BRAF mutations occur in a significant proportion of human tumors, and represent a mechanism of constitutive activation of the MAPK pathway. We have demonstrated that activating mutations of BRAF confer sensitivity to small molecule inhibitors of the pathway. In contrast, we showed that HER2-overexpressing breast carcinomas were resistant to MEK inhibition, despite effective pharmacologic inhibition of MAPK activity. Our preliminary data suggest that tumors with HER kinase activation (and WT BRAF) and those with oncogenic BRAF have similar levels of phosphorylated ERK;however, BRAF mutant tumors have higher levels of phosphorylated MEK and selected MEK-ERK dependent transcripts. Further, MEK inhibitor-induced feedback upregulation of the pathway is seen only in the receptor-activated tumors, but not in tumors with activating BRAF mutation. We hypothesize that increased output of the MAPK pathway in B- RAF mutant tumors compared to HER kinase-activated tumors is due to the impairment of feedback inhibition of the pathway, upstream of, and/or at the level of, RAF. We hypothesize that disabling of upstream feedback in BRAF mutant tumors causes an increase in pathway throughput, resulting in increased expression of ERK effectors, and targets responsible for pathway feedback (DUSP, SPRY proteins). This increase in DUSPs (MAP kinase phosphatases) may be critical for the downregulation of ERK to physiologically tolerated levels. The increase in both feedback and effector proteins may together be responsible for aspects of the transformed phenotype. In this proposal, we describe further preliminary data which support these assertions, and describe a research plan to determine the mechanism of feedback response to MEK inhibition. We will determine whether phosphorylated ERK represents an accurate reflection of pathway activation. We will use small molecule inhibitors of the pathway, as well as RNA interference to determine the role of critical proteins in the feedback program. Finally, we will develop isogenic systems transformed by oncogenic BRAF and activated receptor tyrosine kinases to determine if specific feedback and effector protein expression patterns can be generated. The translational goals of these studies are to identify feedback pathways modulating the response to RAF and MEK inhibitors which will impact the effectiveness of these compounds in clinical trials.

Public Health Relevance

The RAS-RAF-MEK-ERK signaling pathway is an important regulator of tumor cell growth and proliferation. The goals of this research proposal are to understand feedback mechanisms which regulate the activation of this pathway. Understanding this regulatory mechanism will help investigators use drugs which target this pathway to select patients whose tumors are most likely to respond to them. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

National Institute of Health (NIH)
National Cancer Institute (NCI)
Clinical Investigator Award (CIA) (K08)
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Subcommittee G - Education (NCI)
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Myrick, Dorkina C
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Sloan-Kettering Institute for Cancer Research
New York
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Ambrosini, Grazia; Pratilas, Christine A; Qin, Li-Xuan et al. (2012) Identification of unique MEK-dependent genes in GNAQ mutant uveal melanoma involved in cell growth, tumor cell invasion, and MEK resistance. Clin Cancer Res 18:3552-61
Ercan, Dalia; Xu, Chunxiao; Yanagita, Masahiko et al. (2012) Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors. Cancer Discov 2:934-47
Lito, Piro; Pratilas, Christine A; Joseph, Eric W et al. (2012) Relief of profound feedback inhibition of mitogenic signaling by RAF inhibitors attenuates their activity in BRAFV600E melanomas. Cancer Cell 22:668-82
Pratilas, Christine A; Xing, Feng; Solit, David B (2012) Targeting oncogenic BRAF in human cancer. Curr Top Microbiol Immunol 355:83-98
Xing, F; Persaud, Y; Pratilas, C A et al. (2012) Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring (V600E)BRAF. Oncogene 31:446-57
Joseph, Eric W; Pratilas, Christine A; Poulikakos, Poulikos I et al. (2010) The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc Natl Acad Sci U S A 107:14903-8
Dry, Jonathan R; Pavey, Sandra; Pratilas, Christine A et al. (2010) Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244). Cancer Res 70:2264-73
Pratilas, Christine A; Solit, David B (2010) Targeting the mitogen-activated protein kinase pathway: physiological feedback and drug response. Clin Cancer Res 16:3329-34