Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR

TLDR: An RNA-interference-based genetic screen in human cells is performed to search for kinases whose knockdown synergizes with BRAF(V600E) inhibition, and it is reported that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAFs inhibition.

Abstract: Inhibition of the BRAF(V600E) oncoprotein by the small-molecule drug PLX4032 (vemurafenib) is highly effective in the treatment of melanoma. However, colon cancer patients harbouring the same BRAF(V600E) oncogenic lesion have poor prognosis and show only a very limited response to this drug. To investigate the cause of the limited therapeutic effect of PLX4032 in BRAF(V600E) mutant colon tumours, here we performed an RNA-interference-based genetic screen in human cells to search for kinases whose knockdown synergizes with BRAF(V600E) inhibition. We report that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAF(V600E) inhibition. We find in multiple BRAF(V600E) mutant colon cancers that inhibition of EGFR by the antibody drug cetuximab or the small-molecule drugs gefitinib or erlotinib is strongly synergistic with BRAF(V600E) inhibition, both in vitro and in vivo. Mechanistically, we find that BRAF(V600E) inhibition causes a rapid feedback activation of EGFR, which supports continued proliferation in the presence of BRAF(V600E) inhibition. Melanoma cells express low levels of EGFR and are therefore not subject to this feedback activation. Consistent with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to cause resistance to PLX4032. Our data suggest that BRAF(V600E) mutant colon cancers (approximately 8-10% of all colon cancers), for which there are currently no targeted treatment options available, might benefit from combination therapy consisting of BRAF and EGFR inhibitors.

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09 August 2022
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Original Citation:
Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR
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DOI:10.1038/nature10868
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Questa è la versione dell’autore dell’articolo:
Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback
activation of EGFR.
Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, Beijersbergen
RL, Bardelli A, Bernards R.
Nature. 2012 Jan 26;483(7387):100-3. doi: 10.1038/nature10868.
The final version is available at:
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http://www.nature.com/nature/journal/v483/n7387/full/nature10868.html

Unresponsiveness of colon cancer to BRAF
V600E
inhibition through
feedback activation of EGFR
Anirudh Prahallad
1,a
, Chong Sun
1,a
, Sidong Huang
1,a
, Federica Di Nicolantonio
2,3,a
, Ramon
Salazar
4
, Davide Zecchin
2
, Roderick L. Beijersbergen
1
, Alberto Bardelli
2,3
and René
Bernards
1,b
1
Division of Molecular Carcinogenesis, Center for Biomedical Genetics and Cancer
Genomics Centre,
The Netherlands Cancer Institute,
Plesmanlaan 121, 1066 CX Amsterdam,
The Netherlands.
2
Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment, University
of Torino, Medical School, Str prov 142 Km 3.95, 10060 Candiolo, Torino, Italy.
3
FIRC-IFOM Institute for Molecular Oncology, Via Adamello 16, 20100 Milan, Italy.
4
IDIBELL, Institut Català d'Oncologia L'Hospitalet de Llobregat, Spain.
a
These authors contributed equally to this work
b
Corresponding author:
René Bernards, Phone: +31 20 512 1952, E-mail: r.bernards@nki.nl

2
Inhibition of the BRAF
V600E
oncoprotein by the small molecule drug PLX4032
(vemurafenib) is highly effective in the treatment of melanoma
1
. However, colon cancer
patients harbouring the same BRAF
V600E
oncogenic lesion have poor prognosis and show
only a very limited response to this drug
2-4
. To investigate the cause of the limited
therapeutic effect of vemurafenib in BRAF
V600E
mutant colon tumours, we performed a
RNA interference-based genetic screen to search for kinases whose knockdown
synergizes with BRAF
V600E
inhibition. We report here that blockade of the epidermal
growth factor receptor (EGFR) shows strong synergy with BRAF
V600E
inhibition. We
find in multiple BRAF
V600E
mutant colon cancers that inhibition of EGFR by the
antibody drug cetuximab or the small molecule drugs gefitinib or erlotinib is strongly
synergistic with BRAF
V600E
inhibition, both in vitro and in vivo. Mechanistically, we find
that BRAF
V600E
inhibition causes a rapid feedback activation of EGFR, which supports
continued proliferation in the presence of BRAF
V600E
inhibition. Melanoma cells express
low levels of EGFR and are therefore not subject to this feedback activation. Consistent
with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to
cause resistance to PLX4032. Our data suggest that BRAF
V600E
mutant colon cancers
(some 8-10% of all colon cancers
2,3
5
) for whom there are currently no targeted
treatment options available might benefit from combination therapy consisting of
BRAF- and EGFR inhibitors.

