corepressor	
  com plex,	
  epigenetic	
  modification,	
  chromatin	
  remodeling	
  and 	
  
disruption	
  of	
  subnuclear	
  compartments	
  (	
  Tenen	
  DG.,	
  2003) 	
  
Thus,	
  starting	
  from	
  a	
  single	
  cell,	
  HSC	
  or	
  a	
  more	
  differentiated	
  cell,	
  a	
  series	
  of	
  DNA 	
  
demages,	
  genetic	
  and	
  epigenetic	
  changes	
  promote	
  the	
  forma tion	
  of	
  a	
  neoplastic	
  clone, 	
  
that	
  have,	
  against	
  the	
  normal	
  population,	
  a	
  proliferative	
  advantage	
  for	
  which	
  they 	
  
gradually	
  accumulate	
  in	
  the	
  bone	
  marrow.	
  These	
  deranged,	
  immature	
  cells	
  are	
  not	
  able 	
  
to	
  carry	
  out	
  the	
  normal	
  functions	
  of	
  blood	
  cells. 	
  
Either	
   lymphoid	
  and	
  myeloid	
  progenitors	
  can	
  reacquire	
  the	
  ability	
  to	
  self -­‐renewal	
  and 	
  
then	
  accumulate	
  secondary	
  mutation	
  leading	
  to	
  leukemic	
  transformation.	
  Leukemias 	
  
that	
  affect	
  the	
  myeloid	
  lineage	
  are	
  called	
  myelocytic	
  (also	
  myelogenous,	
  myeloblastic, 	
  
or	
  nonlym phocytic)	
  leukemias,	
  when	
  they	
  affect	
  the	
  lymphoid	
  lineage	
  are	
  called 	
  
lymphocytic	
  (also	
  lymphoblastic	
  or	
  lymphogenous)	
  one. 	
  
Each	
  of	
  them	
  include	
  both	
  acute	
  and	
  chronic	
  forms.	
  Acute	
  leukemias	
  are	
  tipically 	
  
associated	
  with	
  functionally	
  cooperating	
  genetic	
  mu tations	
  of	
  the	
  transcriptional 	
  
machinery	
  that	
  lead	
  to	
  a	
  differentiation	
  block	
  and	
  a	
  subsequent	
  expansion	
  of	
  immature 	
  
progenitors	
  unable	
  to	
  generate	
  mature	
  effectors	
  cells.	
  Thus,	
  in	
  the	
  acute	
  myelocytic 	
  
leukemias,	
  the	
  abnormal	
  cells	
  grow	
  rapidly	
  and	
  do	
  not	
   mature.	
  Most	
  of	
  these	
  immature 	
  
cells	
  tend	
  to	
  die	
  rapidly.	
  In	
  the	
  acute	
  lymphocytic	
  leukemias,	
  growth	
  is	
  slower	
  and	
  cells 	
  
tend	
  to	
  accumulate.	
  Untreated,	
  death	
  occurs	
  within	
  weeks	
  or	
  a	
  few	
  months.	
  In	
  the 	
  
chronic	
  leukemias	
  the	
  onset	
  tends	
  to	
  be	
  slow.	
   Tipically,	
  they	
  are	
  associated	
  with 	
  
mutations	
   that	
   constitutively	
   activate	
   either	
   growth	
   factor	
   receptors	
   or	
   their	
  
downstream	
  
signaling	
  components	
  leading	
  to	
  uncontrolled	
  proliferation.	
  In	
  chronic	
  leukemia	
  the 	
  
cells	
  generally	
  mature	
  abnormally	
  and	
  often	
  ac cumulate	
  in	
  various	
  organs,	
  often	
  over 	
  
long	
  intervals.	
  Their	
  ability	
  to	
  fight	
  infections	
  and	
  assist	
  in	
  repairing	
  injured	
  tissues	
  is 	
  
impaired.	
  
