Modeling Proteasome Inhibition in Lymphoma

Abstract 4946

The proteasome inhibitor Bortezomib (Bz) has been widely used to treat multiple myeloma, relapsed mantle cell lymphoma and is undergoing clinical evaluation for other B cell malignancies including non-Hodgkin lymphoma. Despite its initial success, patients treated with Bz eventually relapse due to the development of drug resistance. Therefore, understanding the basis of drug resistance is a critical component for improved therapy. The acquisition of Bz resistance in lymphomas, particularly those with constitutive expression of the B cell-specific DNA mutator, activation-induced cytidine deaminase (AID), has not been previously characterized.

We have utilized the AID-expressing human non-Hodgkin Burkitt's lymphoma as a model system for this study. Burkitt's lymphoma lines (Ramos and BL-2) are suitable because they are highly sensitive to Bz induced apoptosis with an IC₅₀ of approximately 11 nM after 48 hrs of treatment. In order to generate Bz resistant Burkitt's lymphoma lines, Ramos cells were treated weekly with increasing concentrations of Bz for 3 months. Compared to the parental line, this newly formed line displayed an approximately 2.5–3-fold increase in IC₅₀ to Bz as well as to three other proteasome inhibitors (next-generation proteasome inhibitor, MLN 2238, epoxomicin and carfilzomib), while maintaining sensitivity to different chemotherapeutic agents (PD 0332991 cyclin 4/6 dependent kinase inhibitor and melphalan). In this model system, resistance to Bz conferred a general cross-resistance to other proteasome inhibitors, a phenotype that has been stably maintained for 5 months.

We next asked whether AID plays a role in the acquisition of Bz resistance in Ramos cells by promoting hypermutation and genomic instability. In support of this hypothesis, the G322A and C326T mutations in the gene encoding the proteasome subunit and target of Bz, psmb5, occur within AID hotspots raising the possibility that AID could directly mutate psmb5. Furthermore, like many proteins, AID is degraded by the proteasome arguing that proteasome inhibition further stabilizes AID protein resulting in aberrant hypermutation. Consistent with this, we detected by flow cytometry (intracellular staining) a 2-fold increase in AID protein following a 24-hour, 20 nM Bz treatment of Ramos cells. AID normally mutates immunoglobulin (Ig) genes and these mutations are necessary for the production of protective antibodies, while aberrant AID activity leads to mutations in non-Ig genes. Surprisingly, despite the increase in AID protein, we observe reduced mutation frequency within the functional IgH gene following 3-month Bz treatment compared to untreated controls. Despite this reduction, array comparative genomic hybridization (a-CGH) studies indicate copy number abnormalities in Bz resistant cells and details of the chromosomal abnormalities and target genes deregulated will be presented. These data demonstrate that Burkitt's lymphoma cells are sensitive to Bz and drug resistance can be readily achieved in vitro. Furthermore, Bz treatment stabilizes AID protein and promotes increased genomic instability.