		The mechanism of action of vemurafenib (Zelboraf) in inhibiting BRAF activity.

Until recently, the cornerstone of therapy for metastatic melanoma had been chemotherapy with dacarbazine (DTIC) and immunotherapy with high-dose interleukin-2 (HD IL-2) or interferon-α (IFN-α). Although considered standard-of-care treatment, DTIC has shown objective response rates as low as 15.3% for a total of 3356 patients enrolled in more than 20 studies. Additionally, most of these responses were partial. Despite significant toxicity, immunotherapy with highdose IL-2 resulted in a substantial benefit for some patients, leading to a 27% survival rate at 2 years and 16% at 4 years, in a trial involving 684 patients. However, the efficacy of IFN-α remains controversial. Its significant toxicity, especially at high doses, has restricted its use. Due to its easier use and reduced side effects compared with unmodified IFN-α, pegylated IFN-α was approved by the FDA in the adjuvant setting in 2011 for patients with lymph node-positive resected melanoma. Trials remain ongoing to evaluate its optimal dosage, as well as the subset of patients that might benefit from this option.

Frontline treatment options for patients with advanced melanoma have recently improved with the 2011 FDA approval of vemurafenib and ipilimumab, both with indications for metastatic disease. Vemurafenib is an orally available, targeted agent that selectively inhibits the BRAF kinase. It has specificity for the V600E-mutated form of the protein, the oncogenically active variant in 90% of patients with BRAF-mutated melanoma.

 In the randomized phase III BRIM-3 study,vemurafenib was compared with DTIC in 675 patients with previously treated, BRAF

-positive metastatic melanoma. The response rate was substantially improved in patients treated with vemurafenib versus DTIC (48% vs 5%). Patients in the vemurafenib arm also experienced a markedly improved 6-month overall survival (OS) compared with those in the DTIC arm (84% vs 64%). Vemurafenib was also associated with a relative reduction in risk of death (63%), and a 74% reduction in the risk of either death or disease progression compared with DTIC (P< .001).

Ipilimumab is a fully human, monoclonal antibody directed against the T-cell antigen cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). In a multicenter, singlearm, phase II clinical trial, ipilimumab was shown to be active in patients with relapsed metastatic melanoma, with 27% of patients achieving disease control, despite a low overall response rate (5.8%). The 1-year and 2-year survival rates were 47.2% and 32.8%, respectively.

	In late May 2013, dabrafenib, a BRAF inhibitor, received approval for the treatment of patients with unresectable or metastatic melanoma with BRAFV600E mutation. A multicenter, international, open-label, randomized, active- controlled phase III trial evaluated the efficacy and safety of dabrafenib compared with DTIC in 250 patients with previously untreated, histologically confirmed, unresectable stage 3 or 4 melanoma that has been determined to be BRAFV600E mutation-positive by centralized testing. Median progression-free survival (PFS) was significantly improved in patients in the dabrafenib arm compared to the DTIC arm (5.1 vs 2.7 months). The most common drug-related adverse events (AEs) were hyperkeratosis, headache, pyrexia, arthralgia, papilloma, alopecia, and palmar-plantar erythrodysesthesia syndrome. Serious AEs involved development of new primary skin cancers (cutaneous squamous cell carcinomas [SCC], new primary melanomas, and keratoacanseen thomas), febrile drug reactions requiring hospitalization, hyperglycemia, and uveitis/iritis.

Subsequently, in the randomized, prospective phase III MDX010-20 trial, ipilimumab significantly prolonged median OS when administered either as a single-agent (10.1 months) or combined with the gp100 vaccine (10.0 months), versus gp100 alone (6.4 months; 

 < .001 for each comparison, respectively).

 Another phase III trial, Study 024, found that OS was significantly improved in patients in the DTIC plus ipilimumab combination group (11.2 vs 9.1 months; 

 <.001). Survival rates were higher in the ipilimumab—DTIC group at 1 year (47.3% vs 36.3%), 2 years (28.5% vs 17.9%), and 3 years (20.8% vs 12.2%). Treatment with ipilimumab plus DTIC produced a two-fold improvement in the median duration of response compared with the DTIC plus placebo arm (19.3 vs 8.1 months; 

 Both vemurafenib and ipilimumab are recommended by the National Comprehensive Cancer Network (NCCN) as category 1 options for treatment of advanced melanoma. The choice of treatment is individualized: Since patients with less-aggressive, low-volume, or asymptomatic metastatic melanoma may have more time to allow an antitumor immune response to mount, they may be good candidates for ipilimumab therapy; patients with more-aggressive, BRAF mutation-positive advanced melanoma, however, may benefit more from first-line treatment with vemurafenib.

