Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G2/M arrest and apoptosis and resistance by reversible G1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, celltype specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

The PIM family kinases promote growth and survival of tumor cells and are expressed in a wide variety of human cancers. Their potential as therapeutic targets, however, is complicated by overlapping activities with multiple other pathways and remains poorly defined in most clinical scenarios. Here we explore activity of the new pan-PIM inhibitor PIM447 in a variety of lymphoid-derived tumors. We find strong activity in cell lines derived from the activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). Sensitive lines show lost activation of the mTORC1 signaling complex and subsequent lost activation of cap-dependent protein translation. In addition, we characterize recurrent PIM1 protein-coding mutations found in DLBCL clinical samples and find most preserve the wild-type protein's ability to protect cells from apoptosis but do not bypass activity of PIM447. Pan-PIM inhibition therefore may have an important role to play in the therapy of selected ABC-DLBCL cases.

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Diffuse Large B-cell lymphoma (DLBCL) is the most frequent subtype of Non-Hodgkin Lymphoma (NHL) in all countries around the world and in all age groups. Several drug regimens have been used in treatment of DLBCL; however, this disease remains eventually fatal in 30 – 40% of the patients. Chemotherapy resistance can be partly explained by the fact that DLBCL is a heterogeneous group of NHLs, with the two most prevalent subtypes being “Activated B-cell Like” (ABC) and “Germinal Center B-cell like” (GCB). Patients with the ABC subtype have the poorest prognosis under the current treatment regimen. Therefore, there is a pressing need for new therapeutics that can increase survival rates in DLBCL patients. Histone deacetylase inhibitors (HDIs) have proven to be promising drugs in the treatment of blood malignancies. Even though their mechanism of action has not been fully characterized, two HDIs, (Vorinostat and Romidepsin) have been approved for the treatment of cutaneous T-cell lymphoma (CTCL). Therefore the purpose of the current study is to investigate the response of DLBCL subtypes to HDIs, with a particular focus on subtype-specific mechanisms of action. Our current working hypothesis is that a comprehensive analysis of the genomic and proteomic response to histone deacetylase inhibitors (HDIs) including gene expression and transcription factor acetylation will reveal both mechanisms and potential biomarkers of HDI action in lymphomas DLBCL. In the current study we have focused on the cellular and genomic effects of the hydroxamate HDI, Belinostat (PXD101), on cell lines representing the GCB subtype of DLBCL. We show that PXD101 inhibits growth of four GCB-type cell lines with 24 h IC50s in the low micromolar range (SUDHL6 =0.15uM, OCI Ly19 = 0.3uM, SUDHL4 = 0.45uM, DB = 0.77uM). Flow cytometry analysis has shown that three of these cell lines (SUDHL6, OCI Ly19 and DB) arrest in the G2/M phase of the cell cycle by 24 hours of treatment at the IC50 dose and then die by apoptosis. In contrast, the SUDHL4 cell line reversibly arrests in the G1 phase without undergoing cell death. Western blot analysis of PARP and caspase-3 cleavage has further confirmed the presence/absence of apoptosis. We suggest that the SUDHL4 cell line represents DLBCL tumors that are refractory to the apoptosis-inducing effects of HDIs. Thus, we are using this cell line to identify other therapeutics which could be used in combination with PXD101 to induce cell death.The mechanistic basis for the differential cellular response between the GCB type cell lines is currently under investigation using expression profiling data obtained from OCI Ly19 and SUDHL4 cells treated with PXD101. Preliminary data indicates divergent responses in expression of GADD45 and p21, the Myc/Max family of proteins, and the clock genes, Per1 and Cry2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2011-597