Pre-Clinical In Vitro Evaluation Of Bcl-2 Antisense Compound Genasense (Tm) (G3139; Genta, Inc.) As A Targeted Pro-Apoptotic Agent For Leukemias With T(4;11)(Q21;Q23) Translocations

Abstract Background: Chemotherapy resistance from reduced apoptosis may contribute to poor outcome in infant leukemias with t(4;11). G3139 has clinical activity against refractory adult leukemias with minimal toxicity. Methods: We quantified anti-apoptotic BCL-2 and pro-apoptotic BAX mRNAs in 56 leukemias cases (55/56 from infants; 41 ALL, 15 AML; 24 t(4;11), 32 non-MLL), the ALL cell lines RS4:11 and SEM-K2, the AML cell line MV4-11, all of which have t(4;11), and normal CD34+ cells. BCL-2 and BAX mRNA levels were normalized to β-actin and examined by the comparative CT method. The in vitro cytotoxicity and pro-apoptotic activity of G3139 was investigated in the cell lines. Cytotoxicity was assessed by MTT after exposing log phase cells for 6 days to single agent G3139, or to fixed-concentration G3139 combined with anti-leukemia cytotoxic drugs over a range of concentrations; transfection reagent was not used for G3139 delivery. Pharmacostatistical response surface modeling was performed to determine synergy. Apoptosis was assessed by flow cytometry analysis of Caspase-3 activation and a FACS TUNEL assay. Results: BCL-2 and BAX mRNAs were abundant in the leukemias and cell lines with t(4;11), whereas CD34+ cells showed lower BCL-2 expression. The difference in normalized expression ratios of BCL-2:BAX relative to CD34+ cells approached significance when t(4;11) leukemias and cell lines were compared to non-MLL leukemias (p=0.07). The approximate IC50 of single agent G3139 was 10 μM in RS4:11 cells, 100 μM in MV4-11, and 180 μM in SEM-K2. Low, biologically achievable G3139 concentrations (5 μM, 1 μM) sensitized RS4:11 cells to the cytotoxicity of doxorubicin, 6-thioguanine, etoposide and cytosine arabinoside, but not methotrexate or dexamethasone. Despite the higher IC50 of G3139 alone in MV4-11 cells, synergy also was suggested when G3139 (50 μM, 10 μM) was combined with etoposide and 6-thioguanine since response surface modeling showed ability to achieve lower effective doses than projected from either single agent. When RS4:11 cells were assayed for proof of principle that the observed cytotoxicity was due to apoptosis, exposure to G3139 and doxorubicin together increased active Caspase-3 and TUNEL staining in a time and dose dependent manner. Conclusions: The imbalanced BCL-2/BAX expression suggests that an anti-apoptotic genotype forms the basis for the chemotherapy resistance in infant leukemias with t(4;11). These in vitro studies indicate that G3139 has pre-clinical activity, that select G3139-cytotoxic agent combinations are synergistic against cell lines with t(4;11), and that the observed activity occurs through apoptosis. Further studies are warranted to determine the pre-clinical in vivo effects of similar combinations against leukemias with t(4;11), with the goal of advancement to the clinic if results are promising.