Dasatinib is a second-generation BCR-ABL tyrosine kinase inhibitor that is more potent than imatinib and effective against some imatinib-resistant mutations. Unlike imatinib, dasatinib interferes with platelet function by targeting Src family kinases, which may contribute to increased bleeding risk in patients. Clinical trials found dasatinib achieved high rates of cytogenetic and molecular response in chronic myeloid leukemia patients, including those resistant to or intolerant of imatinib. However, dasatinib treatment also carried risks of myelosuppression and bleeding that required dose reductions in some cases.

2. Imatinib and Beyond: Protein Kinase
Inhibitors as Cancer Therapeutics

3. Protein Kinases as Drug Targets
Problem:
There are over 500 protein kinases encoded by the
human genome.
No/yes
sufficient specificity
for clinical benefit
There are at least 120 members of the
tyrosine kinase family alone
Can a drug target
just one?
All of these kinases have presumably
evolved from a common ancestor and have
at least one substrate (ATP) in common

4. Protein Kinases: Targets for Cancer Chemotherapy
enzyme
OH
enzyme
O
P O
O -
-
OPO4
2-
ATP ADPprotein
kinase
phosphatase
PP
Ras-GDP
Ras-GTP
Raf Raf
MEK MEK-P
MAPK
MAPK-P
proliferation
growth factor
growth factor receptor
outside
inside
adaptor
MEK-Ser MEK-Ser-PO4
2-
inactive active
MEK-Ser MEK-Asp -
inactive active
MEK gene mutated MEK gene
OH could be Ser, Thr, Tyr
OFF ON
MEK-

5. HSC MPP
CLP
CMP
B cells
NK cells
T cells
dendritic cells
monocytes/macrophages
neutrophils
basophils
mast cells
eosinophils
erythrocytes
megakaryocytes - platelets
Hematopoiesis in CML
multipotent
stem cells
multipotent
progenitor
oligopotent
progenitor differentiated cell types
symptoms
• fever
• fatigue
• bleeding
Blast cells: transformed
immature precursors

6. Imatinib and Related Drugs Target Philadelphia
Chromosome Positive CML
• BCR-ABL fusion creates a dys-regulated
tyrosine kinase that drives the proliferation
of CML clones
protein kinase active site
bind ATP
bind target protein
substrate
imatinib
specific kinase inhibition
A priori, targeting the protein
substrate binding site might be
expected to provide more
specificity, but…………………
Imatinib competes with ATP for
binding to the BcrAbl Kinase

7. The Imatinib Experience
• 82% of patients achieve a complete cytogenetic response (65-85%)
• majority achieve a major molecular response (40-60%)
• 7 year overall survival rate 90%
• event free survival rate 81% (65-85%)
Positives
results from the International Randomized Study of Interferon
Versus STI571 (IRIS) trial 7-year follow up
Blood (2008) 112, 186various studies from 2003-2008
Gambacorti-Passerini and Piazza
(2014) Am. J. Hematol
Persons with CML can now be expected to have a
normal life expectancy when treated with TKIs.

8. • Imatinib, however, does not destroy cancer stem cells
• If you discontinue imatinib, CML returns
• Imatinib is the perfect drug because once you are on it, you are
on it forever
If you place a cancer cell under selective pressure for long enough without killing
it, what will happen?
The Imatinib Experience (Continued)
Negatives
• Current costs ~$100,000 annually

9. Negatives
• 18% of patients do not achieve a complete cytogenetic response
• 10% of patients who achieve a complete cytogenetic response lose their
response
• 4-8% of patients are intolerant to imatinib
The Imatinib Experience (Farmaco Resistenza)
mutated kinase active site
bind ATP
bind target protein
substrate
imatinib
kinase activity
the mutated kinase must still
bind ATP and have kinase
activity to drive proliferation
resistance can occur via pre-existing or
new mutations in BRC-ABL
Since imatinib and ATP bind to the same site there are a
limited number of degrees of freedom through which an
enzyme can become resistant to imatinib and still bind ATP

