Targeting the -Activated Protein Pathway in the Treatment of Malignant Melanoma David J. Panka, Michael B. Atkins, and James W. Mier

Abstract The mitogen-activated protein kinase (MAPK; i.e., Ras ^ Raf ^ Erk) pathway is an attractive target for therapeutic intervention in melanoma due to its integral role in the regulation of proliferation, invasiveness, and survival and the recent availability of pharmaceutical agents that inhibit the various and GTPases that comprise the pathway. Genetic studies have identified activating mutations in either B-raf or N-ras in most cutaneous melanomas. Other studies have delineated the contribution of autocrine growth factors (e.g., hepatocyte growth factor and fibroblast growth factor) to MAPK activation in melanoma. Still, others have emphasized the consequences of the down-modulation of endogenous raf inhibitors, such as Sprouty family members (e.g., SPRY2) and raf-1kinase inhibitory protein, in the regulation of the pathway. The diversity of molecular mechanisms used by melanoma cells to ensure the activity of the MAPK pathway attests to its importance in the evolution of the disease and the likelihood that inhibitors of the pathway may prove to be highly effective in melanoma treatment. MAPK inhibition has been shown to result in the dephosphorylation of the proapoptotic Bcl-2 family members Bad and Bim. This process in turn leads to activation and, ultimately, the demise of melanoma cells through the induction of . Several recent studies have identified non ^ mitogen-activated protein/ extracellular signal-regulated kinase kinase ^ binding partners of raf and suggested that the prosurvival effects of raf and the lethality of raf inhibition are mediated through these alternative targets, independent of the MAPK pathway. Other studies have suggested that endothelial cells are the primary targets of raf inhibitors in vivo and that the antitumor effect of these agents are largely attributable to angiogenesis inhibition.This article reviews the genetic and biochemical factors contributing to MAPK activation in melanoma, the mechanisms by which inhibition of the pathway might prove deleterious to tumor cells, and the potential of MAPK inhibitors in the treatment of the disease.

Activation of the Mitogen-Activated Protein T1796A transversion that results in the substitution ofa Kinase Pathway in Melanoma glutamate for valine at position 600 (V600E) is by far the most common such mutation. This single change The mitogen-activated protein kinase (MAPK) pathway is renders the kinase constitutively active, resulting in the activated in virtually all melanomas (1). Several factors phosphorylation ofMEK and targets further downstream contribute to this phenomenon, including mutations in the (Fig. 1). This particular genetic alteration is not one predicted genes ofkey kinases or G proteins and the absence of to result from the failure to repair UV light–induced endogenous inhibitors that would otherwise constrain the pyrimidine dimers, and its connection with sun exposure is activity ofthis signaling pathway. For example, f60% of therefore unclear (2). Although these B-raf mutations play cutaneous melanomas have an activating mutation in the an essential role in the ontogeny ofmelanoma, they are catalytic domain ofthe serine-threonine kinase B-raf(2, 3). A found in 80% of benign nevi (4) and are therefore by defi- nition insufficient to cause the disease in the absence of other factors. A few melanomas, primarily those lacking B-raf mutations, have activating mutations in N-ras, a small G protein previously Authors’ Affiliation: Division of Oncology, Beth Israel Deaconess Medical Center identified as a contributing oncogene in neuroblastomas (5). and Harvard Medical School, Boston, Massachusetts When GTP-bound, this GTPase binds and activates various raf Received 11/18/05; accepted12/13/05. isoforms, resulting in a signaling cascade qualitatively similar Grant support: Dana-Farber/Harvard Cancer Center Specialized Programs of Research Excellence in Skin Cancer grant P50 CA93683. to that activated by the aforementioned B-raf mutation. Presented at the First International Conference on Innovations and Challenges in Nonmutated ras and rafalso contribute to MAPK activation Melanoma, July 15-18, 2005, Cambridge, Massachusetts. in melanoma. Ras is GTP-loaded and c-rafis phosphorylated in Requests for reprints: James W. Mier, Beth Israel Deaconess Medical Center, melanoma (6), presumably through the action ofconstitutively 330 Brookline Avenue, Boston, MA 02215. Phone: 617-667-0430; Fax: 617-975- 8030; E-mail: [email protected]. produced growth factors, such as hepatocyte growth factor and F 2006 American Association for Cancer Research. fibroblast growth factor. The receptor for hepatocyte growth doi:10.1158/1078-0432.CCR-05-2539 factor, c-met, is frequently overexpressed on melanomas (7, 8),

