Preclinicalvalidation of Vecabrutinib efficacy againstBTK ... · 6/18/2018 · Rec-1 TMD8 SUDHL4...
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Preclinical validation of Vecabrutinib efficacy against BTK C481S mutated lymphomas Camille Libre 1 , Ludovic Moro-Sibilot 1 , Pietro Taverna 2 , Nathalie Bissay 1 , Gilles Salles 1 , Laurent Genestier 1 , Pierre Sujobert 1 1 Centre de recherche en cancérologie de Lyon, Lyon, France, 2 Sunesis Pharmaceuticals, South San Francisco, United States Background The B cell receptor signaling pathway, and especially the Bruton Tyrosine Kinase (BTK), has become a therapeutic target in B cell neoplasms. Ibrutinib, the first in class BTK inhibitor has proven to be highly efficient in chronic lymphocytic leukemia or mantle cell lymphoma, and is thought to be of potential interest in the Activated B cell subtype of diffuse large B cell lymphomas (ABC- DLBCL). However, the emergence of resistant clones has been described, especially through the C481S mutation of BTK that disrupts the covalent binding of ibrutinib to BTK. Given the very poor prognosis of ibrutinib resistant patients, the development of drugs able to inhibit BTK even in case of C481S mutation is of utmost importance. Vecabrutinib is a noncovalent, reversible inhibitor of BTK that retains activity against the C481S mutation in kinase assays. Conclusion These preclinical data suggest that vecabrutinib is a highly specific and potent drug that is able to overcome the resistance due to BTK C481S mutation. Given its very favorable pharmacokinetic and safety profile, a phase 1b/2 dose escalation and cohort expansion clinical trial has been recently launched in patients with previously treated B-lymphoid malignancies (NCT03037645). Methods First, we have tested in vitro the sensitivity of a panel of 6 lymphoma cell lines to ibrutinib and vecabrutinib. Second, we have genetically modified 2 ibrutinib sensitive lymphoma cell lines (TMD8 and REC1) to overexpress either BTK WT or BTK C481S. We performed 7 days co-culture competitive assays in the presence or absence of ibrutinib or vecabrutinib, and assessed by flow cytometry the clonal composition of the surviving cells. Finally, we tested the effect of vecabrutinib on the BCR signaling pathway in TMD8 BTK C481S cells by western blotting. Results • Vecabrutinib selectively induces apoptosis in BTK dependent lymphoma cell lines • Vecabrutinib overcomes the resistance conferred by C481S mutation • Vecabrutinib inhibits the phosphorylation of BTK and PLC g 2 but has no effect on AKT and ERK phosphorylation 1 10 100 1000 10000 100000 0 20 40 60 80 100 viabilty (% of control) RAJI RL Rec-1 TMD8 SUDHL4 HBL1 Ibrutinib 50 nM viability (% of control) RAJI RL SUDHL4 TMD8 Rec-1 HBL1 0 20 40 60 80 100 0 BTK independent Cell lines BTK dependent Cell lines Ibrutinib 50 nM Vecabrutinib (nM) p-PLC g 2 p-BTK p-AKT p-ERK TMD8 BTK C481S a BCR VECA PLC g 2 BTK AKT ERK actin DMSO IBRU - + - - - - + - - - - + + - - - + - + - + - - + BTK-WT BTK-C481S PF272 Ibrutinib: 50 nM Vecabrutinib: 1 μM IC50 nM WT BTK C481S BTK Fold Change vecabrutinib 4.6 1.1 0.24 Ibrutinib 0.1 6.6 66 Neuman et al, ASH 2016 DMSO ibrutinib vecabrutinib 0 20 40 60 80 100 TMD8 cell line % of surviving cells DMSO ibrutinib 5nM vecabrutinib 1 uM 0 20 40 60 80 100 Rec1 cell line % of surviving cells DMSO ibrutinib vecabrutinib DMSO ibrutinib vecabrutinib 7 days Mix TMD8-BTK WT /TMD8-BTK C481S Mix Rec1-BTK WT /Rec1-BTK C481S ? RAJI: Burkitt’s TMD8: ABC-DLBCL RL: FL Rec1: MCL SUDHL4: GCB-DLBCL HBL1: ABC-DLBCL Lymphoma cell lines:
Transcript of Preclinicalvalidation of Vecabrutinib efficacy againstBTK ... · 6/18/2018 · Rec-1 TMD8 SUDHL4...
