Abstract LB-419: An in-frame deletion in the N-terminal regulatory domain of BRAF(V600E) causes resistance to the RAF inhibitor PLX4032
Authors: Poulikos I. Poulikakos, Yogindra Persaud, May Tal Gabay, Manickam Janakiraman, Eric W. Joseph, Christine A. Pratilas, Paul B. Chapman, Neal Rosen, David B. Solit
Journal: Cancer Research (2011)

Abstract Drugs that compete with ATP for binding to RAF selectively inhibit ERK signaling in tumor cells with BRAF(V600E) mutation. In BRAF wild-type cells, these compounds induce ERK signaling. RAF family members form dimers in a RAS-dependent manner. In BRAF wild-type cells, binding of RAF inhibitors to one member of the dimer transactivates the other, non-bound member. In contrast, in tumors with BRAF(V600E) mutation, RAS activity is too low to support dimer formation and the drug thus inhibits ERK signaling. This property of RAF inhibitors such as PLX4032 is believed to account for the wide therapeutic index of this drug and its remarkable clinical activity in patients with BRAF(V600E) melanomas. However, despite the initial effectiveness of PLX4032, resistance invariably develops. The dimerization model predicts that induction of RAS activity in the tumor will confer resistance. RAS mutation and activation of receptor tyrosine kinases (RTK) including PDGFRß and IGF1R have been implicated, as has induction of expression of the COT kinase, which drives RAF-independent ERK signaling. In order to identify novel mechanisms of RAF-inhibitor resistance, we generated PLX4032-resistant cell lines by prolonged exposure of sensitive BRAF mutant melanoma cells to the RAF inhibitor PLX4032. Analysis revealed that resistance of these clones was associated with failure of the drug to inhibit ERK signaling. PLX4032-resistant cells remained dependent on ERK signaling, as growth was inhibited by a MEK inhibitor, albeit at slightly higher doses. We did not detect RAS mutations, upregulation of receptor tyrosine kinases (RTK), or of other MEK kinases (CRAF, COT, Mos) in these cells. In a subset of resistant clones, we identified the expression of a variant form of BRAF(V600E), with an in-frame deletion within the N-terminal regulatory domain and a molecular weight of about 61KD (p61BRAF(V600E)). The N-terminal domain of RAF has been shown to negatively regulate the C-terminal catalytic domain, at least in part, by preventing dimerization. When expressed in 293H cells with intrincically low levels of RAS.GTP, dimerization of p61BRAF(V600E) is elevated compared to that of full-length BRAF(V600E). Expression of p61BRAF(V600E) in 293H cells rendered ERK signaling insensitive to inhibition by PLX4032, whereas a mutation in the dimerization domain of p61BRAF(V600E) restored sensitivity. Thus, in addition to BRAF gatekeeper mutations, there are at least three potential mechanisms that confer resistance by blunting the inhibition of RAF kinase by the inhibitor, all of which promote RAF dimerization: increased RAS.GTP due to RAS mutation or upstream (RTK) activation, RAF overexpression and deletion of the RAS binding domain within the N-terminus of RAF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-419. doi:10.1158/1538-7445.AM2011-LB-419