Jessie Villanueva

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.

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.

Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations

Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

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.

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.

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.

A Modified HSP70 Inhibitor Shows Broad Activity as an Anticancer Agent

The stress-induced HSP70 is an ATP-dependent molecular chaperone that plays a key role in refolding misfolded proteins and promoting cell survival following stress. HSP70 is marginally expressed in nontransformed cells, but is greatly overexpressed in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor but not normal cells; therefore, there has been great interest in the development of HSP70 inhibitors for cancer therapy. Here, we report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES) binds to the substrate-binding domain of HSP70 and requires the C-terminal helical “lid” of this protein (amino acids 573–616) to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in this region of HSP70, and we identify point mutants that fail to interact with PES. A preliminary structure–activity relationship analysis has revealed a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which shows increased cytotoxicity and ability to inhibit autophagy, along with significantly improved ability to extend the life of mice with pre-B-cell lymphoma, compared with the parent compound (P = 0.015). Interestingly, we also show that these HSP70 inhibitors impair the activity of the anaphase promoting complex/cyclosome (APC/C) in cell-free extracts, and induce G2–M arrest and genomic instability in cancer cells. PES-Cl is thus a promising new anticancer compound with several notable mechanisms of action. Mol Cancer Res; 11(3); 219–29. ©2013 AACR.

Targeting BRAF in Advanced Melanoma: A First Step toward Manageable Disease

Melanoma is the deadliest form of skin cancer and its incidence has been increasing worldwide. The disease manifests itself as clinically and genetically distinct subgroups, indicating the need for patient-specific diagnostic and treatment tools. The discovery of activating mutations (V600E) in the BRAF kinase in approximately 50% of patients spurred the development of compounds to inhibit aberrant BRAF activity, and the first drug candidate to show promising clinical activity is PLX4032 (also known as RG7204). Most recent clinical data from a phase II trial indicate that PLX4032 causes tumor regression and stabilized disease in >50% of advanced melanoma patients harboring BRAF V600E tumors. These data validate the effectiveness of oncogene-targeted therapy against advanced melanoma and offer hope that the disease can be overcome. However, as melanoma is dynamic and heterogeneous, careful treatment strategies and combination therapies are warranted to obtain long-term clinical effects. Clin Cancer Res; 17(7); 1658–63. ©2011 AACR.

Control of Tumor Bioenergetics and Survival Stress Signaling by Mitochondrial HSP90s

Tumors successfully adapt to constantly changing intra- and extracellular environments, but the wirings of this process are still largely elusive. Here, we show that heat-shock-protein-90-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase, inhibition of rapamycin-sensitive mTOR complex 1, induction of autophagy, and expression of an endoplasmic reticulum unfolded protein response. This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial heat shock protein 90s are adaptive regulators of tumor bioenergetics and tractable targets for cancer therapy.