Abstract
Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAFV600E melanoma. Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors. We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC50) and maximal effect (i.e., Emax ≪ 1). Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing. This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug Emax. Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.
Highlights
Activation of BRAF via a V600E mutation is the most prevalent genetic change in human melanoma, found in at least 50% of tumors
To profile adaptive responses in melanoma, we applied single-cell phenotypic and multiplex biochemical assays to nine BRAFV600E and one BRAFV600D lines exposed to four RAF inhibitors and one MEK inhibitor at multiple doses and times (Fig 1A)
To monitor the effect of vemurafenib at the level of signaling, we measured S6 phosphorylation (Ser235/236). pS6 is a marker of TORC1 activity (Magnuson et al, 2012; Corcoran et al, 2013), a multiprotein complex controlled by signaling cascades such as MAPK, PI3K/AKT, and LKB1/AMPK (Roux et al, 2004; Shaw et al, 2004; Magnuson et al, 2012) involved in adaptation to vemurafenib. pS6 levels have previously been proposed as a effective predictor of resistance of melanoma cells to vemurafenib-induced apoptosis (Corcoran et al, 2013), and we found pS6(Ser235/236) levels 24–48 h after drug treatment to be the best single predictor of apoptosis across cell lines, drugs, and doses
Summary
Activation of BRAF via a V600E (or V600D) mutation is the most prevalent genetic change in human melanoma, found in at least 50% of tumors. The BRAFV600E oncoprotein constitutively activates pro-mitogenic RAF/MEK/ERK signaling (Davies et al, 2002; Fecher et al, 2008), and therapy with RAF inhibitors such as vemurafenib (Zelborafâ; PLX4032) causes tumor regression in many patients (Bollag et al, 2010; Chapman et al, 2011; Flaherty et al, 2012; Sosman et al, 2012). Resistance usually involves the activation of progrowth/survival mechanisms that increase BRAFV600E activity (Shi et al, 2012) or bypass the need for it altogether. Many mutations involved in acquired resistance have been identified, including NRASQ61K, MEK1F129L, MEK2Q60P, or AKT1Q79K (Nazarian et al, 2010; Wang et al, 2011; Shi et al, 2014a; Wagle et al, 2014), and aberrant splicing of BRAFV600E (Poulikakos et al, 2011). Resistance is associated with elevated IGF1 receptor/PI3K signaling (Villanueva et al, 2010), COT overexpression (Johannessen et al, 2010), and PDGFRb up-regulation (Nazarian et al, 2010)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
