Dual Targeting With The Selective Histone Deacetylase (HDAC) 6 Inhibitor, ACY-1215, and Bortezomib (BOR) Leads To Marked Disruption Of Protein Degradation Pathways and Apoptosis In Preclinical Models Of Lymphoma

The development of isoform selective HDAC inhibitors has opened the opportunity to better define and target pathways germane to lymphoma. Class IIb HDAC6 binds polyubiquinated, misfolded proteins facilitating their transport to the aggresome for proteosome-independent degradation. The aggresome is a key outlet for the unfolded protein response (UPR), a quality control mechanism that promotes survival during endoplasmic reticulum stress and signals CHOP (C/EBP-homologous protein) mediated apoptosis when homeostasis cannot be reestablished. We investigated the impact of the selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc) in preclinical models of lymphoma, its synergistic potential with BOR, its mechanism of action with respect to inhibiting protein degradation pathways, and potential biomarkers for response.

Single agent concentration-effect curves were generated for 8 diffuse large B-cell lymphoma (DLBCL) (4 GCB: OCI-Ly1, OCI-Ly7, Su-DHL4, Su-HDL6; 4 ABC: OCI-Ly10, Su-DHL2, HBL-1, RIVA), 5 mantle cell lymphoma (MCL) (Maver, JVM-1, JEKO, Rec-1, HBL-2 ), and 4 T-cell lymphoma cell lines (HH, H9, CCL-119, Sup-T1). Maximal cytotoxicity was observed at 48-72 hr with IC₅₀ ranging between 240-3500 nM. Activity was greatest in DLBCL, with 72 hr IC₅₀ values as low as 240 nM. ACY-1215 began to show irreversible activity after 6 hr exposure. Single agent exposure at the IC₅₀led to minimal apoptosis as analyzed by annexin V/propidium iodide (PI) staining, and caspase 3 and PARP cleavage, but led to G1 cell cycle arrest.

The synergistic potential of ACY-1215 in combination with BOR was measured and evaluated for schedule and dose dependency in 6 cell lines. The greatest synergy was observed with simultaneous exposure of ACY-1215 (750 nM) and BOR (2 nM) at 48 hr with synergy coefficients as low as 0.24. Cell death occurred by apoptosis as evaluated by annexin V/PI, and cleavage of caspase 3 and PARP.

Treatment with ACY-1215 led to inhibition of the aggresome as evidenced by increased poly-ubiquitinated proteins and acetylation of α-tubulin. Upregulation of UPR was demonstrated by acetylation of GRP78 and subsequent dissociation of PERK, increased p-eIF2a, spliced XBP-1 and CHOP. These effects were enhanced by treatment with BOR, confirm that accumulation of misfolded proteins activates the UPR response triggering apoptosis, and substantiate blocking two protein degradation systems simultaneously. Minimal acetylation of histone was observed following treatment with ACY-1215, and was considerably less relative to cells treated with pan-HDAC inhibitors confirming selectivity of the drug.

These findings were validated in an in vivomodel of DLBCL (OCI-Ly10). Mice were treated with ACY-1215 50 mg/kg days 1-5, 8-12, 15-19 and/or BOR 1.5 mg/kg days 1, 8, 11. Treatment was well tolerated with no toxicity related deaths with the combination. One cycle of the combination led to statistically significant tumor growth delay (p=0.001) and extended overall survival (p<0.05) compared to either single agent. Tumors from mice treated with ACY-1215 demonstrated acetylation of α-tubulin at 4-8 hr, and increased GRP78, and XBP-1. These pharmacodynamic effects were enhanced by the addition of BOR. Interestingly, these findings were not observed in the spleen, suggesting a preferential effect in malignant lymphocytes.

Establishing biomarkers for response to isoform selective HDAC6 inhibition may lead to the treatment of patients with specific molecular and phenotypic signatures. Immunoblot levels of basal HDAC6 did not correlate with sensitivity to ACY-1215 in 8 cell lines. Resistance was characterized by combined increase in Bcl2 and loss of BIM. All cell lines expressed more GRP78 than normal peripheral blood mononuclear cells. Fifty-one untreated lymphoma patient samples (10 DLBCL, 11 FL, 10 MZL, 7 MCL, 13 TCL) were analyzed for markers of the UPR by immunohistochemistry. HDAC6 had bright expression in 30/41 lymphoma samples compared to 0/11 reactive lymph nodes. A similar pattern was observed for GRP78 (36/46 vs 0/10), and XBP-1 (26/50 vs 2/12).

Selective targeting of HDAC6 with ACY-1215 inhibits aggresome mediated protein degradation inducing the UPR and cell cycle arrest. Dual targeting of protein degradation pathways represents a novel and rational approach for the treatment of lymphoma. Plans to study the clinical efficacy of ACY-1215 in patients with relapsed lymphoma are underway.