Also approved in May 2013, trametinib is a MEK inhibitor and first in its class to receive approval, and is indicated for use in patients with unresectable or metastatic melanoma with BRAFV600E or V600K mutation. A multicenter, international, open-label, randomized, active-controlled phase III trial evaluated the efficacy and safety of trametinib compared to chemotherapy with either DTIC or paclitaxel in 322 patients with histologically confirmed, stage 3c or 4 melanoma determined to be BRAF V600E or V600K mutation-positive by centralized testing. Patients were not permitted to have received more than one prior chemotherapy regimen, and those with previous exposure to BRAF inhibitors or MEK inhibitors were ineligible. Median PFS was significantly improved in patients in the trametinib arm, compared with those receiving chemotherapy (4.8 vs 1.5 months). Trametinib did not demonstrate antitumor activity in patients who had received prior BRAF inhibitor therapy. The most common drug-related AEs were rash, diarrhea, and lymphedema. Serious AEs included cardiomyopathy, retinal pigment epithelial detachment, retinal vein occlusion, interstitial lung disease, and serious skin toxicity.

There are currently numerous experimental therapeutic options in various phases of clinical development that may hold promise for patients with advanced melanoma.

In the MAPK pathway, MEK kinases are situated immediately downstream of the BRAF kinase; once activated, MEK kinases trigger activation of pathways responsible for tumorigenesis. GSK1120212 is one such small molecule that has now reached phase III trials for the treatment of melanoma. In a phase III study, this selective, allosteric inhibitor of MEK 1 and 2 induced an objective response in 22% of patients, and 74% experienced some degree of tumor regression. Most common AEs included diarrhea, rash, and peripheral edema. No cases of SCC occurred.

		RTK=receptor tyrosine kinase signaling pathway, which includes 

 oncogenes. PI3K=phosphatidylinositol 3-kinase pathway activation also occurs in melanoma.

AZD6244 is another MEK inhibitor that selectively binds to MEK 1 and 2. This drug remains in phase II clinical trials to evaluate its efficacy in treating patients with stage III or IV melanoma.

Lenvatinib is an oral, multitargeted receptor tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1-4, RET, KIT, and PDGFR . In a recent phase II study in which lenvatinib was administered to 93 patients with advanced BRAF wild-type melanoma, some clinical benefits were keratoacanseen, with confirmed partial responses in 9% of patients, and a 32% clinical benefit rate. Median PFS was 3.7 months, and median OS was 9.5 months. Dose reduction was required to manage toxicity in 46% of patients. Most frequent AEs included hypertension, fatigue, nausea, diarrhea, and reduced appetite.

Nab-paclitaxel (Abraxane) is a nanoparticle formulation of paclitaxel bound to albumin for injectable suspension. In a randomized, international phase III study in chemotherapy-naïve patients with metastatic melanoma, nab-paclitaxel significantly improved PFS compared to patients receiving DTIC chemotherapy.

Darleukin is a fully-human vascular-targeting immunocytokine, a fusion protein containing interleukin-2 (IL-2), which increases the safety of administration of IL-2, and improves its antitumor activity. Darleukin is currently being tested in a randomized phase IIb study of its efficacy in combination with DTIC, compared to DTIC monotherapy, in patients with metastatic melanoma.

Data have demonstrated clinical benefit of cancer vaccines containing a specific cancer antigen, and a peptide vaccine for melanoma. Toll-like receptor (TLR) agonists have the potential to enhance antitumor T-cell responses, either in combination with incomplete Freund’s adjuvant (IFA) or alone. CD40 ligation at the vaccine site microenvironment may also enhance adjuvant activity. A clinical trial is recruiting patients with resected stage 3B-4 melanoma, and is designed to evaluate the efficacy and safety of a multipeptide vaccine with TLR agonists and IFA.

Talimogene laherparepvec (T-VEC) is an oncolytic immunotherapy (OI) designed to selectively replicate in tumors and produce granulocyte-macrophage colony- stimulating factor (GM-CSF) to increase systemic antitumor immune responses. It is the first OI to demonstrate therapeutic benefit against melanoma, and represents a novel treatment option for melanoma with regional or distant metastases. In the phase III, randomized trial (OPTiM), the efficacy of T-VEC was evaluated in 436 patients with unresected stage 3B, 3C, or 4 melanoma, compared to GM-CSF. Data revealed that the primary endpoint of durable response rate was met, demonstrating a significant difference in the T-VEC arm compared to the GM-CSF arm (16% vs 2%). Most frequent AEs included chills, fatigue, and pyrexia. Serious AEs occurred in 26% of patients in the T-VEC arm, and 13% in the GM-CSF arm.

PD-1 is an inhibitory T-cell co-receptor that may result in suppression of anti-tumor immunity. Various antibodies are being developed to inhibit the PD-1 pathway.

Lambrolizumab is a humanized, monoclonal IgG4 antibody against PD-1. In an ongoing phase Ib study of 135 patients with advanced melanoma, with or without previous ipilimumab treatment, preliminary data showed that even the lowest dose of lambrolizumab administered intravenously resulted in a high rate of sustained tumor regression. In addition to its significant antitumor activity, lambrolizumab was well-tolerated with manageable AEs. The confirmed overall response rate pooled across all dose cohorts was greater than 35%, with 77% of patients demonstrating a reduction in tumor burden, including 8 who had stable disease for more than 24 weeks. The most common drug-related AEs to date include grade 1/2 fatigue, pruritus, and rash. The incidence of grade 3/4 AEs was 10%.

