Growth Inhibition of Human Multiple Myeloma Cells by a CD40 Ligand (CD40L, CD154) Transgene - Oncolytic Virus Construct.

Human multiple myeloma (MM) remains incurable despite recent advances in induction therapy. To explore biotherapeutic approaches that may potentially enhance the efficacy of conventional treatment, we examined the growth regulatory properties of CD40L (CD154), the natural ligand for the myeloma cell surface receptor, CD40. Based on our previous findings that the recombinant CD40L protein effectively inhibited human MM cell growth, a conditional-replicative adenoviral construct, AdEHCD40L was used for targeted delivery of the CD40L transgene. AdEHCD40L incorporates tumor/tissue specific promoters that limit viral and transgene expression to HIF (hypoxia inducing factor)-1α overexpressing cells, which are prevalent in the human bone marrow compartment. Conditional expression of the early adenoviral E1A gene and the CD154 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 (32.68% and 30%, respectively). Further, treatment with AdEHCD40L at a multiplicity of infection (MOI) of 1 resulted in pronounced growth inhibition for both cell lines (95.5±2.1% and 80.5±9.8%, mean±SD, respectively). AdEHCD40L treatment was more effective than the parental construct without the CD154 transgene (AdEHNULL) in both cell lines (p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells (0% at 48hrs) and normal fibroblast cells IMR-90 (2.8±0.3%). The in vivo antitumor activity of AdEHCD40L was examined with a subcutaneous RPMI 8226 heterotransplant model in SCID mice. Intratumoral injection of AdEHCD40L (5x10⁷ pfu, x 5) reduced xenograft growth by 53% at day 29 (4.8±0.9 mm, vs. 10.5±1.2 mm in mock-treated animals; p=0.002), and was more effective than AdEHNULL (7.6±1.1 mm; p=0.03). Adenoviral hexon and CD40L expression was detectable at 29 days post-viral treatment, based on immunohistochemical analysis. Hence intratumoral treatment with AdEHCD40L likely involved oncolytic viral replication. To further characterize cellular events that accompany MM cell growth inhibition, apoptotic activity was measured by annexin V and propidium iodide incorporation. A marked elevation of the annexin V⁺ subset (21.3±6.5%, vs. 7.7±1.9% in untreated culture; p=0.007) was accompanied by decreased cell viability (52.8±1%, vs. 76.1±11% in untreated cultures; p = 0.04) following AdEHCD40L treatment. Cell cycle distribution analysis demonstrated a corresponding increase in the subG_0/G₁ compartment (AdEHCD40L, 23.9±3.6%; vs. untreated, 9.1±2.3%) that was consistent with elevated apoptosis. Further, AdEHCD40L increased S phase accumulation by 72 hrs (68.0±2.4%, vs. 54.0±5.5% in AdEHNULL and 48.9±4.6% in untreated culture; p<0.003). Currently, gene expression array analyses are underway to define molecular events that are pertubated by CD154 transgene activity and by viral oncolysis. These findings will further elucidate the mechanism of action of the CD154+oncolytic viral approach for experimental gene therapy of human MM.