Download Mechanisms of ibrutinib resistance

Mechanisms of Ibrutinib Sensitivity and Resistance
in CLL
Y. Lynn Wang, MD, PhD, FCAP
Dept. of Pathology
University of Chicago
October 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
A
C
CLL patients
Normal donors
CLL patients
t-BTK
p-BTK
GAPDH
GAPDH
t-BTK
p-BTK
GAPDH
GAPDH
B
Normal donors Controls
p-BTK relative expression
p-BTK relative expression
D
CLL
(N=11)
Normal
(N=5)
CLL
(N=1
Normal
(N=5)
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57 4
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”
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
Blood, 117, 6287, 2011
In vivo apoptosis was largely absent in the peripheral
blood of CLL patients treated with ibrutinib
CLL 043
CLL 011
CLL 195
pre D28
pre D28
pre D28
CLL 085
pre 4h D28
BCL2
Cutoff 15%
XIAP
MCL1
Cleaved
Caspase-3
PARP
GAPDH
Again, apoptosis induction may not be the predominant
effect of ibrutinib in CLL patients
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
8
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
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
10
CLL proliferation is targeted by BTK inhibition in
an in vitro model of cell proliferation
Cheng 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
What is the effect of ibrutinib on
signal transduction?
B-cell Receptor Signaling
Activity of BTK (pY223) was inhibited in
treated patients
Cheng S…Wang YL. Leukemia, 2014, 28, 649-57
15
BCR signaling in CLL
• CLL in vivo proliferation is mediated by the pathway LYN-SYK-BTKPLCg2 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.
Pre
Ibrutinib
initiation
Four specimens collected
450
Relapsed S1
Relapsed S2
400
350
250
Responding
ALC
300
200
150
100
50
0
-100
0
100
200
300
400
500
600
700
Day
Furman RR…Wang YL, NEJM, 2014, 370, 2352-4
19
A novel BTK mutation is identified by RNA-Seq
which is present only in relapse samples
Pre-Rx
Relapse s1
Responding
Relapse s2
Nucleotide composition at position 1634 of the BTK gene
CLL
Numbers of the reads
Specimens
A
C
G
T
Pre-Rx
0
0
0
84
Responding
0
0
0
54
Relapse S1
102
8
0
6
Relapse S2
67
5
0
1
%A
0
0
88
92
Furman RR…Wang YL, NEJM, 2014, 370, 2352-4
Before relapse
Pre-Rx
Relapsed
Relapse 1
Responding
Relapse 2
G C
T
G C
C T C
G C
AG
C C T C
20
What is the impact of the
mutation on ibrutinib binding?
The mutation disrupts the covalent binding of
ibrutinib to BTK
WT
C481S
WT
Ibrutinib (nM)
Probe-labeled BTK
Total BTK
-actin
-
1
10
C481S
100
-
1
10
C481A
100
-
1
10
No DNA
100
Inhibition of BTK phosphorylation (%)
Covalent bond
WT BTK
(IC50 =2.2nM)
BTK C481S
(IC50 =1006nM)
0
1
10
100 1000
Ibrutinib (nM)
Furman RR…Wang YL, NEJM, 2014, 370, 2352-4
22
What is functional consequence
of BTKC481S at the molecular
level?
The mutation restores the BCR signaling
pathway
Pre-Rx
Responding
Relapse 1
Relapse 2
Relapsed
Pre-RxResponding
1
2
p-BTK
(Y223)
p-ERK1/2
(T202/Y204)
p-AKT
(S473)
GAPDH
Before relapse
Pre-Rx
Responding
Relapsed
1
2
BCR signature
CCL4
LPL
PDGFA
EGR3
SLAMF7
NAB2
LILRA4
NR4A3
EGR1
CTLA4
CD83
Ly9
IRF4
KLF10
CASP3
DDIT3
DUSP2
PAICS
GFI1
GNPDA1
OAS3
TXNRD1
DDX21
NFKBIE
RGS10
***
BCR Signature Score
•
•
•
•
Before relapse
*
***
Pre-Rx
*
***
RespondingRelapse1 Relapse2
-3 -2 -1 0 1 2 3
Cheng S…Wang YL, Leukemia, 2014, epub
24
What is the functional
consequence of BTK C481S at the
cellular level?
The mutation leads to increased cellular
proliferation in vivo
Before Relapse
Ki67
Pre-Rx
Responding
Relapsed
1
2
CD19
Cheng S…Wang YL, Leukemia, 2014, epub
26
The mutation leads to increased cellular proliferation
in vitro that did not respond well to ibrutinib
+CLL+Stroma + ibrutinib
Iso
0 nM
250nM
500nM
BrdU
Responding
Before Relapse
Pre-Rx
1
Relapsed
2
7-AAD
Cheng S…Wang YL, Leukemia, 2014, epub
27
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
Cheng S…Wang YL, Leukemia, 2014, epub
33
Ibrutinib-resistance CLL cells remain sensitive
to idelalicib
Cheng S…Wang YL, Leukemia, 2014, epub
34
Acknowledgement
• Wang Lab at
Cornell
Shuhua Cheng, PhD
Chunyan Yang, MD
Pin Lu, MD, PhD
Ailin Guo, MD, PhD
Annie Ma, PhD
Zibo Song, PhD
Joelle Racchumi
Acknowledgement
• Cornell CLL
Research Center
Dr. Morton Coleman
Dr. Richard Furman
• Pharmacyclics
Dr. Betty Chang
• MSKCC
Dr. Christina Leslie
Menu Setty
• Harvard University
Dr. Hao Wu
Thank you!