		By binding PD-1 on the surface of T cells, cancer cells can evade the immune system. Therapies that block PD-1/PD-L1 can reactivate the immune system.

Nivolumab is another humanized, monoclonal IgG4 antibody against PD-1. In a phase I trial of 107 patients with melanoma, data showed that nivolumab produced durable OS and responses with an acceptable safety profile. Cohorts of patients received 0.1, 0.3, 1.3, and 10mg/kg. Median OS across all doses was 16.8 months, and 20.3 months at the 3 mg/kg dose that was selected for phase III trials; 44% and 40% of patients remained alive at 2 and 3 years respectively; and of 29 responders who had been treated for at least one year, 16 demonstrated responses lasting one year or more. Drug-related AEs occurred in 82% of patients, and included lymphopenia, fatigue, and elevated serum lipase levels. Grade 3/4 AEs occurred in 21% of patients, and included diarrhea, endocrine disorders, and hepatitis.

Clinical and preclinical studies have begun to demonstrate evidence to support the use of combination therapies as a therapeutic strategy in melanoma. Numerous combined regimens are being evaluated, and include:

	CTLA-4 Blockade and GM Secreting Tumor Vaccine Combinations

CTLA-4 Blockade and PD-1 Blockade Combination

These combinations have shown therapeutic synergy in preclinical studies. GM increases dendritic cell activation, enhances antitumor T and B cell responses, and may activate regulatory immune responses. It has shown benefit in various types of carcinoma, and is also being evaluated in phase III adjuvant trials for melanoma. In a randomized phase II trial involving 245 patients, ipilimumab plus GM significantly increased OS compared to ipilimumab alone (67.9% vs 51.2%), as well as improved OS (17.5 months vs 12.7 months), with no significant differences in toxicity. This combination of GMCSF and ipilimumab therefore may hold promise for improving survival of patients with advanced melanoma.

This combination of drugs also appears to offer potential benefit for treatment of melanoma, compared with monotherapy. Combination immunotherapy with ipilimumab and nivolumab has also been demonstrated to produce durable tumor regression with a manageable safety profile. In a phase I trial involving 53 patients, intravenous doses of nivolumab and ipilimumab were administered every 3 weeks for 4 doses, followed by nivolumab alone every 3 weeks for 4 doses; the combined treatment was then administered every 12 weeks for up to 8 doses. In a sequenced regimen, patients previously treated with ipilimumab were treated with nivolumab every 2 weeks for up to 48 doses. Clinical activity was observed in 65% of patients, and at the maximum doses that were associated with an acceptable level of AEs, 53% of patients had an objective response, with tumor reduction of 80% or more. Drug-related grade 3/4 AEs occurred in 53% of patients in the concurrent-regimen group, but were typically reversible, and in 18% in the sequenced-regimen group. The objective response rate was increased in the concurrent-regimen group compared to the sequenced-regimen group (40% vs 20%).

Metastatic melanoma remains one of the most therapeutically challenging malignancies, and curative treatments remain elusive. However, for the first time in decades, new drugs have resulted in significant clinical responses. Advances in basic and clinical research have produced novel treatment strategies that target specific molecules and pathways expressed in cancer cells. And one of the major focuses in melanoma therapy continues to be the understanding and targeting of signal transduction pathways responsible for the tumor’s capacity for growth and resistance to chemotherapy. 

 Additionally, the molecular heterogeneity of melanoma, and the recognition of numerous mechanisms of resistance to targeted therapies, support the use of a combinatorial approach to treatment. Clinical management of melanoma is therefore improving with increased understanding of the molecular pathogenesis of the tumor.

 Pretto F and Neri D. Pharmacotherapy of metatstatic melanoma: emerging trends and opportunities for a cure. 

 Nikolaou VA, Stratigos AJ, Flaherty KT, et al. Melanoma: new insights and new therapies. 

. Evans MS, Madhunapantula SV, Robertson GP, et al. Current and future trials of targeted therapies in cutaneous melanoma. WS El-Deiry (ed), In: 

Impact of Genetic Targets on Cancer Therapy. Advances in Experimental Medicine and Biology.

. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation.

 O’Day SJ, Maio M, Chiarion-Sileni V, et al. Efficacy and safety of ipilimumab monotherapy in patients with pretreated advanced melanoma: a multicenter single-arm phase II study. 

 Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. 

 Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.

 National Comprehensive Cancer Network. Melanoma. Clinical Practice Guidelines in Oncology. 2012. Available at: http://www. nccn.org/professionals/physician_gls/f_guidelines.asp#melanoma. Accessed June 16, 2013.

 FDA. Darafenib. 2013. Available at: http://www.fda.gov/Drugs/ InformationOnDrugs/ApprovedDrugs/ucm354477.htm. Accessed June 16, 2013.

 FDA. Trametinib. 2013. Available at: http://www.fda.gov/ Drugs/InformationOnDrugs/ApprovedDrugs/ucm354478.htm. Accessed June 16, 2013.

