Some animals were euthanized due to the development of skin necrosis that prevented them from reaching the maximum allowed tumor volume (1000 mm3)

Some animals were euthanized due to the development of skin necrosis that prevented them from reaching the maximum allowed tumor volume (1000 mm3). therapeutics, findings may have implications for other cancers as well. The combination of RAF inhibition with huHER3-8 was also more efficacious than either treatment alone at inhibiting tumor growth and promoting durable responses Assays Female athymic mice (NU/J: Jackson) were injected intradermally with human melanoma cells (~1.0 106) and cells allowed up to 2 weeks to reach appropriate tumor volume. huHER3-8 (100 L of 1 1 mg/mL) was injected intraperitoneally every 3 days of the experiment. For shRNA experiments, mice were given doxycycline (2 mg/mL) in the drinking water 3 days prior to huHER3-8 treatment that was replenished every 3 days for the duration of the experiment. For PLX4720 chow experiments, PLX4720 was formulated into rodent chow at 90 mg/kg (Research Diets Inc., New Brunswick, NJ). Tumors were measured using digital calipers, and volume was calculated using the formula: V = (L W2) 0.52. Some animals were euthanized due to the development of skin necrosis that prevented them from reaching the maximum allowed tumor volume (1000 mm3). At the conclusion of each experiment tumors that were larger than 1.00 (progression), less than 1.00 (regression), or equal to 0.00 (complete regression) were recorded. Animal experiments were performed at Thomas Jefferson University (AAALAC accredited) and approved by the Institutional Animal Care and Use Committee (IACUC). Statistics was performed using a mixed effect model where error bars represent standard HLM006474 error. Results NRG1-ERBB3 signaling in vemurafenib-treated mutant BRAF melanoma cells is inhibited by huHER3-8 We tested the ability of the humanized anti-ERBB3 monoclonal antibody, huHER3-8, to inhibit ERBB3 phosphorylation in BRAFV600E/D melanoma cells. huHER3-8 binds within residues 20 and 342 of ERBB3 with an affinity of 0.17 nM towards human ERBB3 in FACS assay using SKBR3 human breast adenocarcinoma cells (14) and outcompetes NRG1 binding and prevents ERBB3 dimerization with ERBB2. A 10 g/mL dose of huHER3-8 was used for experiments based on dose-dependent inhibition of NRG1-mediated ERBB3 phosphorylation (Fig. 1A). In the mutant BRAF cell lines, 1205Lu, M238, and A375, basal levels of phosphorylated ERBB3 were low (Fig. 1A & 1B). Consistent with our previous findings, NRG1 stimulates phosphorylation of ERBB3, an effect that was dramatically enhanced by overnight pre-treatment with vemurafenib (8). Pre-treatment with huHER3-8 efficiently inhibited NRG1-induced phosphorylation of ERBB3 in both untreated and vemurafenib-treated cells (Fig. 1B). Open in a separate window Figure 1 huHER3-8 blocks NRG1-mediated ERBB3 activation in mutant BRAFV600E melanoma cell linesA) A375 cells were treated with 1 M vemurafenib for 24 hours, followed by 45 minutes of huHER3-8 at the concentrations indicated. Cells were stimulated with 10 ng/mL NRG1 for 15 minutes HLM006474 and then lysed for Western blot analysis. B) M238, 1205Lu and A375 cells were cultured in the presence/absence of 1 1 M vemurafenib for 24 hours. Cells were then treated or untreated with 10 g/mL huHER3-8 for 45 minutes before stimulation with 10 ng/mL NRG1 for 15 minutes. Cell lysates were analyzed by Western blot with the indicated antibodies. To better understand the effects of ERBB3 on mutant BRAF melanoma cells, we performed Reverse Phase Protein Array (RPPA) analysis on 1205Lu and A375 cells treated with vemurafenib HLM006474 and NRG1 in the absence/presence of huHER3-8 (15). PI3K/AKT pathway signaling was most affected by NRG1 treatment in both cell lines (Supp. Table S1 DPC4 & S2). Importantly, pretreatment with huHER3-8 prevented the phosphorylation of AKT induced by NRG1 (Fig. 2A & 2B). Analysis of the RPPA data using Gene Ontology gene sets was performed to determine the pathways affected by NRG1 and huHER3-8 treatment. In 1205Lu cells treated with vemurafenib and NRG1, there was a significant enrichment of cellular pathways involving phosphorylation and receptor signaling (Fig. 2C). By contrast, huHER3-8 pre-treatment effectively inhibited the activation of NRG1-dependent signaling and significantly enriched pathways involved in the regulation of cell death and apoptosis (Fig. 2D). A375 cells treated with vemurafenib and NRG1 exhibited a significant enrichment of pathways involved in PI3K/AKT signaling, as well as other cellular pathways (Supp. Fig. S1). Pretreatment with huHER3-8 in these cells prevented the enrichment of these pathways, but did not result in a significant enrichment of cell death and apoptosis pathways (Supp. Table S1 & S2 for full data set). Taken together, these data suggest that the PI3K/AKT signaling pathway is activated by NRG1 and inhibited by anti ERBB3 antibodies in RAF-inhibited mutant BRAF melanoma cells. Open in a separate.