The Bcl-2—IP3 Receptor Interaction As a Novel Target for Therapeutics in Myeloma

Abstract 4011

The anti-apoptotic Bcl-2 (B-cell lymphoma 2) protein was first identified in follicular lymphoma, where the Bcl-2 gene was dysregulated as a result of chromosomal translocation. The anti-apoptotic activity of Bcl-2 can be attributed to two mechanisms conferred by structurally distinct domains. First, the BH domains 1–3 of Bcl-2 dimerize with pro-apoptotic members of the Bcl-2 family, such as Bax and Bak, and maintain mitochondrial integrity. Small molecules targeting these domains on Bcl-2 (e.g. ABT-737 and ABT-263/Navitoclax®) are under clinical evaluation. A less explored activity of Bcl-2 is that its BH4 domain functionally interacts with the IP3 receptor located on the endoplasmic reticulum (ER) membrane, the major organelle involved in regulating calcium homeostasis. Through this interaction, Bcl-2 inhibits IP3 receptor channel opening and prevents excessive calcium release from ER that can be harmful to cells. Our strategy is to disrupt the Bcl-2—IP3 receptor interaction to induce cell death in lymphoid malignant cells.

Previously, our group has shown that an IP3 receptor derived peptide, or IDP, blocks the Bcl-2—IP3 receptor interaction and thereby induces elevation of cytosolic calcium in lymphoma cell lines. A modified version of the peptide—IDP-DD/AA, with an asparaginase cleavage site removed, is more potent in inducing calcium elevation as well as apoptosis in primary chronic lymphocytic leukemia (CLL) cells. Concentrations of the IDP-DD/AA peptide that killed CLL cells did not affect the viability of normal lymphocytes, suggesting a therapeutic window. We further exploited the cytotoxic activities in a panel of leukemia/lymphoma/myeloma cell lines and found that over 60% of myeloma cell lines tested were sensitive to the IDP-DD/AA peptide but not to the control peptide. Furthermore, in the sensitive myeloma cell lines treated with the IDP-DD/AA peptide, high amplitude cytosolic calcium elevation preceded caspase-3 activation and apoptotic morphology, suggesting the involvement of a calcium-mediated intrinsic apoptotic pathway.

The in vivo anti-tumor activity of the IDP peptide was assessed in a myeloma xenograft model. Subcutaneous tumors were established in nude mice using the human myeloma cell line, NCI-H929. Randomly grouped mice treated by in-tumor injection of the IDP-DD/AA peptide showed prominent inhibition of tumor growth compared to saline-injected control. One of three mice treated with the IDP-DD/AA peptide showed no visible sign of tumor at the end of treatment. The data proffer promise of therapeutic use of the IDP peptide in myeloma disease.

The effect of combinatory treatment of the IDP-DD/AA peptide and current anti-myeloma drugs (Bortezomib/Valcade®, doxorubicin/Adriamycin®, melphalan) were evaluated. Co-treatment of the myeloma cell line NCI-H929 cells with the IDP-DD/AA peptide and each one of the three anti-myeloma drugs induced synergistic cytotoxicity with combinatory indices (CI) less than 1. The data suggest that the IDP-DD/AA peptide augments cell killing activity of current chemotherapy drugs.

In conclusion, the IDP-DD/AA peptide represents a novel anti-cancer agent that targets the anti-apoptotic Bcl-2 protein. The peptide exhibits both in vitro and in vivo anti-myeloma activity. Moreover, the IDP-DD/AA peptide can function as a single agent (in myeloma or CLL) as well as in combination with current anti-myeloma chemotherapy drugs.