In	
  summary,	
  the	
  4	
  main	
  types	
  of	
  leukemia	
  are: 	
  
Acute	
  lymphocytic	
  leukemia	
  (ALL) 	
   affects	
  both	
  children	
  and	
  adults	
  bu t	
  is	
  more 	
  
common	
  in	
  children.	
  It	
  is	
  a	
  cancer	
  that	
  originates	
  from	
  totipotent	
  stem	
  cells	
  or 	
  
progenitors	
  commissioned	
  in	
  the	
  sense	
  B	
  or	
  T	
  lymphocyte.	
  The	
  neoplastic 	
  
 
	
   19	
  
transformation	
  generates	
  a	
  progeny	
  of	
  undifferentiated	
  cells	
  (leukemic	
  blasts)	
  in	
  B 	
  
or	
  T	
  phen otype,	
  that	
  replace	
  the	
  bone	
  marrow	
  hematopoietic	
  tissue,	
  circulate	
  in	
  the 	
  
peripheral	
  blood	
  and	
  infiltrate	
  lymphoid	
  organs. 	
  
Chronic	
  lymphocytic	
  leukemia	
  (CLL) 	
   is	
  essentially	
  an	
  adult	
  disorder	
  and	
  is 	
  
characterized	
  by	
  an	
  excessive	
  proliferation,	
  with	
   consequent	
  accumulation	
  in	
  the 	
  
peripheral	
  blood,	
  of	
  small	
  mature	
  lymphocytes.	
  In	
  98%	
  of	
  cases	
  the	
  phenotype	
  of 	
  
the	
  leukemic	
  cell	
  is	
  B 	
  
Acute	
  myeloid	
  leukemia	
  (AML) 	
   is	
  the	
  most	
  common	
  form	
  of	
  leukemia	
  in	
  adults.	
  It 	
  
is	
  a	
  clonal	
  neoplasm	
  where	
  a	
  chimeric	
  trans cription	
  factor	
  represents	
  the	
  most 	
  
frequent	
  initiating	
  event	
  for	
  oncogenic	
  conversion	
  of	
  normal	
  cells	
  into	
  preLSCs	
  and 	
  
subsequently	
  LSCs	
  (Hong	
  D.	
  et	
  al	
  2008;	
  Yeung	
  J.	
  et	
  al	
  2010). 	
  
Current	
  evidence	
  shows	
  that	
  fusion	
  proteins	
  found	
  in	
  AML	
  induce	
  a	
  pre -­‐leukemic	
  state	
  
in	
  which	
  further	
  genetic	
  and	
  epigenetic	
  mutations	
  are	
  necessary	
  for	
  progression	
  to	
  
leukemia	
  (Yuan	
  et	
  al.	
  2001).	
  All	
  events	
  that	
  are	
  necessary	
  and	
  sufficient	
  for	
  
leukemogenesis	
  to	
  occur	
  are	
  still	
  not	
  clear,	
  and	
  this	
  seems	
  to	
  differ	
  depending	
  on	
  th e	
  
expressed	
  oncoprotein	
  (Huntly	
  et	
  al.	
  2004).	
  However,	
  for	
  all	
  cases	
  within	
  the	
  AML	
  cell	
  
population	
  that	
  carry	
  the	
  mutations	
  implicated	
  in	
  the	
  pathogenesis,	
  leukemogenesis	
  
appears	
  to	
  require	
  a	
  functional	
  heterogeneity.	
  Within	
  the	
  leukemia	
  clone,	
  there	
  is	
  
significant	
  cellular	
  morphologic,	
  phenotypic,	
  and	
  functional	
  heterogeneity	
  analogous	
  to	
  
the	
  hierarchical	
  organization	
  of	
  normal	
  hematopoiesis.	
  Specifically,	
  it	
  has	
  been	
  
suggested	
  that	
  there	
  is	
  a	
  subpopulation	
  of	
  cells	
  with	
  self -­‐renewal	
  capacity	
  that	
  are	
  abl e	
  
to	
  maintain	
  and	
  propagate	
  the	
  AML	
  phenotype,	
  namely,	
  the	
  leukemic	
  stem	
  cells	
  (LSCs)	
  
