Cancer often arises when the control of normal cell function goes awry due to defects in critical signal transduction pathways. The signaling pathway regulated by the RasGTPase is one such pathway, and it functions to modulate vital cellular processes, including proliferation, differentiation, survival, and senescence. Members of the Raf serine/threonine kinase family are key intermediates in the Ras pathway, serving to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. There are three mammalian Raf proteins, A-Raf, B-Raf, and C-Raf (also known as Raf-1). As might be expected for proteins so centrally involved in cell signaling, the Raf kinases can directly contribute to oncogenic transformation and other human disease states. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently results in constitutive signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. In addition, mutations in the Raf proteins themselves can function as disease drivers. Germline-mutations in C-Raf are causative for Noonan and LEOPARD syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) syndromes, with B-Raf mutations occurring in 75% of CFC patients. Moreover, somatic mutations in B-Raf are observed in 70% of malignant melanomas as well as in many colorectal, ovarian, lung and papillary thyroid carcinomas. Our research has elucidated several important mechanisms contributing to the regulation of both normal and mutant Raf signaling. Our studies have revealed that the KSR1 scaffold plays a critical role in modulating the intensity and duration of Raf signaling emanating from the plasma membrane in response to growth factor treatment. In addition, our work has provided insight regarding how the KSR1 scaffold is recruited from the cytosol to the plasma membrane in response to growth factor signals. Through structure/function analysis, our studies showed that the conserved CA1 region of KSR1 forms an extended sterile-alpha-motif domain, which upon growth factor signaling, becomes exposed and binds phospholipids found in the plasma membrane. As a result, KSR1-bound MEK is localized to the cell surface where it can be phosphorylated by activated Raf kinases. Finally, our studies have revealed that the expression levels of KSR1 can alter the effects of ATP-competitive Raf inhibitors on oncogenic Ras to ERK signaling. Specifically, KSR1 competes with C-Raf for inhibitor-induced binding to B-Raf and in doing so attenuates the paradoxical activating effect of these drugs on ERK signaling. In regard to the regulation of the Raf proteins themselves, our studies have shown that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events (e. g., phosphorylation on inhibitory feedback sites and the phosphorylation of residues that mediate 14-3-3 binding) and protein interactions (e.g., 14-3-3 binding and Raf dimerization). Moreover, we have found that Raf dimerization is critical for upregulated signaling induced by human disease-associated Raf mutants with moderate, low or impaired kinase activity, or in cases where the pathway is induced by activated RTK or Ras proteins. Our work has further revealed that somatic mutations which modulate Raf dimerization have the potential to alter the progression and treatment of human disease states with elevated Ras pathway signaling. In addition, our reasearch provided the first 'proof of principle' that inhibiting Raf dimerization can suppress Raf signaling under conditions where dimerization is required. Taken together, these findings have important implications for the treatment of human disease states with elevated Ras pathway signaling and identify the Raf dimer interface as a therapeutic target. Finally, our recent studies have uncovered a previously unknown rote for the inhibition of the Ras/Raf/MEK/ERK signaling that is mediated by the stress-activated JNK cascade. We have found that key Ras pathway compenents, including the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in resonse to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as Rigosertib and Paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and my function as a signaling checkpoint to suppress Ras pathway signaling during conditions of cellular stress.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010329-18
Application #
9556265
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Durrant, David E; Morrison, Deborah K (2018) Targeting the Raf kinases in human cancer: the Raf dimer dilemma. Br J Cancer 118:3-8
Neiswender, James V; Kortum, Robert L; Bourque, Caitlin et al. (2017) KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling. Cancer Res 77:5820-5830
Holderfield, Matthew; Morrison, Deborah K (2016) RAS signaling: Divide and conquer. Nat Chem Biol 13:7-8
Ritt, Daniel A; Abreu-Blanco, MarĂ­a T; Bindu, Lakshman et al. (2016) Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-Induced Phospho-Regulatory Circuit. Mol Cell 64:875-887
Zhou, Bingying; Ritt, Daniel A; Morrison, Deborah K et al. (2016) Protein Kinase CK2? Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2? Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma. J Biol Chem 291:17804-15
Kortum, Robert L; Morrison, Deborah K (2015) Path Forward for RAF Therapies: Inhibition of Monomers and Dimers. Cancer Cell 28:279-81
Freeman, Alyson K; Ritt, Daniel A; Morrison, Deborah K (2013) The importance of Raf dimerization in cell signaling. Small GTPases 4:
Freeman, Alyson K; Ritt, Daniel A; Morrison, Deborah K (2013) Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. Mol Cell 49:751-8
Morrison, Deborah K (2012) MAP kinase pathways. Cold Spring Harb Perspect Biol 4:
Logue, Jeremy S; Morrison, Deborah K (2012) Complexity in the signaling network: insights from the use of targeted inhibitors in cancer therapy. Genes Dev 26:641-50

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