Many enzymes are regulated by covalent attachment of
phosphate, in ester linkage, to the side-chain hydroxyl group
of a particular amino acid residue (serine, threonine, or
tyrosine).
A protein kinase transfers the terminal phosphate of ATP to a
hydroxyl group on a protein.
A protein phosphatase catalyzes removal of the Pi by
hydrolysis.
Protein OH + ATP Protein O P
O
O
O
+ ADP
Pi H2O
Protein Kinase
Protein Phosphatase
Circa 500 serin- o treonin-kinasi,
solo 96 Tirosin-kinasi in tutto il genoma umano.
(funzioni importanti!)
Meccanismi di attivazione delle TKMeccanismi di attivazione delle TK
Amplificazione iperespressione
– (ERB2, MET…..)
Geni di fusione
– (BCR-ABL, Tel-PDGFRb, FIP1L-PDGFRa…)
Alterazioni strutturali interne
– (FLT3 ITD…..)
Mutazioni puntiformi
– (FLT3, JAK2, KIT……..)
Iperespressione Geni di fusione
ITD Mutazioni
Attivazione costitutiva TK,guadagno di funzione e
attività oncogenica
Myeloid
Progenitor
Stem Cell
Proliferation Defects Differentiation defects
CMPDs Acute Leukemias
Myeloid Leukemias Myeloid Leukemias are are caused by caused by a a limitedlimited
numbers numbers of of recurrent molecular defectsrecurrent molecular defects::
TK activation TFs involvementTK
activ
ation
10 11 12 13 14 15 16 17 18 19
TM JM TK1 K1 TK2
FLT3 alterations in AML
ITD D835
ITD and POINT MUTATION ARE FREQUENTLY FOUND IN AML
(36% of adult AML)
Relapse-free survival according to FLT3
(GIMEMA LAM-99P)
0.00
0.25
0.50
0.75
1.00
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25
Years from CR
ITD negative
ITD positive
P=0.0001
KIT KIT activationactivation in AML in AML
Mutazioni presenti in circa il 5% delle LMA
Associate a “CBF leukemias”
Mutazioni in esoni diversi:
– D419X
– V560X
– D816X
Valore prognostico negativo nelle t(8;21), ancoraincerto nelle inv16
Iperespressione CD117 in circa il 20% delle AML
Coexpressione di CD117 e di CD34
Iperespressione di CD117
Prognosis
poor
poor
??
?
Refs:•Cairoli R e t al . Blood. 2006
•Schnittger S et al. Blood. 2006•Boissel N et al. Leukemia 2006
•Paschka P et al. J Clin Oncol 2006
Druker B et al 1996
Bcr-Abl
Y = Tyrosine
P = Phosphate
Substrate
Y
Y
Substrate
Effector
STI571
Y
Bcr-Abl
Substrate
PPP
PY
Substrate
Effector
ATP
Mechanism of action
Imatinib = rivoluzione
Per la prima volta si dimostrava che piccolemolecole competitrici del sito di legamedell’ATP delle TK:
– potevano essere abbastanza selettive e bloccaresolo l’attività di alcune TK;
– il blocco dell’attività TK era efficace comeattività antitumorale senza produrre effettitossici gravi.
Risultati terapia con inibitori TK
nelle neoplasie ematologiche
• Buoni, talora ottimi, nei disordini
mieloproliferativi semplici (LMC, HES,
CMML etc…)
• Molto inferiori e transitori nelle forme
avanzate di trasformazione e nelle LA
Event-free Survival and
Survival Without AP/BC on First-line Imatinib
Progression events:
6.5% AP/BC
4.7% loss of MCyR
2.5% loss of CHR
1.4% CML-unrelated deaths
84%
93% (90-96)
(80-87)
Estimated rate at 54 months (with 95%CI)
Overall Survival on First-line Imatinib (ITT
principle)
10.6%4.6% (2-7)
(8-14)
Estimated rate at 60 months (with 95%CI)
CML-related deaths All deaths
95%
89%
Survival without CML-related deaths
Survival Without AP/BC by Molecular Response at 12
months on First-line Imatinib
n= 136 100%
n= 94 95%
n= 138 89%
Estimated rate at 54 months
} p<0.001} p=0.007
97 99 9993 90 93
81 83 82
0
10
20
30
40
50
60
70
80
90
100
CCyR PCyR No MCyR
12 months
18 months
24 months
Survival Without AP/BC at 60 months
by Level of CyR at 12, 18 and 24 months%
without
AP
/BC
at
60 m
onth
s
% with CCyR after landmark 64 50 38 36 27 26
Kantarjian H. et al. Clin Cancer Res 2004;10:68– 75.
