plx-4720 and Necrosis

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF(V600E) melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF(V600E) melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF(V600E) melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Drug Synergism; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Male; Melanoma; Mice; Mice, Nude; Mutation, Missense; Necrosis; Panobinostat; Proto-Oncogene Proteins B-raf; Receptor-Interacting Protein Serine-Threonine Kinases; RNA, Small Interfering; Sulfonamides; Vemurafenib; Vorinostat; Xenograft Model Antitumor Assays

Carcinomas are comprised of transformed epithelial cells that are supported in their growth by a dedicated neovasculature. How the genetic milieu of the epithelial compartment influences tumor angiogenesis is largely unexplored. Drugs targeted to mutant cancer genes may act not only on tumor cells but also, directly or indirectly, on the surrounding stroma. We investigated the role of the BRAF(V600E) oncogene in tumor/vessel crosstalk and analyzed the effect of the BRAF inhibitor PLX4720 on tumor angiogenesis. Knock-in of the BRAF(V600E) allele into the genome of human epithelial cells triggered their angiogenic response. In cancer cells harboring oncogenic BRAF, the inhibitor PLX4720 switches off the ERK pathway and inhibits the expression of proangiogenic molecules. In tumor xenografts harboring the BRAF(V600E), PLX4720 extensively modifies the vascular network causing abrogation of hypoxia. Overall, our results provide a functional link between oncogenic BRAF and angiogenesis. Furthermore, they indicate how the tumor vasculature can be "indirectly" besieged through targeting of a genetic lesion to which the cancer cells are addicted.

Topics: Alleles; Angiogenesis Inducing Agents; Animals; Antibodies, Monoclonal, Humanized; Bevacizumab; Blood Vessels; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Chickens; Chorioallantoic Membrane; Cytostatic Agents; Down-Regulation; Gene Knock-In Techniques; Humans; Indoles; MAP Kinase Signaling System; Mice; Molecular Targeted Therapy; Mutation; Necrosis; Neoplasms; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Sulfonamides; Xenograft Model Antitumor Assays