Mechanism of Action of G3139

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DESCRIPTION (provided by applicant): Recent exciting pre-clinical and phase II clinical trial data employing G3139 (a.k.a. Oblimersen), an 18mer I antisense phosphorothioate oligodeoxynucleotide targeted to the first six codons of the bcl-2 mRNA, indicates that this novel agent may be clinically active in combination with cytotoxic chemotherapy in the treatment of a relatively large number of human tumors. However, significant questions remain as to the precise mechanism of action of this agent. We and others have ascertained four distinct possible mechanisms of action of G3139: 1) Downregulation of bcl-2 with postulated subsequent specific increase in chemosensitivity; 2) Non-specific effects of the oligonucleotide, including irrelevant cleavage, leading to synergy with mechanism 1; 3) CpG motif-modulated stimulation of immune effector mechanisms, possibly involving Toll-like receptors, and 4) Local (at the level of the tumor) CpG-modulated production of reactive oxygen species (ROS) leading a diminution in the rate of cell growth. In order to demonstrate which mechanisms are responsible for the antitumor effects of G3139 both in vitro and in vivo, we have devised four Specific Aims: Specific Aim 1: We will determine how the production of ROS and H202 by G3139 and related oligos in prostate and bladder cells serves to affect the growth and viability of these cells. We will add small molecules to scavenge ROS and H202, add catalase to the external media to block the effects of H202 on cell growth, and infect cells with an adenoviral-MnSOD vector to reduce ROS production. We will also determine the intracellular concentration of H202, and determine the ability of these oligos to induce lipid-peroxidation in a bcl-2 dependent and independent manner. We will also examine mitochondrial function in the presence of G3139 (e.g., mitochondrial potential, ATP production, oxygen consumption). Specific Aim 2: We will employ novel 2'-O,4'-C-methylene-linked bicyclic ribonucleosides (LNAs) at the 3' and 5' termini of G3139 to increase oligo stability and affinity for its target. The optimal gapmer will be determined, in addition to its ability to downregulate expression of bcl-2 compared to G3139. Specific Aim 3: We will use PC3 cell xenografts in SCID mice to evaluate the roles of immunostimulation downregulation of bcl-2 expression, and the local production of reactive oxygen species in the antitumor effect of G3139 and its LNA gap-mer homolog. Finally, in Specific Aim 4, we will perform Affymetrix gene-chip analysis of G3139 and related oligo-treated prostate and bladder carcinoma cells in order to specifically determine which genes are affected by G3139 treatment.