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
Months
Pro
gre
ssio
n-f
ree
surv
ival
Gimema CML WP Study 002
Late CML unresponsive or intollerant to IFN
The TKI Imatinib “per se” seems
to decrease the progression rate of the CML clone,
probably decreasing its genomic instability
DNA damageROS
Radical Oxigen Species
Imatinib
Ph+
Ph+Ph+
Ph+Ph+
Ph+Ph+
Ph+
Ph+ Ph+Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+
Ph+
Ph+
Ph+
Ph+Ph+
Ph+
Ph+
Ph1
Ph1
Ph1
Ph1
Ph1
Ph1Ph1
Ph1Ph1 Ph1
Ph1Ph1
Ph1
Ph1Ph1
Ph+
Ph+
Ph+
Ph+
Ph+
Ph+
Ph+Ph+
Ph+
Ph+
Ph+
Ph+
Ph+
Ph+Ph+
Ph+Ph+
Ph+Ph+
NN
NN
N
N
N
N
N
N
N
NN
N
N
N
NN
Ph+
N
N
N
NN
N
N
N N N
NN
NN
N
N
NN
N
N
N
N
N
NN
NN
N
NN
NN
N
N
N
NN
N
N
N
N
N
N
N
N
N
N
N
N
N
NN
Ph+
Ph+
Ph+
Ph+
Ph+
Ph1
Ph1
Studio 102 – OVERALL SURVIVAL
% o
f p
ati
en
ts a
live
Median survivalUntreated patients 7.5 months
Pretreated patients 5.6 months
Since diagnosis of blast crisis 9.9 months
p=0.16
3 6 9 12 15 18 21 24 27 30 33 36
Months since start of treatment
0
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
STUDIO 106 – OVERALL SURVIVAL
All deaths
Imatinib
IFN + Ara-C
% a
live
Months since randomization
0 3 6 9 12 15 18 21 24
Imatinib
IFN + Ara-C
Imatinib IFN + Ara-C
CML related deaths 9 16
Deaths after BMT 3 5
Other deaths 8 7
Estimated survival at 18 months (p=0.16) 97.2% 95.1%
CEP-701 (Cephalon)
PKC412 (Novartis)
SU5416 (Pharmacia)
(MLN518) (Millenium)
SU11248 (Pfizer)
L-021649 (Merck)
CLINICAL TRIALS WITH FLT3 INHIBITORS
CEP701
Knapper et al., Blood 2006
ASH 2006
70% remissioni complete – 90-100% nei FLT3 mutati
Primary resistance
Relapse/
progression
Earlychronicphase
Latechronicphase
Acceleratedphase
(600 mg/d)
Blasticphase
(600 mg/d)
0
25
50
75
100
47
20
4
24
60
66
93
Frequency of imatinib resistance
within 3 years
Pe
rce
nt
Hochhaus and La Rosée Leukemia. 2004
Difetto genetico semplice fenotipo leucemico
Più difetti -> fenotipo leucemico complesso
Efficacia TKIs(..e non è solo questione di potenza!)
16/199
8%
0 2 4 6 8 10 12 14 16 18 20
Months
Dasatinib 70 mg BID in CP-CML
Progression-free survival
Progression was defined as confirmed AP / BP, loss of CHR / MCyR,
or increasing WBC count
Pro
po
rtio
n p
rog
ressio
n-f
ree
1.0
0.8
0.6
0.4
0.2
0
40386Total37287Resistant 3 99Intolerant
No. progressedN
0 3 6 9 12 15 18 21
Months
Dasatinib in blast-phase CMLProgression-free survival
Patients who underwent SCT were censored at that time
Pro
po
rtio
n p
rog
ressio
n-f
ree
1.0
0.8
0.6
0.4
0.2
0
48
109
N
3.037CML-LB
6.764CML-MB
Median (mo)No. of deaths
Updated ASH 2006
Clone with
T315I
Clone with
Mutation
Clone without
mutations
Imatinib
Dasatinib or
nilotinib
LBH or
ON012380
Resistenza = fenomeno plastico
Nat Rev Cancer, Dec 2006
• Genetic and epigenetic alterations that are beneficial to a neoplastic
clone are facilitated by natural selection and genetic drift
• The fitness of a neoplastic cell is shaped by its interactions with
cells and other factors in its microenvironment (its ecology),
including interventions to prevent or cure cancer.
