Defining Mechanisms of Sensitivity and Resistance to Histone Deacetylase Inhibitors to Develop Effective Thereaputic Strategies for the Treatment of Aggressive Diffuse Large B-Cell Lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). The current standard of care is the combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but this only results in a 60% over-all 5-year survival rate, thus highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics that is being clinically evaluated for combination therapy. Rational selection of companion therapeutics for HDACi is difficult due to their poorly understood, cell-type specific mechanisms of action. To understand these mechanisms better, we developed a pre-clinical model system of response to the HDACi belinostat. Using this model system, we identified two major responses. Resistance, consisting of a reversible G1 cell cycle arrest with little induction of apoptosis; or sensitivity, consisting of mitotic arrest and high levels of apoptosis. In this dissertation, we determine that the induction of G1 cell cycle arrest is due to the increased expression of cyclin dependent kinase inhibitors (CDKi) that bind to and inhibit the cyclin E/CDK2 complex thereby blocking the final repressive phosphorylation steps of Rb protein. Repression of transcriptional elongation blocked CDKi upregulation and prevented G1 cell cycle arrest in belinostat-resistant cells. Additionally, we identified that belinostat arrests sensitive cells prior to metaphase and belinostat-resistant cells slow-down in mitosis but complete the process prior to arresting in G1. The combination of belinostat with the microtubule-targeting agent, vincristine resulted in strong synergistic induction of apoptosis by targeting mitotic progression. Furthermore, this combination prevents polyploidy, a key mechanism of resistance to microtubule targeting agents. Finally, we utilized selective class one HDAC inhibitors to identify the individual contributions of HDACs in the eliciting the responses observed with belinostat treatment. HDAC1&2 inhibition recapitulated the belinostat-resistant phenotype of G1 cell cycle arrest with little apoptosis, in both belinostat-resistant and sensitive cell lines. HDAC3 inhibition resulted in the induction of DNA damage, increased S phase and the induction of apoptosis in belinostat-resistant cells. Belinostat-resistant cells did not have observable effects to HDAC3 inhibitor alone but when combined with vincristine had significantly increased G2/M population at early time points. This suggests that HDAC3 maintains roles in DNA replication and also in mitotic progression. HDAC3 inhibition combined with vincristine resulted in a significant increase in polyploidy, suggesting that HDAC3 might not regulate the expression of apoptotic regulating factors as belinostat does.