www.aacrjournals.org 2371s Clin Cancer Res 2006;12(7 Suppl) April 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Fig. 1. MAPK activation in melanoma cells. As described in the text, the activity of this pathway is governed by the composite actions of oncogenic mutations in B-raf or N-ras (*) and autocrine growth factors, such as hepatocyte growth factor (HGF). It is further enhanced by the absence of raf inhibitory proteins, such as raf-1kinase inhibitory protein (RKIP) and SPRY2. Survival signals imparted by this pathway are mediated through the constraining effect of c-raf on proapoptotic kinases, such as ASK1and MST2, and the phosphorylation (inactivation) of various BH3-only Bcl-2 family members.When phosphorylated by ERK1and ERK2, BIM is targeted to the proteasome, where it is degraded.When phosphorylated by p90rs6k, Bad binds to14-3-3 and is sequestered in the cytosol.These prosurvival activities of the MAPK pathway are potentially reversible with pharmacologic agents that block raf or MEK. and neutralizing antibodies against hepatocyte growth factor diversity ofstrategies used by these cells to achieve this and fibroblast growth factor substantially reduce the level of objective. ERK phosphorylation in cultured melanoma cells (6). These data suggest that ERK phosphorylation in these cells is driven in part through an autocrine loop involving the interaction Mechanism of Apoptosis Induced by MAPK ofsecreted growth factorswith their respective membrane Inhibition tyrosine kinase receptors. The acquisition ofmutations in B-rafor N-ras is not the only The MAPK pathway regulates numerous cellular activities in strategy used by melanoma cells to ensure that the MAPK melanoma, including survival. Effective disruption of this pathway is constitutively active. In addition to these genetic pathway, either through pharmacologic inhibition or the alterations, melanomas frequently fail to express genes that deletion ofone or more key pathway components, results in encode proteins that negatively regulate ERK phosphorylation. cell death. Studies with small interfering RNAs targeting c-raf For example, raf-1 kinase inhibitory protein is conspicuously and B-rafhave affirmedthe critical role played by the B-raf V600E down-regulated in melanomas (9). The restored expression of oncogene in the maintenance ofmelanoma cell viability (14). this inhibitor reduces ERK phosphorylation in melanoma Other studies have shown the lethality ofMEK inhibitors in cells, indicating that the loss ofthis protein contributes to melanoma (15–17), suggesting that MAPK inhibition may MAPK activation in these cells. Members ofthe Sprouty (SPRY) be an important component ofthe mechanism by which the family are thought to play a similar role. Originally described as B-raf V600E knockdown induces apoptosis. One such study critical participants in the tracheal bifurcation that occurs implicated the dephosphorylation ofthe proapoptotic BH3- during Drosophila development, at least two members ofthe only Bcl-2 family member Bad as the key event leading to family (SPRY2 and SPRY4) have been shown to bind raf-1 and the death ofmelanoma cells fromMAPK inhibition (15). In wild-type B-raf, but not B-raf V600E, and inhibit their kinase this model, the ERK substrate (p90rsk) activity (10–12). A recent microarray study comparing the gene phosphorylates Bad on Ser75 (Fig. 1). This phosphoserine lies expression profiles of melanomas harboring the B-raf V600E within a consensus sequence that binds to the cytoplasmic mutation with those possessing only the wild-type allele protein 14-3-3, sequestering Bad as an inert complex in the specifically identified SPRY2 as a gene down-modulated in cytoplasm. In the presence ofa MEK inhibitor, ERK and p90 rsk melanomas lacking a mutated B-raf(13). This findingsuggests are inactive and Bad is dephosphorylated. Bad then dissociates that absence ofSPRY2 may contribute to the activity ofthe from 14-3-3 and translocates to the mitochondria, where it MAPK pathway in cells lacking a B-rafoncogene. Collectively, binds the antiapoptotic proteins Bcl-2 and Bcl-XL and under- these observations attest to the need ofmelanoma cells to mines their prosurvival function (15, 18, 19). Other studies maintain the activity ofthe MAPK pathway and the consequent suggest that the phosphorylation ofanother BH3-only Bcl-2

