BRAFV600E as a Therapeutic Target in Colorectal Cancer
The BRAF serine/threonine kinase is a member of the Raf kinase family consisting of A-Raf, B-Raf and C-Raf or Raf-1.23
Although all
three Raf isoforms share considerable sequence homology and exhibit the same substrate specificity (MEK1 and MEK2), they differ in their biological functions and regulations, which have not been fully elucidated.24
and subsequent high-throughput Our work
Figure 1: Oncogenic Signalling of the ERK–Mitogen- activated Protein Kinase Pathway in Colorectal Cancer
Ligands
EGFR overexpression (~50%)
Raf remains the best characterised activator of MEK, with BRAF being the most potent MEK activator of the Raf isoforms. The Raf family of genes were first identified as potent retrovirus oncogenes in 1984,25,26
genomic sequencing has identified activating mutations in BRAF as the predominant genetic alterations in human cancers.27,28
and that of others have found that KRAS and BRAF mutations almost never occur in the same tumour, suggesting not only that BRAF is the principal effector of KRAS in the MAPK pathway, but also that they may be equivalent in their tumorigenic effects.16,18,19,29,30
The discovery of BRAF mutations in various human cancers has stimulated further intensive research of this gene. The most common mutation in BRAF, accounting for up to 90% of all BRAF mutations in human cancers, is a thymidine-to-adenine transversion at nucleotide 1799 in the kinase domain of the protein resulting in a V600E amino acid (valine-to-glutamate) exchange.31,32
This mutation leads to constitutive
activation of the MAPK signalling cascade with the mutated protein demonstrating greatly elevated kinase activity, and potently transforms rodent fibroblasts and other cell types.9,27
Furthermore, while mutant
BRAFV600E cells were shown to be dependent on continued BRAF activity for their tumorigenic growth,33–35 for proliferation.27
they do not require Ras function
Subsequent studies have shown that BRAFV600E mutations occur mainly in tumours with hypermethylation of hMLH1 promoter causing transcriptional silencing of the hMLH1 gene and hence defective DNA mismatch repair (dMMR) and the MSI phenotype.17,37
By
contrast, BRAFV600E mutations rarely occur in tumours with dMMR attributable to the presence of germ-line mutations, indicating that BRAFV600E mutations are not a direct result of defective DNA mismatch repair.37
The complex relationships between BRAF, MSI and CIMP are the subject of ongoing investigation and are beyond the scope of this article.
Targeting BRAFV600E in Colorectal Cancer
The unfulfilled promise of targeting KRAS in cancer has shifted the focus of therapeutic development to its downstream effector, BRAF.32 Moreover, KRAS is central to a complicated network of signal transduction pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway, characterised by cross-talks and feedback loops.32 BRAF, on the other hand, would appear to be a ‘purer’ target with a relatively unidirectional MEK–ERK effector pathway. The finding that tumour cells and xenografts harbouring the BRAFV600E mutation were extremely sensitive to MEK inhibition compared with those without this mutation or those bearing the Ras mutation further highlights the dependency of BRAFV600E mutant cancer cells on MEK–ERK signalling.38
Consistent with this is the pre-clinical work with a specific
BRAFV600E inhibitor, PLX4032, which demonstrated the selective sensitivity of melanoma and colorectal cancer cell-lines and xenografts harbouring BRAFV600E mutation to BRAFV600E inhibition compared with BRAF wild-type cells or xenografts.10
The first ‘proof-of-concept’ clinical data validating BRAFV600E mutation as a therapeutic target came from the preliminary result
EUROPEAN ONCOLOGY
Interestingly, BRAFV600E mutations were found to be associated with colorectal cancers exhibiting the microsatellite instability (MSI) phenotype and CpG island methylator phenotype
(CIMP).16,29,36
EGFR
p p
p p
SoS Grb
Raf MEK
ERK Proliferation Survival Migration Angiogenesis
Epidermal growth factor receptor (EGFR) overexpression and mutation in KRAS and BRAFV600E result in constitutive activation of this signalling cascade in colorectal cancer, ultimately affecting nuclear targets involved in regulating cell proliferation, differentiation, survival, migration and angiogenesis. KRAS and BRAFV600E mutation rarely co-exist in the same tumour, suggesting that Raf is the principal effector of Ras in tumorigenesis.
of the phase I study of PLX4032 in advanced melanoma that was presented at the 45th American Society of Clinical Oncology (ASCO) annual meeting.10
Mutually exclusive
BRAFV600E
mutation (10%) Ras KRAS mutation (30–50%)
Notably, patients in the initial cohorts were not
stratified by BRAFV600E mutational status; of the 21 melanoma patients treated at the ≥240mg twice daily dose level (minimum target dose for tumour regression), 16 carried the BRAFV600E mutation and five did not. The efficacy data were extremely promising with nine partial responses, all seen in tumours carrying the BRAFV600E mutation. The interim progression-free survival (PFS) for patients with BRAF-mutated melanoma was six months, with many patients still on treatment, while all five patients with BRAF wild-type melanomas had progressive disease. The safety data were encouraging, with the majority of adverse events being mild and transient. The most common side effects were rash, fatigue and photosensitivity. Cutaneous squamous cell carcinoma following chronic dosing was also observed. Initial data suggesting a favourable therapeutic index and selectivity of this BRAFV600E inhibitor therefore provide an appealing therapeutic strategy for BRAFV600E-mutated cancers. This study is currently being expanded to include advanced melanoma and colorectal cancer harbouring the BRAFV600E mutation.
Unlike melanoma, where BRAFV600E mutations are frequently seen (43–66%), the prevalence of BRAFV600E mutations in colorectal cancer is considerably lower, at about 10%. This poses a unique set of challenges in the clinical development of BRAF inhibitors in the colorectal cancer patient population, as large numbers of patients will need to be screened to select for patients who will be carrying this mutation. Therefore, being able to enrich the population screened by selecting patients with clinical features associated with the BRAFV600E mutation may assist in drug development and allow for more efficient utilisation of resources.
Defining Colorectal Patient Subgroups with Higher Frequencies of BRAFV600E mutation
We designed a study specifically to address this matter.18
Five
hundred and twenty-five colorectal cancer cases were identified from a prospectively collected clinical database and analysed for BRAFV600E, KRAS mutational and MSI status. Patients were selected
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