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Transcript of Mechanisms of Ibrutinib Sensitivity and Resistance in CLL Y. Lynn Wang, MD, PhD, FCAP Dept. of...
Mechanisms of Ibrutinib Sensitivity and Resistance in CLL
Y. Lynn Wang, MD, PhD, FCAPDept. of Pathology
University of ChicagoOctober 24, 2014
Outline
• Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity
• BTKC481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding
• PLCg2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling
• Overcome ibrutinib resistance: Exploration of other kinase inhibitors
Outline
• Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity
• BTKC481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding
• PLCg2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling
• Overcome ibrutinib resistance: Exploration of other kinase inhibitors
BTK is more active in treatment-naive CLL patients than in normal individuals
4
Normal donors
259
259
CLL patients
89 92 95 96
123
11
03127
2078208
45 64 67 74
82
76
11
03127
2078208
t-BTK
GAPDH
t-BTK
GAPDH
A
Controls
ly7+IgM
ly7+IgM
CLL patients Normal donors
ly745 64 67 74
76
4771
47782078
208259
ly789 92 95 96
123
4
7714778
2078208
259
82
p-BTK
GAPDH
p-BTK
GAPDH
C
B
CLL(N=11)
Normal(N=5)
p-B
TK
rel
ativ
e ex
pres
sion
p-B
TK
rel
ativ
e ex
pres
sion
CLL(N=1
Normal(N=5)
D
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
Study Design
• In vivo: Prospective collection of serial samples from patients undergoing clinical trial of ibrutinib
• Enrolled a total of 16 patients– 12
previously treated
– 4 treatment naïve
What is the effect of ibrutinib on apoptosis induction?
Does ibrutinib induce apoptosis?-An ex vivo study
“Apoptosis induced by PCI-32765 in primary CLL cells was significant compared with vehicle treatment alone at 10 uM PCI-32765”
Blood, 117, 6287, 2011
The Cmax values in humans range from 80-200 nM
Cytopenia was uncommon in patients treated at 420 mg/day. Grade 3&4 neutropenia was more common in the 840 mg group-ASCO 2011
In vivo apoptosis was largely absent in the peripheral blood of CLL patients treated with ibrutinib
8
BCL2
XIAP
MCL1
PARP
Cleaved Caspase-3
GAPDH
CLL 085
D28
CLL 195 CLL 043 CLL 011
pre 4hpre D28 pre D28 pre D28
Again, apoptosis induction may not be the predominant effect of ibrutinib in CLL patients
Cutoff 15%
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
What is the effect of ibrutinib on CLL cell proliferation?
In vivo CLL proliferation is targeted by BTK inhibition: Ki-67 was reduced over the treatment course
10Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
11
CLL proliferation is targeted by BTK inhibition in an in vitro model of cell proliferation
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
12
CLL proliferation is targeted by BTK inhibition in an in vitro model of cell proliferation
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
What is the effect of ibrutinib on signal transduction?
B-cell Receptor Signaling
15
Activity of BTK (pY223) was inhibited in treated patients
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
BCR signaling in CLL
• CLL in vivo proliferation is mediated by the pathway LYN-SYK-BTK-PLC2 and downstream signaling through AKT and ERK.
• The data highlight the role of cell proliferation in CLL that has been thought mainly as a disease with apoptotic defects.
Outline
• Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity
• BTKC481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding
• PLCg2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling
• Overcome ibrutinib resistance: Exploration of other kinase inhibitors
The Case • The patient is a 61 year old woman first diagnosed with
CLL in 2000 when she presented with lymphadenopathy. • In September of 2010, she was enrolled in the phase Ib
trial (NCT01105247) of ibrutinib. She achieved an excellent response, with normalization of her CBC, (ALC=3600), but some residual lymphadenopathy remained on CT scan.
• In March of 2012, 19 months after ibrutinib initiation, her ALC and lymphadenopathy rapidly increased.
• In May of 2012, the ibrutinib dose was escalated from 540 to 840 mg. Over the next four weeks, the patient continued to demonstrate disease progression.