10. Dasatinib
• A second generation BRC-ABL inhibitor used to treat chronic myeloid leukemia
• Approved for all phases of Philadelphia-positive CML with resistance or
tolerance to prior therapy, including imatinib

11. 1st
and 2nd
Generation BCR-ABL Inhibitors
Dasatinib
This chemistry added to improve drug’s
pharmacological properties, not
needed for activity
Dasatinib lacks
all this extra
chemistry
destra/rightsinistra/left
chiudi gli occhi e strabismo

12. Comparison of Abl Kinase Structure with Dasatinib
and Imatinib Bound
extra functional groups
of imatinib bind to a
hydrophobic pocket in
Abl kinase not needed
for binding ATP: imparts
specificity
steric clashes with
extra functional
groups block active
conformation
mutations in this
region can interfere
with imatinib
binding without
affecting ATP or
dasatinib binding
ATP binding pocket
dramatic differences
in activation loop
notorious 315 mutation
closer to ATP binding
site affects binding of all
inhibitors
lucky
imatinib
dasatinib
left
right
dasatinib may have less
specificity than imatinib

13. Multikinase Inhibitory Panels of
BCR-ABL Inhibitors
Imatinib use for cancers other than CML, c-KIT/PDGF-R based cancers like
gastrointestinal stromal tumors

14. However, in a single-institution study, 37% of bleeding episodes (any grade)
occurred in patients without thrombocytopenia.[33]
Adverse Events with Dasatinib
Bleeding Related Events (mostly associated with severe thrombocytopenia):
CNS hemorrhages, including fatalities, have occurred. Severe gastrointestinal
hemorrhage may require treatment interruptions and transfusions. Use
SPRYCEL(dasatinib) with caution in patients requiring medications that inhibit
platelet function or anticoagulants.
from full US prescribing information
• myelosuppression
• GI symptoms
• rash
• fluid retention
• bleeding
dealt with by dose reduction or
interruption of treatment
a more specific effect on bleeding beyond reducing platelet number

15. Hemostasis Overview
Vessel injury
Collagen/VWF
exposure
platelet adhesion/release reaction
platelet aggregation
primary hemostatic plug
platelet fusion/contraction
stable hemostatic plug
tissue factor
blood coagulation
cascade
thrombin
fibrin
thromboxane A2
ADP
serotonin
vasoconstriction
reduced blood
flow
platelets –
thrombocytopenia –
bleeding disorder

16. Primary Hemostasis – Platelet Adhesion
endothelium
Von Willebrand factor
collagen
platelet
adhesion molecule
receptor
vessel damage
platelets contain receptors that bind subendothelial proteins
exposed upon vessel damage

17. Primary Hemostasis – Platelet
Activation/Aggregation
platelet activation
release reaction
aggregation
platelet
adhesion molecule
receptor
von Willebrand factor,
fibronectin, vitronectin
ADP
thromboxane A2
clopidogril
aspirin
ADP ADP
Exposure of negatively
charged phospholipids on
the platelet surface
Increased Ca2+

18. Collagen Signaling and Platelet Aggregation
A member of the Src family of
kinases

19. The Effect of Dasatinib on Agonist-Induced Platelet
Aggregation
after 2-5 hr of treatment
dasatinib can reach 150
and 100 nM in the
plasma of patients
treated with 140 and 70
mg regimes, respectively
tyrosine
phosphorylation
specific src-family
phosphorylation

20. Multikinase Inhibitory Panels of
BCR-ABL Inhibitors

21. Imatinib does not Interfere with Collagen-Induced
Platelet Aggregation or Signaling Pathways

22. The Effect of Dasatinib on Collagen-Induced Signaling
or Glycoprotein VI

23. DASATINIB:
• targets the BCR-ABL kinase active site in a manner distinct from imatinib
• is more potent a Abl kinase inhibitor than imatinib and is not affected by some of
the mutations that lead to imatinib resistance
• targets a wider spectrum of protein kinases than does imatinib
• interferes with platelet function by presumably targeting one or more Src kinases
• may be considered as a lead compound for a new class of anti-thrombotic agents
CONCLUSIONS

