AT-101, a Pan-Inhibitor of Bcl-2 Family Anti-Apoptotic Proteins Antagonizes the Protective Effect Conferred by Nurselike Cells on Primary Chronic Lymphocytic Leukemia (CLL) Cells.
Authors: James Danelle F. , Mervis Rebecca M. , Mosadeghi Ruzbeh , Kipps Thomas J. 
Published: October 2019

Despite the longevity of CLL B cells in vivo, in vitro CLL cells readily undergo apoptosis under conditions that support the growth of normal B cells. However, when leukemia cells are co-cultured with non-neoplastic accessory cells, such as those found in the marrow stroma and nurselike cells (NLC) that can differentiate from blood mononuclear cells they become resistant to spontaneous and drug induced cell death. These findings suggest that the defective apoptosis of the CLL B cells is not only ascribed to intrinsic defects in the neoplastic cell, but also to extrinsic factors that influence their behavior provided by the tumor microenvironment.

As such, identification of molecular targets that link malignant B cells to supportive cells in the microenvironment may lead to new therapeutic avenues for CLL patients. For example, we have found that co-culture with NLC induces expression of Mcl-1, an anti-apoptotic protein of the Bcl-2 family in CLL cells. [Nishio M 2005] Induction of such prosurvival proteins by accessory cells may play a role disease aggressiveness and leukemia cell resistance to chemotherapy in vivo.

AT-101 is a small molecule that mimics the inhibitory BH3 domain of endogenous antagonists of the Bcl-2 family anti-apoptotic proteins negating their cytoprotective role. Fluorescence polarization assays demonstrate that AT -101 binds to Bcl-2, BcL-XL, Bcl-W, and Mcl-1. AT-101 is cytotoxic to primary CLL cells in vitro and has been shown to have single agent activity in high risk CLL patients.

Therefore, we hypothesized that inhibition of Mcl-1 by AT-101 may diminish the protection of NLC on CLL cells and may render the leukemia cells more sensitive to spontaneous and drug induced apoptosis.

Primary CLL cells from 20 different patients were plated with or without NLCs, and cell viability was assessed overtime. Co-culture of CLL cells with NLC protected the CLL cells from undergoing spontaneous apoptosis. After 48 hours the average viability (+/− SEM) of CLL cells with NLC was 75% (+/− 4%) whereas the mean viability of CLL cultured alone was significantly lower at 59% (+/−5%) (p<0.02). In addition, we found that co-culture of CLL cells with NLC protected the leukemia cells from the cytotoxic effects of fludarabine (F-ara-A). For instance, after 48 hours after treatment with 10 μM F-ara-A the mean viability of the CLL cells was 28% (+/− 4%) whereas the viability of CLL cells cultured with NLC was 51% (+/−4%), a difference that was highly significant (p<0.001). In contrast, NLC could not protect leukemia cells from apoptosis induced by AT-101.

For example, treatment of CLL cells with AT-101 at 5 μM, with or without NLC, resulted in CLL cell viability at 48 hours of only 40% (+/−7%.) or 30% (+/−7%.), respectively, a difference that was not statistically significant. AT-101 and F-ara-A were titrated in the setting of NLC and appeared to act additively in inducing apoptosis of CLL cells. In fact, NLC protection of CLL cells from fludarabine mediated cytotoxicity was negated in the presence of AT-101.

Together our results describe a new therapeutic mechanism for targeting CLL interaction with the microenvironment. Specifically, a pan-inhibitor of Bcl-2 family anti-apoptotic proteins AT-101 antagonizes the protection of CLL by NLC to both spontaneous and fludarabine mediated apoptosis.