ClinCancerRes2006;12(7Suppl)April1,2006 2372s www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. MAPK Inhibitors in theTreatment of Melanoma family member Bim accounts for the prosurvival effect of MAPK inhibition and tumor involution (29), establishing the para- activation. When active, ERK1 and ERK2 phosphorylate Bim, digm of raf inhibition as a potentially effective treatment which targets the phosphoprotein to the proteasome where it is modality. Preclinical studies with the rafinhibitor sorafenib degraded (20, 21). Inhibition ofthe MAPK pathway results in have shown antitumor activity in vivo using tumor cells that are the accumulation ofBim and its association with the completely resistant to the drug in vitro (17), suggesting that, proapoptotic Bcl-2 family member Bax, resulting in the death in these cases, the antitumor activity might be entirely based ofthe cell. on the antiangiogenic activity ofthe drug. The extent to Although these models are consistent with the toxicity of which the inhibition of raf is responsible for these effects is MEK inhibitors, they do not provide an adequate explanation unclear, because the drug directly inhibits several membrane forthe lethality ofrafinhibitors such as sorafenibfor tyrosine kinase receptors (e.g., vascular endothelial growth melanoma cells. Studies by Panka et al. (22), for example, factor receptors I and II, and platelet-derived growth factor have shown that the knockdown ofBad offersno protection receptor-h) involved in angiogenesis (17). Recent studies with against sorafenib-induced apoptosis in most melanoma cell melanoma cells engineered to conditionally express a small lines examined, suggesting that the lethal effects of raf interfering RNA for B-raf V600E suggest that in established inhibition may not be entirely due to the inactivation ofthe tumors, the dominant antitumor effect associated with the loss MAPK pathway and the dephosphorylation and mitochondrial ofB-rafactivity may be the result ofdiminished vascular translocation ofBad. This notion is supported by the recent endothelial growth factor production by the tumor cells and identification of several raf-binding proteins unrelated to MEK reduced angiogenesis (30). Thus, it is likely that the adminis- (23–25) and numerous studies suggesting that rafcan function tration ofrafinhibitor to cancer patients will have both direct independently ofMEK (26, 27). For example, in developing effects on endothelial cell viability and indirect effects based on murine cardiac muscle cells, the prosurvival effect of c-raf is impaired growth factor production by tumor cells. attributable to its ability to bind and suppress apoptosis signal- regulating kinase-1 (ASK1), a kinase upstream ofc-Jun-NH 2- Discussion kinase (23), rather than its ability to activate MEK. The proapototic consequences ofa selective cardiac-targeted c-raf The remarkable diversity ofmolecular mechanisms enlisted deletion can be prevented by the concurrent deletion ofASK1, by melanoma cells to ensure the constitutive activation ofthe clearly implicating this stress-associated kinase in the myocyte MAPK pathway attests to the extent to which the integrity ofthis death observed in these genetically modified mice. Mammalian signaling cascade is essential to tumor cell growth and viability. sterile 20-like kinase 2 (MST2), another kinase whose activity is The critical role played by the MAPK pathway in cell survival is negatively regulated by raf, induces apoptosis through an further supported by multiple studies with small interfering elaborate signaling cascade involving the recently characterized RNAs and pharmacologic inhibitors demonstrating the adverse tumor suppressor Lats1 (28). The enhanced susceptibility of consequences ofrafor MEK blockade. Several agents that target À À raf-1 / cells to apoptosis is largely abrogated by MST2 this pathway are already undergoing clinical testing (reviewed depletion (24). Although these observations show that ASK1 elsewhere in this supplement) and some have already shown and MST2 play a role in the apoptosis induced by the deletion promise in clinical trials involving melanoma patients. ofc-raf,it must be kept in mind that the kinase activity ofc-raf Despite our increasing knowledge ofthe regulation ofthe is not required for the suppression of either ASK1 or MST2. It is MAPK pathway, it remains unclear how its inhibition results in therefore unclear if either of these kinases plays a role in the melanoma cell death. In the case ofrafinhibitors such as apoptosis induced by the pharmacologic inhibition ofraf sorafenib, it is unclear whether the critical lethal event induced activity. by the drug involves MEK/ERK, one or more ofthe recently identified non-MEK raf-binding kinases, or an as yet unchar- Angiogenesis Blockade through raf Inhibition acterized factor. It is even unclear whether the in vivo antitumor activity ofsorafenibis based on its antineoplastic or anti- Independent of their direct effects on tumor cells, drugs that angiogenic activities and to the extent its effects on the tumor inhibit the MAPK pathway may prove highly effective in vivo neovasculature predominate, whether they are mediated because oftheir ability to inhibit angiogenesis. The notion that through the inhibition ofrafor upstream tyrosine kinase this signaling pathway might be essential for vascular develop- receptors, such as those for vascular endothelial growth factor ment arose from the results of knockout experiments demon- and platelet-derived growth factor. It is hoped that ongoing strating early embryonic lethality, largely due to angiogenic laboratory and clinical studies will not only lead to the À À failure, in raf-1 / mice (26). These studies, however, showed clarification of these mechanistic details but also place us in a that the raf-1–deficient phenotype could be rescued through position to take optimal advantage ofthis emerging class of the introduction ofa mutant raf-1that does not activate the novel therapeutic agents. MAPK pathway (i.e., raf-1–YY340/341FF), suggesting that the effect of raf on endothelial cell survival could not be mediated Open Discussion through the conventional MAPK (i.e., Raf-MEK-ERK) pathway. Regardless ofthe identity ofthe particular rafsubstrate involved Dr. Atkins: What would you consider combining a raf in the promotion ofendothelial cell viability, it is clear that inhibitor with ifthe mechanism ofaction is, forexample, c-rafinhibition affectsnot only normal vascular development through apoptosis-inducing factor or the platelet-derived but tumor angiogenesis as well. Investigators in the Cheresh growth factor receptor? laboratory showed that a dominant-negative rafconstruct Dr. Mier: It may be best to answer that through blind high- selectively targeted to the endothelium results in angiogenesis throughput screening, in which drug X plus sorafenib are