Four specimens collected
19
-100 0 100 200 300 400 500 600 7000
50
100
150
200
250
300
350
400
450
Day
ALC
Re
lap
sed
S
2
Pre
Re
spo
nd
ing
Re
lap
sed
S
1
Ibru
tinib
initi
atio
n
Furman RR…Wang YL, NEJM, 2014, 370, 2352-4
A novel BTK mutation is identified by RNA-Seq which is present only in relapse samples
20
RelapsedBefore relapse
Pre-Rx
Responding
Relapse 1
Relapse 2
G C T G C C T C G C A G C C T C
Responding
Pre-Rx Relapse s1
Relapse s2
Nucleotide composition at position 1634 of the BTK gene
CLL Numbers of the reads %A
Specimens A C G T
Pre-Rx 0 0 0 84 0Responding 0 0 0 54 0Relapse S1 102 8 0 6 88Relapse S2 67 5 0 1 92
Furman RR…Wang YL, NEJM, 2014, 370, 2352-4
What is the impact of the mutation on ibrutinib binding?
The mutation disrupts the covalent binding of ibrutinib to BTK
22
Probe-labeled BTK
Ibrutinib (nM) - 1 10 100 - 1 10 100 - 1 10 100
No DNA
Total BTK
-actin
WT C481S C481A
WT C481S
Covalent bond
Furman RR…Wang YL, NEJM, 2014, 370, 2352-4
WT BTK(IC50 =2.2nM)
BTK C481S (IC50 =1006nM)
Inhi
bitio
n of
BT
K p
hosp
hory
latio
n (%
)
10 10 100 1000
Ibrutinib (nM)
What is functional consequence of BTKC481S at the molecular
level?
The mutation restores the BCR signaling pathway
24
p-BTK(Y223)
p-ERK1/2(T202/Y204)
Before relapse RelapsedPre-RxResponding 1 2
p-AKT(S473)
GAPDH
3210-1-2-3
Pre-Rx Responding 1 2
RelapsedBefore relapse
EGR1
PAICSDUSP2
KLF10
CD83
CASP3
NFKBIEDDX21
GFI1
IRF4
CTLA4
GNPDA1
Ly9
RGS10
CCL4LPLPDGFAEGR3SLAMF7NAB2LILRA4NR4A3
DDIT3
OAS3TXNRD1
BCR signature
BC
R S
ign
atu
re S
core
***
***
*
* ***
Pre-Rx Responding Relapse1 Relapse2
• Pre-Rx• Responding• Relapse 1• Relapse 2
Cheng S…Wang YL, Leukemia, 2014, epub
What is the functional consequence of BTK C481S at the
cellular level?
The mutation leads to increased cellular proliferation in vivo
26
CD19
Ki6
7
Pre-Rx Responding
Before Relapse
1
Relapsed
2
Cheng S…Wang YL, Leukemia, 2014, epub
The mutation leads to increased cellular proliferation in vitro that did not respond well to ibrutinib
27
Iso 0 nM 250nM 500nM
7-AAD
Brd
U
+CLL+Stroma + ibrutinib
Befo
re Relap
seR
elapsed
1R
espo
nd
ing
2P
re-Rx
Cheng S…Wang YL, Leukemia, 2014, epub
BTK Mutation Is Recurrent
Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94
Outline
• Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity
• BTKC481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding
• PLCg2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling
• Overcome ibrutinib resistance: Exploration of other kinase inhibitors
Reactivation of BCR signaling in patient with PLCg2R665W mutation
Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94
Reactivation of BCR signaling in patient with PLCg2R665W mutation
Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94
Outline
• Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity
• BTKC481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding
• PLCg2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling
• Overcome ibrutinib resistance: Exploration of other kinase inhibitors
Ibrutinib-resistance CLL cells remain sensitive to other BCR inhibitors
33Cheng S…Wang YL, Leukemia, 2014, epub
34
Ibrutinib-resistance CLL cells remain sensitive to idelalicib
Cheng S…Wang YL, Leukemia, 2014, epub
Acknowledgement
• Wang Lab at Cornell
Shuhua Cheng, PhD
Chunyan Yang, MDPin Lu, MD, PhD Ailin Guo, MD, PhDAnnie Ma, PhDZibo Song, PhDJoelle Racchumi
Acknowledgement
• Cornell CLL
Research CenterDr. Morton Coleman
Dr. Richard Furman
• MSKCCDr. Christina Leslie
Menu Setty
• PharmacyclicsDr. Betty Chang
• Harvard UniversityDr. Hao Wu
Thank you!