Mechanisms of Sensitivity and Resistance to Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma

Abstract 1359

Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin Lymphoma (NHL) throughout the world. DLBCL is an aggressive, heterogeneous disease with two major recognized cell-of-origin subtypes: “germinal center” (GCB) and “activated B-cell like” (ABC), the latter having the worse prognosis. Overall, DLBCL remains fatal for about 30% patients due to relapse or lack of response to initial therapy. Resistant/relapsed DLBCL patients could benefit from the addition of new promising antiproliferative drugs, such as histone deacetylase inhibitors (HDACIs), to current chemotherapy regimens. So far, Vorinostat and Romidepsin, two structurally different HDACIs, have been approved for the treatment of hematological cancers. Despite their proven antiproliferative, pro-apoptotic effects, response to these drugs against DLBCL in clinical trials have been variable, ranging from complete/partial responses to stable disease to no response. The mechanisms of action of these drugs are still poorly understood, mainly because the function of their target deacetylases are cell context-specific. Therefore, characterization of the specific anticancer mechanisms of action of HDACIs in DLBCL could potentially lead to development of novel combinatorial drug regimens effective against resistant/relapsed DLBCL patients.

To define HDACI action in DLBCL, we treated DLBCL-derived cell lines with PXD101, (Belinostat); a hydroxamate HDACI, like Vorinostat. We demonstrated that PXD101 is able to produce 24h growth inhibition (IC₅₀) at submicromolar concentrations regardless of the DLBCL subtype. The 24h IC₅₀values were used in all the subsequent experiments. Cell cycle and apoptosis analysis by flow cytometry indicated that PXD101 produces cytotoxic effects on two of the GCB cell lines; DB and OCILY19 underwent G2/M cell cycle arrest at 24 hours followed by apoptosis at 48 and 72 hours of treatment. Immunoblotting of PARP and caspase-3 cleavage further confirmed apoptosis. More importantly, when cells were treated for only 8 hours with PXD101 and then the drug was removed for 24 hours, cells showed apoptosis rates similar to those observed with 48h of continuous treatment; suggesting that once that these cell lines are exposed to the drug they rapidly commit to cell death. Thus, we have classified the DB and OCILY19 cell lines as models for sensitivity to the apoptotic effects of HDACI.

In contrast, PXD101 induced cytostatic effects on the GCB cell line SUDHL4 and ABC cell lines U2932 and SUDHL8. All three cell lines showed G1 phase cell cycle arrest with little apoptosis. The G1 arrest is reversible after 48 hours of drug removal. Because of the lack of cell death and the reversibility of cell cycle arrest, we have classified these cell lines as models of HDACI resistance. Previous studies have shown that induction of p21 is responsible for G1 arrest in cells treated with HDACIs. Western blot analysis showed that none of the cell lines, except U2932, express p21, but upon PXD101, p21 protein levels were induced at 24, 48 and 72 hours of PXD101 treatment in SUDHL4 and U2932. In contrast, p21 was induced to a lesser extent in OCILY19 and DB, but its expression was not sustained beyond 24 hours of treatment. Since we also observed a corresponding loss in Rb phosphorylation, we tested the effect of PXD101 on cyclin dependent kinase 2 (CDK2) activity. This enzyme complex is responsible for entry into S phase and is inhibited by association with p21. In all three resistant cell lines CDK2 activity was reduced after only 24 hours of treatment with PXD101. The loss in activity was correlated with increased association with p21, as determined by immunoprecipitation.

These results indicate that sustained upregulation of p21 by HDACIs such as PXD101 plays a role in bringing about G1 arrest that may protect DLBCL cells from apoptosis. Combined treatment with therapeutics that prevent p21 upregulation and G1 arrest may work synergistically with HDACIs to trigger apoptosis in HDACI-resistant cell lines. To that end, we have begun analysis of the cyclin-dependent kinase inhibitor, flavopiridol, and have shown that it prevents both p21 upregulation and G1 arrest in the HDACi-resistant DLBCL cell lines. Studies to measure synergism with PXD101 in bringing about cell death are currently underway.