Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation

Abstract

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ~7% of human malignancies and ~60% of melanomas1. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses2. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways3–5. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistantsubsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Reliable detection of subclonal single-nucleotide variants in tumour cell populations

“…Together, these findings provide compelling evidence for clonal evolution as a general mechanism in cancer development. Quantifying subclonal diversity in tumours is important for understanding the driving forces of their evolution, and sensitive methods are required for detecting low-frequency drug-resistant mutations before treatment 28 .…”

Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma

“…5 Finally, it is important to underline that NRASQ61R has been reported to correlate with resistance to BRAF inhibitors in preclinical and clinical data. 8,16 Because testing for NRAS mutations currently requires the use of molecular techniques that are not available in most diagnostic pathology laboratories, testing may require a dispending laborious process before NRAS testing can commence. Furthermore, these molecular techniques are time consuming and Figure 4 Intratumoral heterogeneity shown by N-Ras (Q61R) antibody (original magnification, Â 2.5; square boxes original magnification, Â 40).…”

“…8 Hence, testing for NRAS may be an integral part in the assessment of metastatic melanoma patients. Several molecular techniques have been developed to detect NRAS mutation such as conventional Sanger sequencing, pyrosequencing, or expensive and more sophisticated techniques including high-throughput multiplexed mass spectrometry-based platforms or targeted next-generation sequencing technologies.…”

BRAF mutations in non-small cell lung cancer: has finally Janus opened the door?

“…Several mechanisms of resistance to B-Raf inhibitors have been described in melanoma, such as activation of PI3 K and COT pathways, and N-Ras mutations (Villanueva et al, 2010;Nazarian et al, 2010;Johannessen et al, 2010). By activating parallel pathways involved in cell growth, cancer cells are able to overcome the blockade at MAPK pathway.…”