24. A Second Second Generation BCR-ABL Inhibitor

25. Multikinase Inhibitory Panels of
BCR-ABL Inhibitors

26. Binding Characteristics of BCR-ABL TKI’s
common imatinib
resistance mutations

27. Application of 2nd
Generation TKIs in a Stepwise
Approach to CML Treatment
A – may respond to inhibitor, evidence
lacking
B – reduced sensitivity to inhibitor
C – compelling clinical evidence
suggests an alternative inhibitor
should be used
D – forget about second generation
inhibitors, try emerging therapies

28. The experience with certain second generation BCR-Abl
inhibitors has been sufficiently positive to consider their use in
replacing imatinib as a frontline therapy for ECP-CML

29. Study Protocol: Methods
Rosti et al Blood (2009) 114, 4933 & Cortes et al. JCO e-pub December 14, 2009
Patient eligibility:
• 18 years of age or older
• diagnosis of CP (Ph+
) – CML within less than 6 months
• untreated or treated only with hydroxyurea or anagrelide
Patient exclusion:
• WHO performance status 2 or more
• uncontrolled serious medical conditions
• received prior treatment with any investigational drug

30. Cytogenetic response (CyR) :
• complete, zero Ph+
chromosomes in 20 metaphases
• partial, 0-35% Ph+
metaphases
• minor, 35-95% Ph+
metaphases
Study Protocol: Methods
Rosti et al Blood (2009) 114, 4933 & Cortes et al. JCO e-pub December 14, 2009
Molecular response (MR) :
• complete, undetectable BRC-ABL transcripts in 105
cells
• major, BCR-ABL/ABL transcript ratio of less than 0.1%
Complete Hematological Response (CHR) :
• normalization of peripheral blood counts
• normal differential
• no splenomegaly

31. BCR
ABL
CML: Complete Molecular Remission
polymerase chain reaction (PCR)

32. Hematological, Cytogenetic, and Molecular
Responses in the European Study
primary endpoint CCgR at 12 year
expectation for imatinib (50-70%)
< 50% response to nilotinib (no interest)
70% response to nilotinib (interest)
nilotinib dose: 400 mg twice daily

33. major molecular response obtained in 52
% of patients by 3 months and 85% by
12 months
Kinetics of Molecular Responses to Nilotinib
adverse effects manageable with does reductions

34. Cytogenetic, and Molecular Responses in the MD
Anderson Study
compare with historical data from MD Anderson patients treated with imatinib
• 6-month CCyR: 96% with nilotinib v 45% with sd imatinib
• 12-month CCyR: 97% with nilotinib v 70/80% with hd/sd imatinib, respectively
• 12-month MMR: 81% with nilotinib v 46/54% with sd/hd imatinib, respectively
major difference between nilotinib and imatinib is the earlier occurrence of the
response with nilotinib and an improved molecular response
Do either of these parameters matter in terms of patient performance and overall
survival??

35. September 1, 2011 — The US Food
and Drug Administration (FDA)
recently granted accelerated
approval to crizotinib (Xalkori, Pfizer)
for the treatment of patients with
advanced-stage nonsmall-cell lung
cancer (NSCLC) that is anaplastic
lymphoma kinase (ALK)-positive.
Only 4-5% of NSCLC is ALK-positive
In PROFILE 1005 (n = 136), the
objective response rate (ORR) was
50%, and that included 1 complete
response and 67 partial responses.
Like many targeted therapies,
crizotinib comes with a hefty price tag.
According to Geno Germano,
president and general manager of
specialty care and oncology at Pfizer,
the drug will cost $9,600 per month, or
$115,000 per year.
Kinase Inhibitors as “Smart” Drugs for Treating Cancer