3
Activating mutations in the BRAF oncogene (BRAF
V600E
) are seen in some 70% of
primary melanomas
6
, some 10% of colorectal cancers
7
and in some 30-70% of papillary
thyroid carcinoma
8-10
. However, the clinical responses to the highly selective small molecule
inhibitor PLX4032 (vemurafenib) of the BRAF
V600E
oncoprotein differ widely, ranging from
some 80% in melanoma to only 5% in BRAF mutant colorectal cancer
2-4
. To investigate the
molecular mechanism responsible for the intrinsic resistance of BRAF
V600E
colorectal cancers
(CRC) to PLX4032, we first tested a panel of BRAF
V600E
mutant melanoma and CRC cell
lines for their response to PLX4032. We found that the sensitivity of melanoma and colon
cancer cells in both short term (Fig. 1A) and long term (Fig. 1B) proliferation assays in vitro
mirrors the clinical experience, with melanoma cells being more sensitive to PLX4032 than
CRC cells.
RNA interference genetic screens have been used successfully to identify genes that
enhance a phenotype
11
. We therefore set out to screen a short hairpin RNA (shRNA) library
representing the full complement of 518 human kinases
12
(the “kinome”) and 17 additional
kinase-related genes (Table S1) for genes whose inhibition confers sensitivity to PLX4032 in
BRAF
V600E
mutant CRC. WiDr cells were infected with the lentiviral kinome shRNA
collection and cultured in the absence or presence of PLX4032 for 10 and 18 days,
respectively. After this, the relative abundance of shRNA vectors was determined by next
generation sequencing of the bar code identifiers present in each shRNA vector (Fig. 1C, see
methods). We arbitrarily considered only shRNA vectors that had been sequenced at least 300
times and which were depleted at least five-fold by the drug treatment. Fig. 1D shows that
only very few of the 3388 shRNA vectors in the library met this stringent selection criterion,
among which were three independent shRNA vectors targeting the Epidermal Growth Factor
Receptor (EGFR, see Table S2 for all selected shRNAs). This suggested that suppression of
EGFR synergizes with BRAF inhibition in these CRC cells. To validate this finding, we
infected WiDr cells with each of these three EGFR shRNA vectors (all of which reduced
EGFR levels (Fig. 1F)) and cultured these cells with or without PLX4032 for two weeks. Fig.

Frequently asked questions

Q1. What have the authors contributed in "Unresponsiveness of colon cancer to braf inhibition through feedback activation of egfr" ?

Use of all other works requires consent of the right holder ( author or publisher ) if not exempted from copyright protection by the applicable law. Availability: This is the author 's manuscript 

Q2. What is the effect of tovemurafenib on EGFR?

Since melanomas arederived from the neural crest, the authors reasoned that the favourable response of melanomas tovemurafenib might result from the paucity of EGF receptors on these tumours and hence the6absence of the feedback activation of EGFR by BRAF inhibition. 

Q3. What is the effect of EGFR on melanoma cells?

The authors transduced EGFR-negative A375 and SK-MEL-28 melanoma cells with a retroviral EGFR expression vectorand subjected these cells to treatment with PLX4032 monotherapy or combination withEGFR drug. 

Q4. What is the effect of PLX4032 on EGFR?

PLX4032 treatmentinhibited MEK and ERK activation downstream of BRAF but activated AKT, which actsdownstream of EGFR in a pathway parallel to BRAF. 

Q5. What drugs are available to treat EGFR?

two classes of anti EGFR drugs are clinically available; these include themonoclonal antibodies cetuximab and panitumumab and the small molecule kinase inhibitorsgefitinib and erlotinib. 

Q6. What is the reason for combining a targeted agent in a clinical trial?

Given that resistance to monotherapy with these targeted therapies oftendevelops rapidly, there is a trend towards combining targeted agents in clinical trials. 

Q7. What is the mechanism of EGFR inhibition in colon cancer?

To address the molecular mechanism underlying the synergy between BRAF andEGFR inhibition in colon cancer, the authors tested lysates of drug-treated cells with phosphoprotein-specific antibodies that identify the activated state of components of the EGFR signallingpathway. 

Q8. What is the reason for the poor clinical outcome of BRAFV600E mutants?

As BRAFV600E mutations are also common in thyroid papillary carcinomas and hairycell leukemias, EGFR expression levels may also help guide the selection of EGFRcombination therapy in these cancers 8-10,19. 

Q9. What was the funding for this project?

Major financial support for thiswork was provided by the EU Seventh Framework Programme, grant agreement 259015 (toA.B. and R.B.). 

Q10. What is the molecule that causes the rapid feedback activation of EGFR?

the authors findthat BRAFV600E inhibition causes a rapid feedback activation of EGFR, which supportscontinued proliferation in the presence of BRAFV600E inhibition. 

Q11. What is the reason for the poor clinical outcome of BRAFV600E mutant colon cancers?

The strong synergistic interaction betweeninhibition of BRAF and EGFR described here is explained by an unexpected and powerfulfeedback activation of EGFR caused by BRAF inhibition, providing a rationale for the poorclinical response of BRAFV600E mutant colon cancers to vemurafenib monotherapy 4. Thefeedback activation of EGFR also implies that EGFR expression levels may be a clinically-useful biomarker to predict the response to vemurafenib monotherapy in BRAF mutanttumours. 

Q12. What is the way to test the EGFR levels in melanoma cells?

Tendays after injection of tumour cells, palpable tumours were present in all animals and cohortsof mice were treated with vehicle, the EGFR targeted drugs cetuximab or erlotinib, the BRAFinhibitor PLX4720 (highly related to PLX4032, but easier to formulate for in vivo use), or thecombination of EGFR inhibitor drug plus PLX4720 (see methods). 

Q13. What is the role of CDC25C in the phosphorylation of EGFR?

treatment of WiDr cells with PLX4032 inhibited phosphorylation of CDC25C atThr48 (Fig. 2F), which has been shown to be required for its phosphatase activity 15. 

Q14. What is the role of CDC25C in the feedback regulation of EGFR?

these data are consistent with a model in which BRAF inhibition leads to inhibitionof MEK and ERK kinases, which in turn leads to a reduced activation of CDC25C. 

Q15. What is the role of MEK in the feedback activation of EGFR?

Inagreement with a central role for MEK in mediating the feedback activation of EGFR, thecombination of MEK and EGFR inhibitors also synergized to inhibit growth of VACO432 orWiDr cells (Fig. S3 and data not shown).