 ClinicalTrials.gov. MEK inhibitor AZD6244 in treating patients with stage III or IV melanoma. Available at: http://clinicaltrials.gov/ct2/ show/study/NCT00866177. Accessed June 16, 2013.

O’Day S, Gonzalez R, Kim K, et al. A phase II study of the multitargeted kinase inhibitor lenvatinib in patients with advanced BRAF wild-type melanoma. 

Celgene Corporation. ABRAXANE meets primary endpoint of progression-free survival in phase III chemotherapy-naïve metastatic melanoma study. Available at: http://ir.celgene.com/phoenix.zhtml? c=111960&p=irol-newsArticle&ID=1740496&highlight. Accessed June 16, 2013.

 ClinicalTrials.gov. A phase 3 pivotal trial comparing Allovectin-7 alone vs chemotherapy alone in patients with stage 3 or stage 4 melanoma. Available at: http://clinicaltrials.gov/show/NCT00395070. Accessed June 16, 2013.

Slingluff CL, Petroni GR, Chianese-Bullock KA, et al. A multipeptide vaccine plus toll-like receptor agonists in melanoma patients, with evaluation of the vaccine site microenvironment and sentinel immunized node (Mel58; NCT01585350). 

Andtbacka RHI, Collichio FA, Amatruda T, et al. OPTiM: a randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous (SC) granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment (tx) of unresected stage IIIB/C and IV melanoma. 

Ribas A, Robert C, Daud A, et al. Clinical efficacy and safety of lambrolizumab (MK-3475, Anti-PD-1 monoclonal antibody) in patients with advanced melanoma. 

 Sznol M, Kluger HM, Hodi S, et al. Survival and long-term follow-up of safety and response in patients (pts) with advanced melanoma (MEL) in a phase I trial of nivolumab (anti-PD-1; BMS-936558; ONO-4538).

 Hodi FS, Lee SJ, McDermott DF, et al. Multicenter, randomized phase II trial of GM-CSF (GM) plus ipilimumab (Ipi) versus Ipi alone in metastatic melanoma. 

 Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. 

 Kwong LN, Davies MA. Targeted therapy for melanoma: rational combinatorial approaches. 

Articles

Until recently, the cornerstone of therapy for metastatic melanoma had been chemotherapy with dacarbazine (DTIC) and immunotherapy with high-dose interleukin-2 (HD IL-2) or interferon-Î± (IFN-Î±).

Novel Agents Revolutionize Melanoma Treatment

		Professor of Medicine and Preventive Medicine, Associate Director, Clinical Research, Chair, GI Oncology Program, Co-director, Colorectal Center, Scientific Director, Cancer Genetics Unit, USC Norris Comprehensive Cancer Center Los Angeles, CA

	Heinz-Josef Lenz, MD, has focused his research on a range of scientific and translational questions, including the regulation of gene expression in drug resistance and targeted therapies for the treatment of metastatic colorectal cancer. A recipient of the ASCO Young Investigator Award and other honors, Lenz has served on several National Cancer Institute committees. His current clinical trial work includes serving as the principal investigator on studies evaluating panobinostat in stage IV colorectal cancer and dasatinib in advanced liver cancer.

	At the USC Center for Molecular Pathways and Drug Development, Lenz is collaborating with Michael Kahn, PhD, on exploring cancer cell-signaling pathways. Lenz, along with other USC Norris researchers, developed PRI-724, a small molecule that inhibits the Wnt pathway. Two clinical trials are under way, including a phase I/II study in advanced myeloid malignancies and a phase I trial in advanced solid tumors.

	He discussed the Wnt pathway in this interview with 

	Can you describe your research and its focus on the Wnt pathway?

	The focus of my research is to identify predictive and prognostic markers in order to identify patients who benefit most from chemotherapy, especially focusing on the EGF and VEGF pathways. We also are very interested in early drug development, which is one of the reasons we are interested in the Wnt pathway since one of the first-in-class medications, called PRI-724, being tested in the clinic is being developed by a colleague of mine, Dr. Kahn, here at USC.

	This agent has completed the first-in-human clinical trial and is a novel inhibitor that modulates the Wnt pathway in a very significant way. It inhibits CBP-catenin, which modifies the Wnt activated pathway in cancers in a way that pushes the pathway into differentiation.

	What is the role of the Wnt pathway in colon cancer?

	Wnt is a very complicated pathway that is particularly important in colon cancer, and is present in over 95% of tumors. The reason this pathway is complex is because the regeneration of the intestinal mucosum is driven by the colon stem cells; in order for the intestinal mucosa to rejuvenate every 24 hours, it first undergoes proliferation and then differentiation to result in a mature intestinal mucosa. The Wnt pathway is one of the most important pathways involved in this process. Obviously, if the Wnt pathway is alteredâŽ¯and particularly the proliferation pathway is changedâŽ¯colon cancer can develop.

	Can you discuss how the Wnt pathway functions in other cancer types?

	The Wnt pathway is activated in many cancers, including liver cancer, breast cancer, leukemias, and liquid tumors. It is a very fundamental pathway because it is one of the pathways that are activated first in the development of a human being because it is involved in the decision point of proliferation and differentiation. It is the first pathway to be activated to decide what the cells should become—whether to proliferate and then differentiate into a specific target organ.

	So because of this fundamental role, this pathway is altered in many cancers. It is also involved in many other degenerative diseases because weakening the proliferation and differentiation functions can also promote degenerative disease, chronic inflammatory diseases, aging disorders, etc.

	How will a drug designed to target the Wnt pathway affect normal cells?

	What usually happens in the aging process of human beings is that the process of stem cell rejuvenation does not work as well. This is the reason we lose hair, get wrinkles, get constipationâŽ¯because our organs are not rejuvenating in the way they should. A Wnt pathway inhibitor can actually rejuvenate the ability for an asymmetric division to take place in normal cells, so it actually will turn back the clock and will make sure cells will do what they are supposed to do. That is the reason our Wnt inhibitor works as an anti-aging, anti-wrinkle, and hair cream, and we have a license for some cosmetic applications. So it makes sense.

	The first worry about this pathway is toxicity, and the problem is pharmaceutical companies have in the past tried to develop an inhibitor because of its crucial role, but the complexity and maybe the lack of understanding of the specific differences can result in significant toxicities. So I think it depends how these drugs are developed. However, if it is possible to harness the potential of this pathway, there may be significant benefits in healthy patients, not only because of its rejuvenating potential but also in patients who are undergoing degenerative changes associated with disease, including cancer.

	When do you expect that Wnt inhibitors will move into clinical practice?

	There are now widespread clinical trials ongoing on colon cancer, pancreatic cancer, and liquid tumors, but it may be a while. I think the Wnt pathway bears unique opportunities to really impact cancer. The ultimate problem in our inefficacy to cure cancers, particularly metastatic disease, is the inability to really kill colon cancer or any cancer stem cells.

	The only way to succeed with targeting stem cells, which are ultimately resistant to any of the treatments we have, including the targeted treatments, is really to differentiate these cancer stem cells out, and then kill them with chemotherapy. I think that will revolutionize the way we do treatment in the future. I think this drug, as a single agent, will probably not be effective, but in combination with chemotherapy, absolutely it will be. We see that in our preclinical models.

Heinz-Josef Lenz, MD, has focused his research on a range of scientific and translational questions, including the regulation of gene expression in drug resistance and targeted therapies.

Five Wnt-Related Questions for Heinz-Josef Lenz, MD

The Wnt signaling pathway was first characterized in the 1970s in 

 development. It was later recognized in mammalian systems for its importance in cancer. Specifically, core components of this pathway were shown to be dysregulated in colorectal disease. It has since been shown to play a role in other forms of cancer, where it promotes proliferation and survival. It also plays an important role in the maintenance of the pool of tumor-initiating cells, which promote the regrowth of tumors after an insult like surgery or chemotherapy. Tumor-initiating cells are thought to be the source of metastasis. For these reasons, the Wnt pathway is viewed as a strong candidate for therapeutic intervention.

The Wnt pathway takes on many forms that fall under the broad classifications of canonical and noncanonical. The canonical pathway deals with the regulation of β-catenin protein levels. Under normal conditions, a cytosolic scaffold known as the destruction complex binds and phosphorylates β-catenin, resulting in its ubiquitylation and degradation. The destruction complex includes the adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase-3β. When Wnt ligand binds to the Frizzled receptor, its coreceptor LRP5/6 is recruited and phosphorylated in the intracellular domain, promoting the binding of Dishevelled protein and the sequestration of axin. This disintegrates the destruction complex, resulting in accumulation of β-catenin in the cytosol and upregulated trafficking into the nucleus. β-catenin promotes transcription of genes related to proliferation and survival by acting as a coactivator for the Tcf/Lef family of transcription factors in the nucleus.

Aside from canonical Wnt signaling, two major noncanonical pathways have been studied. In the first of these two pathways, Wnt ligand binding to the Frizzled receptor induces recruitment of Dishevelled protein and the Dishevelled-associated activator of morphogenesis 1 (Daam1). This complex initiates a cascade that activates the Rac and Rho GTPases to mediate cell polarity. The other most widely studied noncanonical Wnt signaling pathway is related to calcium signaling. Wnt ligand binding to the Frizzled receptor promotes the recruitment of Dishevelled in complex with a G protein. This complex promotes intracellular calcium levels to mediate other signaling pathways.

Biological systems tightly regulate the Wnt signaling pathway to prevent aberrant cell growth. It has been known for decades that dysregulation of Wnt signaling leads to cancer, where it was first recognized in familial colorectal disease with mutations in the 

 gene. Since then, Wnt signaling has been found to act prominently in breast, liver, skin, and prostate cancers.

		This illustration depicts the best-elucidated cancer-promoting routes of Wnt cell signaling, which draws its name from the wingless mutation in the fruitfly 

Aberrations in canonical Wnt signaling can manifest in many ways. For example, proteins involved in the destruction complex can become nonfunctional through mutations or truncations, inhibiting β-catenin downregulation. The β-catenin protein itself can be mutated to inhibit its recognition by the destruction complex. In addition, the production of Wnt ligand or receptors can be upregulated, resulting in excessive signaling. These different routes of activation complicate the use of a single therapeutic against the Wnt pathway.

Wnt signaling is of great interest to cancer researchers because it is linked to many different forms of the disease. Preclinical models throughout the last decade have established this pathway as an attractive drug target. However, to date, therapies meant to attenuate the Wnt pathway have remained largely theoretical and preclinical. Thankfully, compounds are now starting to enter clinical trials.

Vitamin D has been postulated as a suitable anti-Wnt therapy. The vitamin D receptor binds and sequesters β-catenin at the plasma membrane, inhibiting its nuclear translocation. Mice bearing an 

 mutation that promotes spontaneous colon cancer developed more disease when the vitamin D receptor was knocked out. Additionally, the association between sunlight exposure and decreased risk of colon cancer implies that the inhibition of Wnt signaling by vitamin D may be conserved in humans. This phenomenon has yet to be tested in a systematic trial, however.

High-throughput screens have been used extensively to identify small-molecule targeted inhibitors of different Wnt pathway constituents. The binding of β-catenin to its nuclear targets T-cell factor (Tcf) or Creb-binding protein (CBP) is a prototypical example of study in this realm. Screens were performed to identify specific inhibitors of this interaction. This work has identified useful hits in cell-based assays, but translation into the clinic has remained difficult due to the potential off-target effects. First, the drugs identified so far fail to discriminate between the binding of β-catenin to Tcf or APC, so the drug may prevent degradation of β-catenin in addition to its intended effect on transcription. Disruption of the destruction complex binding to β-catenin could lead to severe side effects in normal tissue. Another deleterious effect identified with blockers of β-catenin binding is the inhibition of complex formation with E-cadherin at cell-cell junctions. The net effect of this phenomenon could be to impair cell adhesion on a grand scale. As a result of these challenges, only one compound that directly disrupts β-catenin function has moved into clinical trials.

Other drug candidates inhibit Wnt aberrations upstream of β-catenin function. Several of these compounds have recently begun clinical trials. They block either Wnt ligand secretion or recognition, and preclinical evidence has been encouraging so far. Targeting at this level can also lead to side effects, however. By blocking Wnt signaling at the level of the membrane, it is possible to inhibit noncanonical in addition to canonical signaling. The effect this will have on the body is unknown. In addition, blocking Wnt ligand-receptor interactions may not be sufficient to inhibit Wnt signaling since the activating event may be mutation within the destruction complex or β-catenin itself. In these settings, Wnt signaling can be rendered constitutive and independent of ligand, and the therapy would likely fail.

No approved compounds exist for the treatment of Wnt signaling. Phase I trials for these inhibitors should be illustrative in the coming years. While many cancers are addicted to this signaling for long-term growth and renewal, high-turnover tissues like the gastric epithelium and hair follicles are similarly reliant. Therefore, the field is cautious when utilizing any blockade of Wnt signaling, as significant toxicity may result.

					This monoclonal antibody targets the Frizzled receptors to block association with Wnt ligands. It was recently shown to potently block the capabilities of pancreatic tumor-initiating cells in a serial dilution assay. In xenograft models of breast, lung, pancreatic, and colon cancer, OMP-18R5 demonstrated significant inhibition of tumor growth, and it synergized with standard-of-care treatment in these models (paclitaxel in breast cancer, for example). (NCT01345201)

					This agent is a fusion protein of the Frizzled8 ligand-binding domain with the Fc region of a human immunoglobulin. It binds and sequesters soluble Wnt ligand, impairing its recognition by receptors on tissues. (NCT01608867)

					This is a small-molecule inhibitor of the interaction between β-catenin and CBP. Disrupting the interaction prevents activated transcription by aberrant Wnt signaling at many levels. It is being studied in both solid tumors and myeloid malignancies. (NCT01606579, NCT01302405)

					This small molecule inhibits acyltransferase porcupine. Preclinical work demonstrated this enzyme's action is crucial in the secretion of Wnt ligands out of the cell; therefore, inhibiting porcupine can be a small-molecule—based method for inhibiting Wnt ligand-mediated activation. (NCT01351103)

					This small-molecule poly(ADP ribose) polymerase (PARP) inhibitor has demonstrated efficacy in cellular models of cancer survival. In Wnt signaling, PARPs like the tankyrases promote the ribosylation and subsequent degradation of axin, a key scaffolding protein of the destruction complex. By inhibiting tankyrase, the axin protein is stabilized and can promote the degradation of β-catenin.

					This selective tankyrase1/2 inhibitor has been shown to inhibit the growth of colon cancer cells in both cell and animal models.

		Sources: ClinicalTrials.gov website, company websites.

If Wnt pathway inhibitors can be proven safe, it may represent a milestone in cancer research given the strong preclinical evidence for cancer cell cytotoxicity. Since this pathway is also crucial in the maintenance of tumor-initiating cells, inhibition could represent a powerful tool in our arsenal to target cells that are resistant to traditional chemotherapy and promote metastasis.

			Chen B, Dodge ME, Tang W, et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer [published online ahead of print January 4, 2009]. 

. 2009;5(2):100—107. doi: 10.1038/nchembio.137.

			Clevers H, Nusse R. Wnt/β-catenin signaling and disease. 

			Eckhardt SG. Targeting the WNT Pathway for Cancer Therapy. Presented at: 10th International Congress on Targeted Therapies in Cancer; August 17-18, 2012; Washington, DC.

			He B, Reguart N, You L, et al. Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations. 

			Ichii S, Horii A, Nakatsuru S, et al. Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis (FAP). 

			Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. 

			Rubinfeld B, Souza B, Albert I, et al. Association of the APC gene product with beta-catenin. 

Tumor-initiating cells are thought to be the source of metastasis. For these reasons, the Wnt pathway is viewed as a strong candidate for therapeutic intervention.

Wnt Signaling Inhibition: Will Decades of Effort Be Fruitful at Last?

The clinical subtyping of breast cancer appears to be coming into a golden era as an increased understanding of subtyping promises to help advance treatments and outcomes for patients. A comprehensive review published this year by Matthew J. C. Ellis, MB BChir, PhD, and colleagues with The Cancer Genome Atlas (TCGA) Research Network has provided new information about these subtypes in addition to identifying a new classification of HER2-positive disease, and has also suggested a rationale for new treatment options in each subtype. The study builds on one of the seminal works in breast cancer subtyping, published in 2000 by Charles M. Perou, PhD, and colleagues, which at the time provided a much more detailed understanding of gene signatures in malignant breast tissue than had previously existed, although the initial characterization was limited due to the technique of choice (DNA microarray) and sample size.

Currently, clinicians recognize the following forms of disease: luminal A and luminal B, two cancer subtypes that express the estrogen and progesterone receptors (ER and PR, respectively); human epidermal growth factor receptor 2 (HER2)-positive tumors, which express the HER2 protein; and basal-like tumors, which lack HER2, ER, and PR (often referred to as triple-negative breast cancers [TNBCs], but approximately 25% of TNBCs fall into other micro-RNA subtypes).

Surgery and radiation represent frontline options for patients with breast cancer. Typically, a chemotherapeutic regimen including a taxane is then given to systemically suppress the regrowth of the tumor and prevent metastasis. During initial diagnosis, hormone receptor and HER2 expression are also determined. When the ER and PR status is positive, anti-hormone drugs such as tamoxifen are often indicated. Trastuzumab (Herceptin), a monoclonal antibody against HER2, can be used in HER2-positive disease. In addition, targeted therapeutics have improved the prognosis of patients with these specific cancer subtypes.

	An Expanded Analysis of Breast Cancer Subtypes

Despite the advances in targeted therapy, significant limitations persist. Anti-hormone compounds in hormone receptor-positive disease are able only to suppress the growth of cancer cells. They do not kill these cells, and this gives the tumor time to circumvent hormone signaling and resume unchecked growth, often leading to relapse and resistance. Anti-HER2 therapy is successful in treating a large number of patients, but the development of resistance is a common feature of HER2-positive disease. In addition, roughly half of HER2-positive patients with previously untreated metastatic disease do not respond to trastuzumab when used in combination with chemotherapy . The reason for this lack of response is unclear. No approved targeted therapies yet exist for the basal-like breast cancers, since they do not express any known targetable proteins. This leaves only the nonspecific chemotherapy option for patients with this disease.

The TCGA network, through its pivotal new work, ultimately may help to expand the treatment options for breast cancer by increasing the understanding of the subtypes. The network, launched in 2006 through a collaboration of the National Cancer Institute and the National Human Genome Research Institute, brings together researchers from approximately 15 institutions, including centers at Washington University in St. Louis, Missouri, where Ellis is based.

In the breast cancer study, the researchers combined a variety of techniques using tumor samples from 825 patients in an effort to definitively characterize the breast cancer subtypes. They also hoped to elucidate specific pathways of disease to improve the current standards of care. Six features of genes across the entire exome were analyzed, including DNA copy number, mutation, DNA methylation, mRNA expression, micro-RNA expression, and protein expression.

		Snapshot of Mutated Genes in Breast Cancer Subtypes

The TCGA study included an analysis of the significantly mutated genes in 498 tumor samples correlated with genomic subtype.

 Adapted from Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours [published online ahead of print September 23, 2012]. 

. 2012;490(7418):61-70. doi: 10.1038/nature11412.

	New Findings in HER2-Positive Breast Cancer

Overall, the team concluded that diverse genetic and epigenetic alterations in breast cancers could be classified into the four major biological subtypes, and that “much of the clinically observable plasticity and heterogeneity occurs within, and not across,” those subtypes.

The study elucidated distinct subtypes of HER2-positive cancer. Nearly half of the samples revealed the classically-defined HER2-positive disease where mRNA and protein levels were elevated. In the other half, the gene expression signature indicated strong similarity to the luminal subtype.

Given that nearly half of the cases of HER2-positive disease show little or no effect, response to trastuzumab treatment has been difficult to predict. Breast cancer expressing HER2 but with the luminal gene signature may account for this phenomenon. Indeed, the luminal gene signature for HER2-positive disease may prove to be a useful predictor of trastuzumab efficacy.

					Imatinib, nilotinib, bafetinib (INNO-406)

					Tofacitinib (CP-690550), ruxolitinib (INCB018424 or INC424)

					Foretinib, tivantinib (ARQ 197), MetMAb (PRO143966)

					Cisplatin, carboplatin, PARP inhibitors

In the HER2-overexpressing disease, a marked upregulation of other receptor tyrosine kinase-related phenomena were noted. This provides more rationale for the targeting of both HER2 and other receptors such as the epidermal growth factor receptor (EGFR). Small molecules such as lapatinib (Tykerb) could be used in this context along with trastuzumab to potentially increase the efficacy of treatment. The high rate of PIK3CA mutations in this subtype also suggests that AKT pathway inhibition may be a viable route of therapy for HER2-positive disease.

Many features of the luminal subtype were confirmed upon analysis of the gene signature results. The ER signaling pathway was intact in both types, for example. In addition, the luminal subtypes were given the novel distinction of being “reactive” through analysis of protein expression. This referred to the expression of proteins likely provided by the tumor microenvironment. The finding implies that luminal tumors are especially sensitive to changes in the ecosystem of the disease.

	New Clues to Treating Basal-Like Breast Cancer

The study also highlighted several potential drug targets in addition to the current standard of care (Table). Mutations in the PIK3CA gene were common in many luminal tumors, a sign that drugs affecting the AKT signaling pathway could prove useful to hormone-positive breast cancer treatment. The luminal B subtype, which relies less on hormone signals to grow, was also identified as having a significant number of TP53 mutations, along with elevated MDM2 copy number. Both of these observations suggest an interference with the p53 tumor suppressor pathway in luminal B that helps to explain its more aggressive phenotype. Treating with inhibitors of the p53-MDM2 interaction could therefore help reactivate this tumor suppressive pathway in luminal B cancers and improve prognosis.

Because no targeted therapeutics have been approved for the basal-like phenotype, there is a critical need to identify new drug targets for this subgroup in particular. The study found a strong correlation between p53 pathway suppression and basal-like breast cancer. The AKT pathway was also strongly upregulated in basal-like disease.

Most surprisingly, the gene signature of basal-like breast cancer was found to be highly similar to serous ovarian carcinoma. The work confirmed that other subtypes, by contrast, did not share the similarity. Furthermore, the BRCA1 gene is inactivated in both basal-like breast cancer and serous ovarian carcinoma alike. Platinum-based drugs may prove effective in treating basal-like disease. In addition, upregulation of receptor tyrosine kinase signaling was observed and suggests that targeting receptors other than HER2 or EGFR could prove useful in the treatment of basal-like disease.

Although the TCGA study findings are not intended for direct translation to drug development, researchers hope that the comprehensive mapping of the genomic characteristics of breast cancer (and at least 20 other tumor types) eventually will advance the practice of personalized medicine. The treatment of breast cancer has been moving toward more personalized approaches since 1977, when tamoxifen became the first molecularly targeted therapeutic to gain FDA approval. Tamoxifen acts as an ER modulator that suppresses hormone signaling in breast tissue and is approved for metastatic breast cancer, ductal carcinoma in situ, and systemic inhibition of node growth following radiation and surgery.

The next breakthrough came in 1998 with the approval of Herceptin for use in women with metastatic breast cancer who have tumors that overexpress the HER2 protein. Herceptin is a monoclonal antibody that targets the HER2 protein extracellular domain. Subsequently, the armamentarium of targeted therapeutics both as monotherapy and in various combinations has been expanded with aromatase inhibitors, lapatinib, and, in recent months, pertuzumab (Perjeta) and everolimus (Afinitor).

Everolimus targets the mammalian target of rapamycin (mTOR) protein, one of the downstream targets of the PI3K/AKT pathway. Interestingly, the TCGA study found that, in each breast cancer subtype, the PI3K/AKT pathway was activated in some way. Therefore, the use of AKT inhibitors may be an effective secondary therapeutic strategy to prevent cells from engaging compensatory signaling pathways that help them survive the primary treatment.

Furthermore, activation of receptor tyrosine kinases such as fibroblast growth factor receptor (FGFR) has not typically been associated with breast cancer, but recent studies have highlighted the importance of this protein in breast cancer. This work, in particular, found that FGFR signaling was upregulated in each subtype, providing an attractive, novel candidate for therapeutic intervention.

	Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours [published online ahead of print September 23, 2012 

	Herceptin [prescribing information]. South San Francisco, CA: Genentech, Inc; 2010.

	National Cancer Institute. Targeted cancer therapies [fact sheet].

 http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted

. Reviewed October 15, 2012. Accessed November 1, 2012.

	Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumors. 

The clinical subtyping of breast cancer appears to be coming into a golden era as an increased understanding of subtyping promises to help advance treatments and outcomes for patients.

Emma Hitt, PhD

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