(Bonnet	
  et	
  al.	
  1997).	
  Conversely,	
  the	
  majority	
  of	
  cells	
  are	
  either	
  transitional	
  cells	
  with	
  
limited	
  proliferative	
  capacity	
  or	
  more	
  differentiated	
  end	
  cells	
  (Mackillop	
  et	
  a l.,	
  1983;	
  
Kummermehr,	
  2001).	
  Evidence	
  for	
  a	
  hierarchical	
  cellular	
  organization	
  of	
  human	
  AML	
  
derives	
  from	
  studies	
  showing	
  that	
  only	
  a	
  small	
  proportion	
  of	
  AML	
  cells	
  are	
  clonogenic	
  in	
  
vitro	
  culture	
  (Buick	
  et	
  al.	
  1977),	
  and	
  that	
  an	
  even	
  smaller	
  fraction	
  of	
  AML 	
   blood	
  blasts,	
  
defined	
  by	
  a	
  CD34+	
  CD382	
  surface	
  phenotype,	
  can	
  transfer	
  disease	
  to	
  immune -­‐deficient	
  
mice	
  (Lapidot	
  et	
  al.1994;	
  Bonnet	
  and	
  Dick,	
  1997).	
  Since	
  normal	
  human	
  hematopoietic	
  
stem	
  cells	
  (HSCs)	
  are	
  also	
  CD34+	
  CD382,	
  these	
  and	
  other	
  observations	
  (Miy amoto	
  et	
  al.	
  
2000;	
  Hope	
  et	
  al.	
  2003)	
  have	
  been	
  taken	
  to	
  suggest	
  that	
  AML	
  LSCs	
  originate	
  from	
  and	
  
 
	
   20	
  
reside	
  exclusively	
  within	
  the	
  most	
  immature	
  bone	
  marrow	
  (BM)	
  progenitor	
  
compartment.	
  
However,	
   this	
   lineal	
   relationship	
   which	
   has	
   important	
   pathogenic	
   and	
   clinical	
  
implications,	
   may	
   not	
   always	
   hold	
   true.	
   For	
   example,	
   a	
   recent	
   study	
   of	
   blastic	
  
transformation	
  of	
  chronic	
  myeloid	
  leukemia	
  proposed	
  that	
  cells	
  with	
  a	
  granulocyte -­‐
macrophage	
  progenitor	
  (GMP)	
  cell	
  phenotype	
  were	
  candidate	
  LSCs	
  (Jamieson	
  et	
  al.	
  
2004).	
  In	
  addition,	
  frequent	
  LSCs	
  have	
  been	
  identified	
  in	
  a	
  mouse	
  model	
  of	
  human	
  MLL -­‐
AF9	
   AML,	
   showing	
   that	
   LSCs	
   are	
   not	
   only	
   located	
   within	
   the	
   stem	
   compartment	
  
(Somervaille	
  et	
  al.	
  2006).	
  In	
  accordance	
  with	
  several	
  studies	
  in	
  mouse	
  models	
  of	
  APL,	
  
the	
   identified	
  LSCs	
   in	
   MLL -­‐AF9	
   AML	
   are	
   phenotypically	
   myeloid	
   cells	
   that	
   have	
  
aberrantly	
   acquired	
   self -­‐renewal	
   capacity	
   rather	
   than	
   undifferentiated	
   stem	
   cells	
  
(Somervaille	
  t	
  al.	
  2006,	
  Guibalet	
  al.	
  2009).	
  Taken	
  together,	
  the	
  current	
  data	
  suggest	
  that	
  
LSCs,	
  which	
  establis h	
  a	
  leukemia	
  cell	
  hierarchy,	
  may	
  arise	
  from	
  mutations	
  occurring	
  in	
  
HSCs	
  and	
  also	
  in	
  committed	
  progenitors	
  (Figure	
  8)	
  (Cozzio	
  et	
  al.	
  2003,	
  So	
  et	
  al.2004).