The emergence of epigenetic therapies has identified histone deacetylase(HDAC) inhibitors as effective therapeutic agents for the treatment of refractory lymphoma. While pan-class I/II HDAC inhibitors have led to treatment ramifications, recently goals have shifted to the development of discrete HDAC selective inhibitors to further define and better target pathways germane to specific subtypes of lymphoma. We investigated the therapeutic impact of the selective HDAC6 inhibitor rocilinostat (ACY-1215) (Acetylon Pharmaceuticals, Inc) in a panoply of lymphoma cell lines. HDAC6, a class IIb histone deacetylase, binds polyubiquinated, misfolded protein aggregates and facilitates their transport to the aggresome. The aggresome then sequesters these aggregates for degradation. The aggresomal pathway is proteasome-independent and is a key outlet for the unfolded protein response (UPR). The purpose of this study was to determine whether inhibition of HDAC6 with rocilinostat leads to an accumulation of misfolded proteins, activates the UPR stress response and ultimately induces apoptosis. These results have been confirmed in preclinical models of multiple myeloma, and the drug is currently being evaluated in clinical trials for that disease . We evaluated the single agent activity of rocilinostat, its synergistic potential when combined with bortezomib, and the mechanism of action of this combination in lymphoma with respect to inhibiting two separate protein degradation pathways.
Single agent dose-effect curves were generated for 8 diffuse large B-cell lymphoma (DLBCL) cell lines (4 germinal-center type: OCI-Ly1, OCI-Ly7, Su-DHL4, Su-HDL6; 4 activated B-cell type: OCI-Ly10, Su-DHL2, HBL-1, RIVA), 4 mantle cell lymphoma (MCL) cell lines (Maver, JVM-1, JEKO, Rec-1), and 2 T-cell lymphoma cell lines (HH and H9). The inhibitory concentration 50% (IC50) values were calculated after exposure for 24, 48, and 72 hours. Maximal cytotoxicity was observed in all cell lines at 48–72 hours with IC50 values ranging between 240–3500 nM. Activity was greatest in the cell lines OCI-Ly10 and OCI-Ly7, with 72-hour IC50 values of 240 nM and 255 nM respectively. Rocinlinostat began to show irreversible activity after 6 hours of exposure. The cytotoxicity of rocilinostat in combination with bortezomib was measured and evaluated for schedule and dose dependency in OCI-Ly10. The greatest synergy was observed with simultaneous exposure and was first established at the concentrations rocilinostat750 nM and bortezomib 3 nM (CI=0.576). Synergy did not depend on the concentration of one drug over the other. Eight additional cells lines were evaluated for synergy with similar results. Cell death occurred by apoptosis as evaluated through FACS analysis for annexin V/propidium iodide, and cleavage of caspase 3 and PARP by immunoblotting. Treatment of OCI-Ly10 and Su-DHL6 cells with rocilinostat led to acetylation of a-tubulin, increased poly-ubiquitinated proteins, PERK, GRP78, p-eIF2a, and spliced XBP-1 as detected by immunoblotting. These effects were enhanced by treatment with bortezomib, and confirm that accumulation of misfolded proteins activates the UPR response triggering apoptosis, and substantiates blocking two protein degradation systems simultaneously. In addition, p65 NF#x2610;#x0025;B subunit was decreased with the combination. Slight acetylation of H3 was observed following treatment with roclinostat, but this was considerably less compared with cells treated with pan-Class I/II HDAC inhibitors and confirms selectivity of the drug.
These are the first results to indicate that a selective HDAC inhibitor can have marked activity across a panel of lymphoma cell lines. Future studies will evaluate efficacy and tolerability in in vivo models of lymphoma, and further define mechanism of action. Taken together, these findings raise the prospect that dual targeting of the ubiquitin-proteasome and aggressomal protein degradation pathways can be synergistically effective and provide excellent pre-clinical rationale for expanding the use of rocilinostat in combination with bortezomib for patients with relapsed or refractory lymphoma.
Amengual:Acetylon Pharmaceuticals, Inc: Research Funding. Jones:Acetylon Pharmaceuticals, Inc: Employment. O'Connor:Millenium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics, Inc: Consultancy; Seattle Genetics, Inc: Membership on an entity's Board of Directors or advisory committees; Allos Therapeutics, Inc: Consultancy.
Asterisk with author names denotes non-ASH members.