Adina Vultur

Acquired Resistance to BRAF Inhibitors Mediated by a RAF Kinase Switch in Melanoma Can Be Overcome by Cotargeting MEK and IGF-1R/PI3K

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)⁶⁰⁰(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.

A Temporarily Distinct Subpopulation of Slow-Cycling Melanoma Cells Is Required for Continuous Tumor Growth

Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.

Overcoming Intrinsic Multidrug Resistance in Melanoma by Blocking the Mitochondrial Respiratory Chain of Slow-Cycling JARID1Bhigh Cells

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

Hypoxia Induces Phenotypic Plasticity and Therapy Resistance in Melanoma via the Tyrosine Kinase Receptors ROR1 and ROR2

UNLABELLED An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance. SIGNIFICANCE These data show for the fi rst time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib.

SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells

Src family kinase activity is elevated in many human tumors, including breast cancer, and is often associated with aggressive disease. We examined the effects of SKI-606 (bosutinib), a selective Src family kinase inhibitor, on human cancer cells derived from breast cancer patients to assess its potential for breast cancer treatment. Our results show that SKI-606 caused a decrease in cell motility and invasion of breast cancer cell lines with an IC50 of ∼250 nmol/L, which was also the IC50 for inhibition of cellular Src kinase activity in intact tumor cells. These changes were accompanied by an increase in cell-to-cell adhesion and membrane localization of β-catenin. By contrast, cell proliferation and survival were unaffected by SKI-606 at concentrations sufficient to block cell migration and invasion. Analysis of downstream effectors of Src revealed that SKI-606 inhibits the phosphorylation of focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), and Crk-associated substrate (p130Cas), with an IC50 similar to inhibition of cellular Src kinase. Our findings indicate that SKI-606 inhibits signaling pathways involved in controlling tumor cell motility and invasion, suggesting that SKI-606 is a promising therapeutic for breast cancer. [Mol Cancer Ther 2008;7(5):1185–94]

Dasatinib (BMS-354825) inhibits Stat5 signaling associated with apoptosis in chronic myelogenous leukemia cells

Dasatinib (BMS-354825) is a novel, oral, potent, multi-targeted kinase inhibitor of Bcr-Abl and Src family kinases (SFK) and is a promising cancer therapeutic agent. Preclinical data indicate that dasatinib is 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl, and that dasatinib is active against 18 of 19 Bcr-Abl mutations known to cause imatinib resistance. Phase I clinical data show that dasatinib is well tolerated and highly effective for the treatment of imatinib-resistant/imatinib-intolerant chronic myelogenous leukemia (CML) and Philadelphia chromosome–positive acute lymphoblastic leukemia. However, the molecular mechanism of action of dasatinib is not fully understood. In this study, we confirm that dasatinib inhibits tyrosine phosphorylation of SFKs, including Src, Hck, and Lyn, in K562 human CML cells. Significantly, downstream signal transducer and activator of transcription 5 (Stat5) signaling is also blocked by dasatinib as shown by decreases in levels of phosphorylated Stat5 and Stat5 DNA-binding activities. In addition, dasatinib down-regulates expression of Stat5 target genes, including Bcl-x, Mcl-1, and cyclin D1. Consistent with these results, blockade of Stat5 signaling by dasatinib is accompanied by inhibition of cell proliferation and induction of apoptosis. Surprisingly, Stat5 DNA-binding activities are enhanced with increasing cell density, which is associated with resistance to apoptosis by dasatinib. Our findings indicate that inhibition of Stat5 signaling downstream of Bcr-Abl/SFKs contributes to the action of dasatinib, and, conversely, that increasing cell density up-regulates Stat5 activation and confers resistance to dasatinib. Moreover, the level of phosphorylated Stat5 in CML cells represents a mechanistically relevant biomarker for monitoring inhibition of Bcr-Abl signaling by dasatinib in CML patients using convenient immunocytochemical assays. [Mol Cancer Ther 2007;6(4):1400–5]

Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

Cell-to-cell adhesion modulates Stat3 activity in normal and breast carcinoma cells.

Stat3 (signal transducer and activator of transcription-3) activity is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. Although in most instances Stat3 is growth-promoting, the impact of cell density on Stat3 activation status and the biological importance of Stat3 during growth arrest have not been characterized. Previous results indicated that cell density alters tyrosine phosphorylation levels of cultured cells. Since signalling through Stat3 is determined by a key phosphorylation at tyr705, we examined the effects of cell density upon Stat3 activity in normal breast epithelial cells, breast carcinoma lines and normal mouse fibroblasts. Intriguingly, the results revealed a dramatic increase in Stat3, tyr705 phosphorylation and activity with cell density, which gradually declined at later stages. This activation was dependent upon cell–cell contact, since it was eliminated if cell adhesion was disrupted through calcium chelation, while it was reinstated through cell aggregation. Furthermore, this activation was suppressed following inhibition of JAKs (Janus kinases) but not inhibition of Fer, IGF1-R, or kinases of the c-Src family. On the other hand, constitutively active Stat3 in carcinoma lines, known to harbor activated Src, was blocked by pharmacological inhibitors of Src as well as JAKs. These results point to the existence of two distinct pathways of Stat3 activation in breast carcinomas, based on Src dependence. More importantly, our results suggest that Stat3 activity is upregulated during the confluence-mediated growth arrest by a signalling mechanism that requires JAKs.

Inhibition of Src family kinases with dasatinib blocks migration and invasion of human melanoma cells.

Src family kinases (SFK) are involved in regulating a multitude of biological processes, including cell adhesion, migration, proliferation, and survival, depending on the cellular context. Therefore, although SFKs are currently being investigated as potential targets for treatment strategies in various cancers, the biological responses to inhibition of SFK signaling in any given tumor type are not predictable. Dasatinib (BMS-354825) is a dual Src/Abl kinase inhibitor with potent antiproliferative activity against hematologic malignancies harboring activated BCR-ABL. In this study, we show that dasatinib blocks migration and invasion of human melanoma cells without affecting proliferation and survival. Moreover, dasatinib completely inhibits SFK kinase activity at low nanomolar concentrations in all eight human melanoma cell lines investigated. In addition, two known downstream targets of SFKs, focal adhesion kinase and Crk-associated substrate (p130CAS), are inhibited with similar concentrations and kinetics. Consistent with inhibition of these signaling pathways and invasion, dasatinib down-regulates expression of matrix metalloproteinase-9. We also provide evidence that dasatinib directly inhibits kinase activity of the EphA2 receptor tyrosine kinase, which is overexpressed and/or overactive in many solid tumors, including melanoma. Thus, SFKs and downstream signaling are implicated as having key roles in migration and invasion of melanoma cells. (Mol Cancer Res 2008;6(11):1766–74)

Resistance to BRAF Inhibitors: Unraveling Mechanisms and Future Treatment Options

The mitogen-activated protein kinase (MAPK) pathway has emerged as a central target for melanoma therapy due to its persistent activation in the majority of tumors. Several BRAF inhibitors aimed at curbing MAPK pathway activity are currently in advanced stages of clinical investigation. However, their therapeutic success is limited by the emergence of drug resistance, as responses are transient and tumors eventually recur. To develop effective and long-lasting therapies for melanoma patients, it is essential to understand the mechanisms underlying resistance to BRAF inhibitors. Here, we briefly review recent preclinical studies that have provided insight into the molecular mechanisms of resistance to BRAF inhibitors and discuss potential strategies to treat drug-resistant melanomas.