Abstract A38: Novel oncolytic adenovirus (ILV-19) induces apoptosis in BRAF inhibitor resistant (V600E) mutant melanoma cell lines 451lu-R and Mel1617-R

Abstract

Abstract Over 50% percent of melanomas carry activating BRAF mutation with a vast majority of these mutations (>90%) resulting from a single nucleotide switch at amino acid 600 resulting in a glutamic acid substitution for valine (V600E). As a result of this mutation BRAF Kinase is constitutively expressed and results in disregulation of the RAS-RAF-MEK-ERK protein kinase pathway that controls cell proliferation and apoptosis. Although, Vemurafenib, a specific inhibitor of the V600E BRAF mutant, has shown to have initial positive clinical response in clinical trials, drug resistant develops after several months of continuous exposure resulting in the recurrence of the treated cancer. We have developed a modified adenovirus vector (AdILV-19) that effectively kills vemurafenib resistant cells. We demonstrate that AdILV-19 suppresses expression of c-Myc, survivin, cyclin D1, B-catenin, and Mcl-1 in BRAF V600E mutated cells that are resistant to vemurafenib. This novel vector, AdILV-19 is derived from our previously published adenoviral vector TAV-255 through an additional modification of the adenoviral genome. In order to facilitate and maximize recombinant adenoviral induced apoptosis by this new vector, we removed the first 200 base pair of the adenoviral E1b 19k protein, a human BCL-2 homolog. Using this newly modified virus in two highly Vemurafenib resistant melanoma cell lines (451lu-R, Mel1617-R) we were able to not only infect and express viral proteins i.e. E1a, but also induce a significant cell killing in these resistant BRAF mutant cell lines by MTT as well as crystal violet assays. MTT assay showed 35% to 45% cell killing at an MOI as low as 15 to 80 to 90% killing at an MOI of 50. Crystal violet assay on the other hand showed a significant cytotoxicity in these resistant cells at a low MOI of 15. Additionally, we were able to show using Immunoblot analysis a postinfection reduction in the expression of major pro-survival proteins in time dependent manner. We observed as early as 24hrs postinfection c-Myc (an oncogenic transcription factor) and survivin (Pro-survival protein) expression were turned off while expression of cyclin D1 (a regulatory subunit of holoenzyme) which shown to be amplified in BRAF inhibitor resistant melanoma cells and Mcl-1 (a pro-survival protein) were turned off as early as 48hs postinfection in both 451lu-R and Mel1617-R melanoma cells. Furthermore, colony formation assays showed a substantial reduction in the ability of single cells to form colonies after infection at MOI of 20 with AdILV-19 in both of these resistant melanoma cell lines. As an extension of these studies we also studied the activity of AdILV-19 to treat a xenograft nude mouse model with subcutaneous injection of 451lu-R melanoma cells. When the tumor volume reached 100 mm3 the mice received 3 intratumoral injections at an MOI of 20. Following treatment, we observed a significant inhibition of tumor growth (p≤0.02) in treated mice (n=4) when compared with PBS treated mice controls. Taken together these data we show this newly modified adenovirus vector AdILV-19 alters and silences cellular oncogenic protein synthesis in vemurafenib resistant BRAF mutant melanomas and has a great potential as therapeutic for the treatment of these human resistant melanomas. Citation Format: Farah Hedjran, Lingzhi Zhang, Jason Sicklick, Tony Reid. Novel oncolytic adenovirus (ILV-19) induces apoptosis in BRAF inhibitor resistant (V600E) mutant melanoma cell lines 451lu-R and Mel1617-R. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A38.