Growth inhibition of human multiple myeloma cells by a conditional-replicative, oncolytic adenovirus armed with the CD154 (CD40-ligand) transgene.
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Rodrigues, Margret S.
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Human multiple myeloma (MM) remains incurable despite successes in induction therapy. Based on our previous findings that treatment with the recombinant CD40L protein inhibited the growth of CD40+ human myeloma cell lines, we examined the targeting of the myeloma cell surface receptor, CD40 by its natural ligand, CD40L, using a gene transfer approach. For targeted delivery of CD40L, we have constructed a conditional-replicative adenoviral for delivery of the CD40L transgene (AdEHCD40L). AdEHCD40L incorporates tumor/tissue specific promoters limiting transgene expression and viral replication to HIF (hypoxia-inducing factor)-1α and/or estrogen overexpressing cancer cells. Conditional expression of the early adenoviral E1A gene and the CD40L transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.7% and 30%, respectively). Treatment with AdEHCD40L resulted in pronounced growth inhibition (RPMI 8226: 95.5 ± 2.1%; Kas-6/1: 80.5 ± 9.8%, mean ± SD), and was more effective than the parental construct without the CD40L transgene (AdEHNULL)(p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells and IMR-90, normal fibroblast cells (2.8 ± 0.3%). We also observed a 53% reduction in myeloma xenograft growth, following AdEHCD40L intratumoral injections (4.8 ± 0.9 mm, vs. 10.5 ± 1.2 mm in mock-treated animals; p=0.002), which was more effective than AdEHNULL (7.6 ± 1.1 mm; p=0.03). Evidence of adenoviral hexon and CD40L transgene expression within the treated tumors was demonstrated. In the study of cellular events leading to myeloma growth inhibition, we observed increased apoptotic activity at 72 hrs following treatment with AdEHCD40L infection (21.3 ± 6.5% vs. 7.7 ± 1.9% in untreated; p=0.007) which was accompanied by increased S phase accumulation (68.0 ± 2.4%, vs. 54.0 ± 5.5% in AdEHNULL and 48.9 ± 4.6% in untreated culture; p<0.003). High throughput gene array expression analysis identified pro-apoptotic TNF superfamily members, Fas and TRAIL and cytokines, IL-8 and RANTES to be uniquely upregulated by AdEHCD40L. These findings confirm the growth inhibitory activity of AdEHCD40L, through the induction of apoptosis and is a promising approach for the experimental gene therapy of human multiple myeloma.