1. Mutations in BCR-Abl associated with resistance to imatinib
BCR-ABL Kinase Domain
High Throughput Screen for Inhibitor-Resistant Mutations of Bruton's Tyrosine Kinase
Kevin K. Chang, Ethan Ahler, Douglas M. Fowler
Department of Genome Sciences, University of Washington
Abstract
Bruton’s Tyrosine Kinase (BTK) is a non-receptor tyrosine kinase that is
crucial for B-cell maturation and proliferation. BTK is linked to the
development and maintenance of B-cell malignancies. A small molecule
inhibitor of BTK, ibrutinib, shows impressive response rate in chronic
lymphocytic leukemia and mantle cell lymphoma. Early results from
clinical trials identified only the C481S resistance mutation; however,
other mutations in BTK could also confer resistance to ibrutinib.
Understanding the full spectrum of BTK resistance mutations could have
profound clinical applications, including personalizing treatment by
combining several different inhibitors to make a drug cocktail that
reduces the chance of resistance. I will use deep mutational scanning to
quantify the level of resistance of all possible single mutations in BTK. I
propose to select for ibrutinib-resistant BTK variants using a modified
version of the Yeast 2 Hybrid system (Y2H), a system used to test
protein-protein interaction. Here, I will link the expression of a reporter
gene to the phosphate-dependent interaction between an universal
protein tyrosine kinase substrate and an SH2 domain. Under these
conditions, yeast will only grow when the universal substrate has been
phosphorylated by BTK, enabling the interaction with the SH2 domain.
The effect of each variant on resistance will be proportional to the
growth rate of yeast expressing the variant in the presence of inhibitor.
Variants that increase in frequency during the selection have a higher
level of inhibitor resistance. Profiling of BTK inhibitor resistance is
critical for future research and therapies targeting BTK.
Yeast 3 Hybrid System
Growth assay is not compatible for functional assay, so we designed a
yeast 3 hybrid system (Y3H). With Y3H system, we will be able to
measure and quantify the effect of the mutation on inhibitor resistance.
AATC
TATC
ATTC
AATG
AATCAATC ATTCATTC
TATCTATC AATGAATG
A BTK variants expressed in yeast B
AATCAATCAATC ATTCATTCATTCATTCATTCATTC
TATC AATGAATGAATGAATG
Select for variants resistance to
inhibitor
C
AATC
AATC
AATC
TATC
ATTC
ATTC
ATTC
ATTC
ATTC
ATTC
AATG
AATG
AATG
AATG
Identify variants using
high-throughput sequencing
D
Variant Input Output Ratio Response
3 3 1 neutral
3 6 2 resistance
3 1 1/3 sensitive
3 4 4/3 neutral
Data Analysis by comparisons of input
and output frequencies
Large-Scale Assessment of BTK Variants
There are thousands of single mutations in the kinase domain of BTK that can potentially
affect inhibitor resistance. How can we test on and analyze all of the variants within
reasonable amount of time? We will use deep mutational scanning, a novel assay that
can test up to 1 million variants in a single experiment.
A. Create a library of nearly all single mutation variants, and express them in yeast. B. Grow yeast in
presence or absence of inhibitor, inhibitor sensitivity of BTK will cause yeast to grow at different rate. C.
High-throughput sequence both input and output libraries to identify frequencies of each variant. D.
Compare the input and output frequencies to calculate resistance score.
OD600(growth)
Time (hours)
Wild type Kinase-dead Inhibitor-resistant Empty vector
The wild type, kinase-dead, and inhibitor-resistant variants have no significant
difference in growth rate.
Growth Assay
Using deep mutational scanning to identify and measure the effect of the mutation on
resistance, we will need an assay to select for the resistant variants in the library. In
finding of a suitable functional assay, we first started with a simple growth assay.
Phosphorylated tyrosine is known to be toxic to yeast, so we suspected the expression
of BTK might affect yeast growth.
A. Y3H system with presence of ibrutinib. Ibrutinib will inhibit BTK activity, so
universal substrate remains unphosphorylated, and no transcription will occur.
B. Y3H without ibrutinib, BTK is able to phosphorylate universal substrate, and thus
enables URA3 transcription.
Reference
Soverini S, Hochhaus A, Nicolini FE, et al. BCR-ABL kinase domain mutation analysis in chronic myeloid
leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on
behalf of European LeukemiaNet. Blood. 2011;118(5):1208-15.
Soverini, et al., 2011
Acknowledgements
Professor Douglas M. Fowler, Ethan Ahler, and the members of Fowler Lab
No
transcription
UAS URA3
DBD
SH2
Domain
AD
Y
A
ibrutinib
Illustration of BTK pathway and ibrutinib inhibition
Proliferation and
transcription regulation
P
BTK BTK
ibrutinib
Assay for BTK Activity
Impact
1. Help with the design of BTK
inhibitors to circumvent resistance
mechanisms.
2. Provide guidance in BTK inhibitor
treatments to avoid relapses.
P
transcription
UAS URA3
DBD
SH2 Domain
AD
B
ibrutinib
Resistant
BTK
Y3H
+inhibitor
+Resistant
BTK
+WT BTK
-BTK
time
growth
Yeast with resistant BTK will growth faster
than yeast with wild type BTK and yeast
without BTK.
Universal
substrate
Universal
substrate
Universal
substrate
Universal
substrate
Expected Results: A Complete Resistance Map for BTK
BTK expressed yeast growth in presence of inhibitor
Y3H Illustration with WT and Resistant BTK
Growth Curve of BTK Expressed Yeast in InhibitorResistance Heatmap
A heatmap illustrating intensities of resistance of
mutations on each positions of the protein.
WT
BTK