Preclinical validation of Vecabrutinib efficacy against BTK C481S mutated lymphomas
Camille Libre1, Ludovic Moro-Sibilot1, Pietro Taverna2, Nathalie Bissay1, Gilles Salles1, Laurent Genestier1, Pierre Sujobert1
1Centre de recherche en cancérologie de Lyon, Lyon, France, 2Sunesis Pharmaceuticals, South San Francisco, United States
BackgroundThe B cell receptor signaling pathway, and
especially the Bruton Tyrosine Kinase (BTK), has
become a therapeutic target in B cell neoplasms.
Ibrutinib, the first in class BTK inhibitor has proven
to be highly efficient in chronic lymphocytic
leukemia or mantle cell lymphoma, and is thought
to be of potential interest in the Activated B cell
subtype of diffuse large B cell lymphomas (ABC-
DLBCL). However, the emergence of resistant
clones has been described, especially through the
C481S mutation of BTK that disrupts the covalent
binding of ibrutinib to BTK. Given the very poor
prognosis of ibrutinib resistant patients, the
development of drugs able to inhibit BTK even in
case of C481S mutation is of utmost importance.
Vecabrutinib is a noncovalent, reversible inhibitor
of BTK that retains activity against the C481S
mutation in kinase assays.
ConclusionThese preclinical data suggest that vecabrutinib is a highly specific and potent drug that is able to overcome the resistance
due to BTK C481S mutation. Given its very favorable pharmacokinetic and safety profile, a phase 1b/2 dose escalation and
cohort expansion clinical trial has been recently launched in patients with previously treated B-lymphoid malignancies
(NCT03037645).
MethodsFirst, we have tested in vitro the sensitivity of a
panel of 6 lymphoma cell lines to ibrutinib and
vecabrutinib. Second, we have genetically
modified 2 ibrutinib sensitive lymphoma cell lines
(TMD8 and REC1) to overexpress either BTK WT or
BTK C481S. We performed 7 days co-culture
competitive assays in the presence or absence of
ibrutinib or vecabrutinib, and assessed by flow
cytometry the clonal composition of the surviving
cells. Finally, we tested the effect of vecabrutinib
on the BCR signaling pathway in TMD8 BTK C481S
cells by western blotting.
Results
• Vecabrutinib selectively induces
apoptosis in BTK dependent lymphoma
cell lines
• Vecabrutinib overcomes the resistance
conferred by C481S mutation
• Vecabrutinib inhibits the
phosphorylation of BTK and PLCg2 but
has no effect on AKT and ERK
phosphorylation
1 10 100 1000 10000 1000000
20
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Vecabrutinib (nM)
viab
ilty
(% o
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trol
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Rec-1TMD8SUDHL4
HBL1
Ibrutinib 50 nMvi
abili
ty (%
of c
ontr
ol)
RAJI RL SUDHL4 TMD8 Rec-1 HBL10
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0
BTK independent
Cell lines
BTK dependent
Cell lines
Ibrutinib 50 nM Vecabrutinib (nM)
p-PLCg2
p-BTK
p-AKT
p-ERK
TMD8 BTK C481S
a BCR
VECA
PLCg2
BTK
AKT
ERK
actin
DMSO
IBRU
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BTK-WTBTK-C481S
PF272
Ibrutinib: 50 nM
Vecabrutinib: 1 µM
IC50 nM WT BTK C481S BTK Fold Change
vecabrutinib 4.6 1.1 0.24
Ibrutinib 0.1 6.6 66
Neuman et al, ASH 2016
DMSO
ibrutin
ib
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brutin
ib 0
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TMD8 cell line
% o
f sur
vivi
ng c
ells
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ib 5nM
veca
brutin
ib 1 uM
0
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Rec1 cell line
% o
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DM
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tinib
veca
brutin
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DM
SO
ibru
tinib
veca
brutin
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7 days
Mix TMD8-BTK WT /TMD8-BTK C481S
Mix Rec1-BTK WT /Rec1-BTK C481S
?
RAJI: Burkitt’s TMD8: ABC-DLBCL
RL: FL Rec1: MCL
SUDHL4: GCB-DLBCL HBL1: ABC-DLBCL
Lymphoma cell lines:
