Nikolas K. Haass

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors.

Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (MEK)/extracellular signal–regulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a three-dimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions. [Mol Cancer Ther 2006;5(5):1136–44]

The Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase Inhibitor AZD6244 (ARRY-142886) Induces Growth Arrest in Melanoma Cells and Tumor Regression When Combined with Docetaxel

Purpose: Disseminated melanoma is highly therapy resistant. The finding that 66% of melanomas harbor the activating BRAFV600E mutation has raised expectations for targeting the Ras/RAF/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway in melanoma. This study addresses the anti-melanoma activity of the MEK inhibitor AZD6244 (ARRY-142886). Experimental Design: We recently have shown that growing melanoma cells as three-dimensional collagen-implanted spheroids enhances resistance to the MEK inhibitor U0126. Here, we investigated the anti-melanoma activity of AZD6244 in two-dimensional cell culture, the three-dimensional spheroid model, and an in vivo model. Results: In two-dimensional cell culture, AZD6244 was cytostatic and reduced the growth of melanoma cells in a concentration-dependent fashion through the induction of G1-phase cell cycle arrest. In our three-dimensional spheroid model, the effects of AZD6244 were largely cytostatic and reversible, with drug washout leading to spheroid regrowth. Finally, 1205Lu cells were grown as tumor xenografts in severe combined immunodeficient mice. After tumor establishment, mice were dosed twice daily with 0, 10, or 30 mg/kg AZD6244 p.o. AZD6244 treatment decreased phospho-ERK in the tumors and significantly suppressed tumor growth. The original tumors remained viable, suggesting that AZD6244 monotherapy was largely cytostatic, and not proapoptotic in this model. Further studies showed that co-administration of AZD6244 (30 mg/kg) with docetaxel (15 mg/kg) led to tumor regression, indicating the potential for MEK inhibitor/chemotherapy drug combinations. Conclusions: Inhibition of MEK is cytostatic as a monotherapy in melanoma, but cytotoxic when combined with docetaxel.

An Organometallic Protein Kinase Inhibitor Pharmacologically Activates p53 and Induces Apoptosis in Human Melanoma Cells

Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mechanisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 suppressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3beta (GSK3beta) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53-WT in the cytotoxic effects of the GSK3beta inhibition. Mechanistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochondrial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviral-mediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy.

The Role of Altered Cell–Cell Communication in Melanoma Progression

Under normal homeostasis, melanocyte growth and behaviour is tightly controlled by the surrounding keratinocytes. Keratinocytes regulate melanocyte behaviour through a complex system of paracrine growth factors and cell–cell adhesion molecules. Pathological changes, leading to development of malignant melanoma, upset this delicate homeostatic balance and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. In particular, there is a switch from the E-cadherin-mediated keratinocyte–melanocyte partnership to the N-cadherin-mediated melanoma–melanoma and melanoma–fibroblast interaction. Other changes include the alteration in the gap junctions formed between the melanocyte and keratinocyte. Changes in the connexin expression, in particular the loss of connexin 43, may result in a reduction or a loss of gap junctional activity, which is thought to contribute towards tumour progression. In the current review we describe the alterations in cell–cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.

PLX4032, a Potent Inhibitor of the B-Raf V600E Oncogene, Selectively Inhibits V600E-positive Melanomas

Targeted intervention of the B‐Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next‐generation inhibitor with exquisite specificity against the V600E oncogene and striking anti‐melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E‐positive cells in a variety of in vitro experimental systems; follow‐up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.

Up-Regulated Expression of Zonula Occludens Protein-1 in Human Melanoma Associates with N-Cadherin and Contributes to Invasion and Adhesion

During the process of malignant transformation, nascent melanoma cells escape keratinocyte control through down-regulation of E-cadherin and instead communicate among themselves and with fibroblasts via N-cadherin-based cell-cell contacts. The zonula occludens (ZO) protein-1 is a membrane-associated component of both the tight and adherens junctions found at sites of cell-cell contact. In most cancers, levels of ZO-1 are typically down-regulated, leading to increased motility. Here we report the novel observation that ZO-1 expression is up-regulated in melanoma cells and is located at adherens junctions between melanoma cells and fibroblasts. Immunofluorescence and co-immunoprecipitation studies showed co-localization of ZO-1 with N-cadherin. Down-regulation of ZO-1 in melanoma cells through RNA interference produced marked changes in cell morphology--leading to a less-dendritic, more rounded phenotype. Consistent with a role in N-cadherin-based adhesion, RNAi-treated melanoma cells were less adherent and invasive when grown in a collagen gel. These data provide the first evidence that increased ZO-1 expression in melanoma contributes to the oncogenic behavior of this tumor and further illustrate that protein products of genes, such as ZO-1, can function in either a pro- or anti-oncogenic manner when expressed in different cellular contexts.

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAFWT (C8161 and WM852) and BRAFV600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAFWT and BRAFV600E melanoma.

Ki67 expression levels are a better marker of reduced melanoma growth following MEK inhibitor treatment than phospho-ERK levels

The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies.

Modulation of NOXA and MCL-1 as a Strategy for Sensitizing Melanoma Cells to the BH3-Mimetic ABT-737

Purpose: Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Inhibiting antiapoptotic BCL-2 and its relatives is an attractive strategy for sensitizing lymphoid malignancies to drugs but it has been largely unsuccessful for melanoma and other solid tumors. ABT-737, a small-molecule BH3-mimetic, selectively inhibits BCL-2, BCL-XL, and BCL-w and shows promise for treating leukemia, lymphoma, and small-cell lung cancer. Melanoma cells are insensitive to ABT-737, but MCL-1 inhibition reportedly increases the sensitivity of other tumors to the compound. Experimental Design: The efficacy of MCL-1 and BFL-1 inhibition for sensitizing melanoma cells to ABT-737 was investigated by short hairpin RNA–mediated knockdown or overexpression of their antagonist NOXA in two-dimensional cell culture, a three-dimensional organotypic spheroid model, and an in vivo model. Results: MCL-1 downregulation or NOXA overexpression strongly sensitized melanoma cells to ABT-737 in vitro. NOXA-inducing cytotoxic drugs also strongly sensitized melanomas to ABT-737 but, surprisingly, not vice versa. The drugs most suitable are not necessarily those normally used to treat melanoma. Resistance to ABT-737 occurred quickly in three-dimensional melanoma spheroids through reduced NOXA expression, although experiments with both xenografts and three-dimensional spheroids suggest that penetration of ABT-737 into tumor masses may be the principal limitation, which may be obviated through use of more diffusible BH3-mimetics. Conclusion: Sensitization of tumors to BH3-mimetics by cytotoxic drugs that induce NOXA is a therapeutic strategy worth exploring for the treatment of melanoma and other solid cancers. Clin Cancer Res; 18(3); 783–95. ©2011 AACR.