Genetic Instability may increase the evolution speed
Acquired From the beginning
Blood 2007
CCRCCR
Not Not
detecteddetected6 6 9 9 12 12 1818PrePre 33 2424 2727 3030
4.04.0
3.03.0
2.02.0
1.01.0
Base lineBase line
Lo
g r
edu
ctio
n o
f L
og
red
uct
ion
of BCR-ABL
BCR-ABL
92%92%
BCR-BCR-
ABLABL
positivepositive
Molecular response in IRIS trial
MonthsMonths
I TKIs non sembrano in grado da soli
di eliminare le cellule staminali leucemiche
Michor et al., Nature 2005
Differentiated
Progenitors
Dynamics of CML response to imatinib
Holyoake Holyoake TL, TL, Blood Blood 20022002
Primitive quiescent BCR-ABL+ leukemic stem cells
are less sensitive to imatinib
HolyoakeHolyoake TL, TL, BloodBlood 2004 2004
Punish the Parent not the Progeny
Bcr-Abl off? Bcr-Abl on?
In CML progenitor cells (lin-, CD34+, CD38- cells):
A) Decreased Imatinib concentration:
- decreased influx (OCT-1) (White DL et al., Blood 2006)
- increased efflux (ABCB1/MDR1, ABCG2)
(Zhou et al , 2001)
(Burger et al, 2004)
(Jordanides et al., 2006)
…………
B) Increased expression of BCR-ABL
Blood 2006
Ph+ cells that
survive and return
to “normality”
BCR-ABL
inhibition
BCR-ABL
inhibition
Ph+ cells
Apoptosis
The persistence of Ph-positive cells may be due to:
- cells more resistant to imatinib
- cells in which the BCR-ABL TK activity may be suppressed
without a great damageImatinib
Imatinib
The persistence of Ph-positive cells may be due to:
- cells more resistant to imatinib
- cells in which the BCR-ABL TK activity may be suppressed
without a great damage
Significant Improvement in Rate of
CMR with Continuous Imatinib Therapy
7
24
34
41
0
5
10
15
20
25
30
35
40
45
50
24 months 36 months 48 months 69 months
Branford et al. Blood, 108 (11): Abstract 430
Months of Imatinib Therapy
Rousselot et al., Blood 2007
Can cure be achieved with imatinib alone?
0,0001
0,001
0,01
0,1
1
10
10001/11/00
01/03/01
01/07/01
01/11/01
01/03/02
01/07/02
01/11/02
01/03/03
01/07/03
01/11/03
01/03/04
01/07/04
01/11/04
01/03/05
Imatinib
stop
(30
months)
Nested PCR-negative
Pazient CS: RQ PCR analysis on PB and Bone Marrow samples
CCR
SMR
GMR
NoCCRImatinib 400 mg
Why?
• Stem cell senescence?
• Reactivation of an autologous immune
control on the CML stem cells?
The strategy to eradicate the persistence of this
tricky Ph+ population must be appropriate
• Immunotherapy ?
• Specific targeted therapies for “stem cells”?
• Vaccines?
Conclusioni• I TKIs sono entrati nell’ armamentario
terapeutico delle neoplasie ematologiche e lohanno arricchito
• Sono molto efficaci nei CMPDs, meno nelle formeavanzate di malattia e nelle forme acute
• I meccanismi di resistenza appaiono molto piùcomplessi di quanto originariamente immaginato.
• Per le forme avanzate ed acute apparei dis s bil l t i di mbi i
• University of Turin
• Daniela Cilloni
• Giovanna Rege Cambrin
• Francesca Messa
• Carmen Fava
• Francesca Arruga
• Ilaria Defilippi
• Emanuela Messa
• Alessandro Morotti
• Enrico Gottardi
• Emilia Giugliano
• Anna Serra
• Milena Fava
San Luigi Hospital-University of San Luigi Hospital-University of TurinTurin
ROS01001.PPT
CML CML ––GIMEMAGIMEMA WP WP
1973 1973 –– 2007 2007
v