www.aacrjournals.org 2373s Clin Cancer Res 2006;12(7 Suppl) April 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. simply thrown in together and you pick a few variables, such as inhibition is not known to be influenced by the kinase function Annexin staining, and look for the combination that maximally ofany ofthe RAFs, but it is a definiteproapoptotic pathway. affects the chosen variable. That kind of unbiased approach One ofthe reasons why it may not be terribly important in may lead to an answer long before a reasoned approach would. melanoma is that many ofthe downstream components are What we found to be most helpful and what amplifies the genes that are epigenetically silenced in melanoma such as lethality ofthis drug most significantlyare things that block LATS. survival pathways that are activated by the drug. Dr. Flaherty: As we develop new RAF inhibitors, we need a Dr. Gajewski: What is the structural basis for sorafenib readout ofwhat each one does and a profileofwhat the blocking RAF function, and why do the constitutively active downstream effects are. It is clear that when we look at MAPK RAF mutants remain susceptible to inhibition? and ERK, we’re probably not seeing the full spectrum of what Dr. Mier: As far as I know, sorafenib goes after the ATP- they can do. binding domain. Dr. Mier: One ofthe potential Achilles’ heel ofsorafenibis Dr. Flaherty: The crystal structure was solved and reported that it’s a p38 blocker. The activation ofp38 is an absolutely with the drug bound in the ATP-. It pushes the essential component to the lethality ofcytokines. Here we’ve activation loop ofthe kinase out ofthe foldthat it would got a drug that blocks p38 with nearly the same IC50 as the RAF otherwise sit in. The question that has not been answered is isoforms. Some of these other raf inhibitors don’t have that how RAF interacts with its substrates; in other words, what feature and may work better for that reason alone. domains ofRAF interact with which substrates. Dr. Sosman: What happens to these different cell lines when Dr. Sondak: I understand that BRAF is also important in you knockout MEK, ERK, BRAF, and CRAF? Do you actually kill mitosis and mitotic spindle assembly, which would obviously all those cell lines? point a finger directly at things such as taxanes and other Dr. Mier: The ones we’ve chosen to knockout are ones that mitotic spindle assembly inhibitors. we felt might be involved in the lethality of the drug, for Dr. Mier: has a similar association. It likes to example, apoptosis-inducing factor and BAD. If you use a flow associate with microtubules, especially the spindle apparatus. cytometer to gage lethality by assessing Annexin or propidium Survivin is easily down-modulated by sorafenib. In fact, if you iodide staining, you can cotransfect with red fluorescent protein transfect in enough Survivin to get a thumb-wide band on your to identify the cells that pick up your small interfering RNA gel and you throw sorafenib into the culture, it all vanishes and just transfect in red fluorescent protein alone for the within minutes. control cells. Dr. Atkins: Is there a way that we can potentially distinguish Dr. Sosman: Ifwe get the ultimate drug, is it going to be one RAF inhibitor from another by a pattern of proteins that it effective in the most simplistic system? If we really knockout all might activate in vitro that might allow us to better select which ofBRAF or MEK, is that enough? ones should be studied? Can we learn enough from this type of Dr. Mier: I would think not. There’s plenty ofreason to think cell line data to help us determine which drug to use? that the lethality ofa RAF knockdown has nothing to do with Dr. Mier: I hope so, but the easiest way ofgetting at that the disruption ofthe MEK pathway. The experiment that most information may be just to do a high-throughput screen with a illustrates this is the fact that if you knockout CRAF, you get an spheroid model or even in vitro cultures where the cells are embryonic lethality at around day 9. The critical abnormality, growing on polystyrene. We can randomly add drug combina- when a pathologist looks at the embryos, is failure to develop tions and see which is the most effective. Those that look blood vessels. The problem is that you can completely rescue especially promising could then be put into an animal model the knockdown with a CRAF that has no MEK activating and validated. The things that work best are the drugs that capability whatsoever. At least in the heart, for example, there is antagonize survival pathways that are activated when the cell is no effect on MEK when you introduce a CRAF knockdown. The stressed with a RAF inhibitor, such as the JNK pathway. CRAF is lethality ofthe knockdown is entirely due to the loss of known to suppress the kinase MST2. There is a possibility that suppression ofASK1. I’m not sure that the MEK pathway is the this may be more important than we know. However, this only thing that is relevant to RAF.

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David J. Panka, Michael B. Atkins and James W. Mier

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