acy-1215 and carfilzomib

Proteasome inhibition induces the accumulation of aggregated misfolded/ubiquitinated proteins in the aggresome; conversely, histone deacetylase 6 (HDAC6) inhibition blocks aggresome formation. Although this rationale has been the basis of proteasome inhibitor (PI) and HDAC6 inhibitor combination studies, the role of disruption of aggresome formation by HDAC6 inhibition has not yet been studied in multiple myeloma (MM). The present study aimed to evaluate the impact of carfilzomib (CFZ) in combination with a selective HDAC6 inhibitor (ricolinostat) in MM cells with respect to the aggresome-proteolysis pathway. We observed that combination treatment of CFZ with ricolinostat triggered synergistic anti-MM effects, even in bortezomib-resistant cells. Immunofluorescent staining showed that CFZ increased the accumulation of ubiquitinated proteins and protein aggregates in the cytoplasm, as well as the engulfment of aggregated ubiquitinated proteins by autophagosomes, which was blocked by ricolinostat. Electron microscopy imaging showed increased autophagy triggered by CFZ, which was inhibited by the addition of ACY-1215. Finally, an in vivo mouse xenograft study confirmed a decrease in tumour volume, associated with apoptosis, following treatment with CFZ in combination with ricolinostat. Our results suggest that ricolinostat inhibits aggresome formation, caused by CFZ-induced inhibition of the proteasome pathway, resulting in enhanced apoptosis in MM cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Synergism; Endoplasmic Reticulum Stress; Female; Heterografts; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Multiple Myeloma; Oligopeptides; Phagosomes; Proteasome Inhibitors; Pyrimidines

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2-M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34(+) cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib-ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; DNA Repair Enzymes; DNA-Binding Proteins; Drug Interactions; Drug Synergism; Female; Gene Knockdown Techniques; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lymphoma, Non-Hodgkin; Oligopeptides; Oxidative Stress; Proteasome Inhibitors; Pyrimidines; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays