Abstract

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as

BRAF) are found in 5070% of malignant melanomas 1. Pre-clinical studies
have demonstrated that the B-RAF(V600E) mutation predicts a dependency on
the mitogen-activated protein kinase (MAPK) signalling cascade in
melanoma2,3,4,5,6an observation that has been validated by the success of
RAF and MEK inhibitors in clinical trials 7,8,9. However, clinical responses to
targeted anticancer therapeutics are frequently confounded by de novo or
acquired resistance10,11,12. Identification of resistance mechanisms in a manner
that elucidates alternative druggable targets may inform effective long-term
treatment strategies13. Here we expressed 600 kinase and kinase-related open
reading frames (ORFs) in parallel to interrogate resistance to a selective RAF
kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a
MAPK pathway agonist that drives resistance to RAF inhibition in B-
RAF(V600E) cell lines. COT activates ERK primarily through MEK-
dependent mechanisms that do not require RAF signalling. Moreover, COT
expression is associated with de novo resistance in B-RAF(V600E) cultured
cell lines and acquired resistance in melanoma cells and tissue obtained from
relapsing patients following treatment with MEK or RAF inhibitors. We
further identify combinatorial MAPK pathway inhibition or targeting of COT
kinase activity as possible therapeutic strategies for reducing MAPK pathway
activation in this setting. Together, these results provide new insights into
resistance mechanisms involving the MAPK pathway and articulate an
integrative approach through which high-throughput functional screens may
inform the development of novel therapeutic strategies.

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Download references

Acknowledgements

We thank members of the Broad Institute/Novartis Cancer Cell Line

Encyclopedia (CCLE) for contributing cell line genomic data, expression data
and pharmacological cell line sensitivity data; J. Thibault and A. Shipway for
CCLE related tissue culture; R. Depinho, G. Dunn, S. Ethier, H. Greulich, A.
Henderson, D. Kaplan, R. Levine, C. Miller, H. Piwnica-Worms, H. Suzuki,
M. Vigny, D. Vollrath and the Harvard Institute of Proteomics for contributing
templates for the kinase collection; J. Du and D. B. Wheeler for assistance with
functional testing of kinases; D. A. Barbie for helpful discussions, S. E. Moody
and H. W. Cheung for technical assistance, and J. K. Grenier and S. J. Silver
for compiling and annotating the list of kinases. This work was supported by
the NIH Directors New Innovator Award (L.A.G.), grants from the Novartis
Institutes for Biomedical Research (L.A.G.), Melanoma Research Alliance
(L.A.G.), Starr Cancer Consortium (L.A.G.) the US National Cancer Institute
(R33 CA128625, RC2 CA148268) (W.C.H.), NIH (CA134502) (J.J.Z) the
Swiss National Foundation (310040-103671) (R.D.), the Gottfried and Julia
Bangerter Rhyner Stiftung (R.D.) the Ellison Foundation (M.V. and CCSB)
and institute sponsored research funds from the DFCI Strategic Initiative
(M.V. and CCSB).

Author information
Author notes
1.
o Haley Hieronymus

Current address: Human Oncology and Pathogenesis Program,

Memorial Sloan-Kettering Cancer Center, New York, New York 10065,
USA
2.
o Cory M. Johannessen
o & Jesse S. Boehm

These authors contributed equally to this work.

3.
o So Young Kim

Present address: Duke Institute for Genome Sciences and Policy, Duke
University Medical Center, Durham, North Carolina 27710, USA
(S.Y.K).
Affiliations
1. Broad Institute of Harvard and Massachusetts Institute of
Technology, 7 Cambridge Center, Cambridge, Massachusetts 02142,
USA
o Cory M. Johannessen
o , Jesse S. Boehm
o , So Young Kim
o , Sapana R. Thomas
o , Laura A. Johnson
o , Nicolas Stransky
o , Jordi Barretina
o , Haley Hieronymus
o , Xiaoping Yang
o , Ozan Alkan
o , David E. Root
o , Todd Golub
o , William C. Hahn
o & Levi A. Garraway
2. Department of Medical Oncology, Dana-Farber Cancer Institute,
Harvard Medical School, 44 Binney Street, Boston, Massachusetts
02115, USA
o Cory M. Johannessen
o , So Young Kim
o , Sapana R. Thomas
o , Leslie Wardwell
o , Laura A. Johnson
o , Caroline M. Emery
 o , Jordi Barretina
o , William C. Hahn
o & Levi A. Garraway
3. Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer
Institute, Boston, Massachusetts 02115, USA
o So Young Kim
o , Ryan R. Murray
o , Kourosh Salehi-Ashtiani
o , David E. Hill
o , Marc Vidal
o & William C. Hahn
4. Division of Surgical Oncology, Medical Oncology and Dermatology,
Massachusetts General Hospital, 55 Fruit Street, Boston,
Massachusetts 02114, USA
o Alexandria P. Cogdill
o , Keith T. Flaherty
o & Jennifer A. Wargo
5. Center for Cancer Genome Discovery, Dana-Farber Cancer Institute,
Harvard Medical School, 44 Binney Street, Boston, Massachusetts
02115, USA
o Jordi Barretina
o , Todd Golub
o , William C. Hahn
o & Levi A. Garraway
6. Novartis Institutes for Biomedical Research, 250 Massachusetts
Avenue, Cambridge, Massachusetts 02139, USA
o Giordano Caponigro
o , Christopher J. Wilson
o , Vic E. Myer
o , Peter M. Finan
o , Barbara L. Weber
o , William R. Sellers
o & Robert Schlegel
7. Department of Pediatric Oncology, Dana-Farber Cancer Institute,
Harvard Medical School, 44 Binney Street, Boston, Massachusetts
02115, USA
o Haley Hieronymus
o & Todd Golub
8. Department of Cancer Biology, Dana-Farber Cancer Institute,
Boston, Massachusetts 02115, USA
o Ryan R. Murray
o , Kourosh Salehi-Ashtiani
o , David E. Hill
o , Marc Vidal
o , Jean J. Zhao
o & Thomas M. Roberts
 9. Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115, USA
o Ryan R. Murray
o , Kourosh Salehi-Ashtiani
o , David E. Hill
o & Marc Vidal
10. Department of Pathology, Harvard Medical School, Boston,
Massachusetts 02115, USA
o Jean J. Zhao
11. Genomics Institute of the Novartis Research Foundation, San Diego,
California 92121, USA
o Sungjoon Kim
o & Jennifer L. Harris
12. Department of Dermatology, University Hospital of Zurich, Zurich
CH-8091, Switzerland
o Reinhard Dummer

Contributions
C.M.J., J.S.B. and L.A.G. designed the experiments, with help from C.M.E.
C.M.J., S.Y.K. and L.W. performed the primary screen, supervised by W.C.H.
L.A.J. helped perform drug sensitivity profiling. J.S.B. designed and created
the CCSB/Broad Institute Kinase ORF Collection in collaboration with S.R.T.,
H.H., R.R.M., K.S.-A., J.J.Z., M.V., T.M.R., T.G., D.E.H. and W.C.H.
Additional help with ORF experiments was provided by X.Y., D.E.R. and O.A.
Clinical samples were collected or experiments performed by C.M.J., A.P.C.,
K.T.F., R.D. and J.A.W. Large scale cell genomic and expression profiling
along with pharmacological screening efforts were designed and data analysed
by N.S., J.B., G.C., S.K., J.H., C.J.W., V.E.M., P.M.F., B.L.W., W.R.S., R.S.
and L.A.G. C.M.J. and L.A.G. wrote the manuscript. All authors discussed
results and edited the manuscript.

Competing interests
C.M.E., G.C., S.K., J.L.H., C.J.W., V.E.M., P.M.F., B.W., W.R.S. and R.S. are
employees of Novartis Pharmaceuticals, Inc. J.J.Z., T.M.R., W.C.H. and
L.A.G. are consultants for Novartis Pharmaceuticals, Inc. L.A.G. is a
consultant and shareholder of Foundation Medicine. All other authors declare
no competing financial interests.

Corresponding author
Correspondence to Levi A. Garraway.

Supplementary information
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1. 1.

Supplementary Information
This file contains Supplementary Figures 1-19 with legends and
Supplementary Tables 1-3.

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Editorial Summary
Drug-resistance mechanism in melanoma
Clinical trials in melanoma patients carrying B-RAF gene mutations have
shown promising results with the B-RAF kinase inhibitor show more

Associated Content
Nature | News and Views
Drug discovery: How melanomas bypass new therapy

David Solit
& Charles L. Sawyers

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1. Abstract
2. References
3. Acknowledgements
4. Author information
5. Supplementary information

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