AT 101, an Inhibitor of Bcl-2 Family Members Is Cytotoxic to a Heterogeneous Group of CLL Samples and Synergistic with Rituximab.
Authors: James Danelle F. , Prada Carlos E. , Castro Januario E. , Kipps Thomas J. 
Published: April 2020

Abstract
Bcl-2 family proteins include both pro-survival and pro-apoptotic factors. The balance of these regulatory proteins determines a cell’s threshold for death. Bcl-2, Bcl-XL, and Mcl-1 are structurally related anti-apoptotic proteins of the Bcl-2 family. Constitutive expression of Bcl-2 is characteristic of CLL and has been implicated in the leukemia’s pro-survival tendency. In addition, high level expression of Mcl-1 and Bcl-2 have been shown to correlate with important clinical outcomes in CLL such as poor response to chemotherapy and decreased overall survival. Bcl-2 prevents mitochondrial cytochrome c release and inhibits apoptosis through direct regulation of caspases. The BH3-only proteins dimerize and neutralize their pro-survival counterparts via interaction with the BH3 binding grove on the anti-apoptotic proteins. This interaction results in the release of cytochrome c, subsequent caspase activation, and resultant cell death. Gossypol is a naturally occurring product found in cottonseed oil. AT 101, an orally bio-available derivative of gossypol is currently being evaluated in cancer clinical trials. AT 101 is a small molecule that mimics the inhibitory BH3 domain of endogenous antagonists of Bcl-2 negating its cytoprotective role. AT 101 induces apoptosis via caspase activation in cancer cells that over-express Bcl-2 or Bcl-XL, but had no significant cytotoxic effect on normal blood mononuclear cells (Mohammad, 2005). Fluorescence polarization assays demonstrate that AT 101 binds to Bcl-2, BcL-XL, and Mcl-1 with greater affinity than gossypol (Prada,ASH 2005). We hypothesized that inhibiting Bcl-2 and Mcl-1 by AT 101 may allow the CLL cells to enter apoptosis more readily and render them more sensitive to standard CLL therapeutics. We tested 10 different primary CLL patient samples in vitro and found gossypol to be cytotoxic in a dose and time dependent manner. We observed 50% reduction in CLL cell viability at a concentration of 2μM after 48 hours of treatment. Significant cytotoxicity was observed irrespective of ZAP-70 expression or IgVH mutational status. Increased ratios of Bcl-2/Bax and Mcl-1/Bax have been demonstrated in CLL patients who do not respond to rituximab (Bannerji 2003). To examine the ability of gossypol to enhance the cytotoxic effects of rituximab in CLL we evaluated samples from 6 different patients affected with the leukemia in vitro. CLL cells treated with rituximab at 10μg/ml showed no significant decrease in viability compared to untreated cells. When AT 101 was added at 5μM concentrations in combination with rituximab 10μg/ml for twelve hours the average CLL viability was decreased by 67.2% over that of cells treated with rituximab alone (P&lt;0.001 by Bonferroni multiple comparison test) and by 45.3% over cells treated with AT 101 alone (P&lt;0.001). The combined effect of AT 101 and rituximab appears synergistic and is displayed throughout CLL subtypes. In our studies we were unable to reach the IC50 of rituximab on CLL using doses up to 100μg/ml. However, the addition of AT 101 to rituximab 10μg/ml surpassed the IC50 and demonstrated similar cytotoxicity to that of high dose rituximab100μg/ml when used in conjunction with AT 101. Together our results suggest that AT 101 may have therapeutic potential in CLL as a single agent or in concert with other known CLL therapeutics. In particular, AT 101 was found to have synergistic cytotoxicity when combined with rituximab. Currently a phase I clinical trial is underway to evaluate the activity of AT 101 in previously untreated patients with CLL.