Alterations In Mitochondrial Priming May Be a Mechanism For Acquired Resistance To Therapy In Diffuse Large B-Cell Lymphoma

Molecular profiling and real-time detection of therapy-resistant lymphoma cells have the potential to improve clinical outcomes by identifying biomarkers capable of predicting treatment efficacy, providing insight into key regulatory signaling pathways suitable for therapeutic targeting, and tailoring treatment selection. Czuczman et al developed and characterized several cell-line models to study the mechanisms involved in the acquirement of rituximab-resistance in B-cell lymphoma. Rituximab-resistant cell lines (RRCLs) were found to be also resistant to multiple chemotherapy agents when compared to rituximab-sensitive cell lines (RSCL). RRCLs have decreased expression of Bax and Bak, key pro-apoptotic members of the Bcl-2 family of proteins. Expression of Bcl-2 family members favoring cell survival has been seen in other lymphoma cell lines that are known to be chemotherapy resistant.

It is important to validate the role of Bax/Bak expression in rituximab/chemotherapy resistance in a more clinically relevant setting and to develop assays that detect rituximab/chemotherapy resistant cells in real-time, ideally before therapy is started in a given patient. To this end, we studied 1) correlations in clinical outcomes in lymphoma patients based on pre-treatment Bax/Bak levels an 2) differences in BH3 profiling, a technique that allows for functional assessment of how “primed” cells are (that is, how capable they are of undergoing apoptosis) between RSCL and RRCL.

To study the pattern of expression of Bax/Bak in lymphoma and its clinical significance, we retrospectively evaluated differences in the expression these pro-apoptotic proteins in patients evaluated at the Dana-Farber/Brigham and Women’s Cancer Center using a tissue micro-array (TMA) containing samples from 180 patients with different subtypes of B-cell lymphoma. Demographic, clinical and pharmacological characteristics were obtained for each patient. Bax/Bak staining was reported as negative, weak, moderate, or intense.

In addition, RSCL and RRCL underwent BH3 priming studies. Briefly, RSCL or RRCL were exposed to a panel of BH3 only, pro-apoptotic proteins, and the amount of mitochondrial outer membrane permeabilization (MOMP) was measured over time as previously described.

In highly primed cells, MOMP occurs quickly after addition of BH3 peptides, indicating that balance of pro- and anti-apoptotic members of the Bcl-2 family is in favor of apoptosis. This technique permits the assessment of the apoptotic potential in real-time.

Generally, there were high levels of Bax and Bak expression amongst the lymphoma specimens represented on the TMA. Of those that could be analyzed, 90% of samples stained moderately or intensely for Bax, and 70% stained moderately or intensely for Bak. Those specimens with decreased expression did not reliably correspond with a more refractory clinical course based on the data available. Ten (10) of the patients included in the TMA had been previously treated with rituximab. Of these patients, 90% stained moderately or intensely for Bax. Nine (9) of these patient samples could be stained for Bak, and 88.9% of them stained moderately or intensely. Significant differences were observed in the BH3 profiling of RSCL and RRCL. Raji 2R and Raji4RH (RRCLs) were less primed than Raji cells (RSCL), suggesting a decrease in the mitochondrial potential.

In summary, while Bcl-2 family members play an important role in the development, maintenance, and progression of B-cell lymphoma, the level expression of each individual Bcl-2 family member (i.e. Bax or Bak) appears to have less of an effect on clinical outcome that the overall balance between pro- and anti-apoptotic members. This underscores the need to develop and validate a real-time assay to define the apoptotic threshold of a cancer cell. BH3 priming further demonstrated that RRCL have impaired MOMP. Potentially, this could be adapted for the clinical setting to identify patients most likely to require alternative therapeutic strategies.

¹Czuczman MS et al. Clin Cancer Res. 2008;14:1561-70.

²Olejniczak S. et al. Clin Cancer Res. 2008;14:1550-60.

³Deng J et al. Cancer Cell. 2007;12:171-185

⁴ Ryan J, et al. PNAS. 2010; 107(29):12895-12900.