Unique Apoptotic Effects Of Panobinostat and Marizomib In Acute Myeloid Leukemia and Bortezomib-Resistant Models
Authors: Corrales-Medina Fernando F. , Egas Bejar Daniela E. , Manton Christa , Johnson Blake , Irwin Mary E , Orlowski Robert Z. , Chandra Joya 
Published: October 2019

Abstract
Panobinostat, a potent pan-histone deacetylase inhibitor (HDACi) is emerging as a valuable therapeutic option for cancer treatment. Compared with the FDA-approved vorinostat, it displays a more potent and broader spectrum of inhibitory activity (inhibiting all class I, II and IV HDACs) at clinically achievable concentrations. Combinatorial therapies with proteasome inhibitors (PI) have been recently evaluated.
Marizomib, a second-generation irreversible PI, exhibits excellent pre-clinical activity against numerous hematologic malignancies. Indeed, published work from our laboratory has shown that marizomib is more effective than the FDA approved PI bortezomib in inhibiting the activity of the proteasome. Additionally, marizomib has been shown to induce higher caspase-8 and reactive oxygen species (ROS) dependent cell death than bortezomib, alone and in combination with HDACi in acute lymphoblastic leukemia (ALL) models.
Despite progress in the treatment of acute myeloid leukemia (AML), 40% of patients die from disease recurrence or treatment toxicities; therefore, targeted approaches allowing for low drug doses are needed to increase clinical efficacy. The objectives of this study were to (1) determine if a panobinostat/PI regimen displayed synergy; (2) whether cell death by this combination triggers an unique profile of caspase activation; and (3) assess utility of this combination in a bortezomib refractory setting.
Human-derived AML cell lines, ML-1 and AML3, were exposed to increasing concentrations of HDACi, (panobinostat or vorinostat), and PI (bortezomib or marizomib) alone and in combination. Panobinostat had an IC50 within the nanomolar range at 24 hours of treatment; in contrast, an IC50 was not achievable with vorinostat until after 48 hours of treatment. Marizomib was found to have a much lower IC50 and higher DNA fragmentation capacity than bortezomib. Calcusyn software was used to determine synergistic combinations. Synergistic cytotoxicity was observed with the combination treatment of panobinostat with both PIs. No synergy was observed with combinations involving vorinostat. Interestingly, the panobinostat + marizomib combination demonstrated earlier and higher ROS induction.
To assess the differences in apoptotic mechanisms between these agents, caspase activities were measured. The combination of panobinostat + marizomib induced an earlier and 2.5x higher induction of caspase-3 activation than the combination of panobinostat + bortezomib. Caspase-8 and caspase-9 dependence, were evaluated using pre-treatment with specific caspase-8 (IETD-fmk) and caspase-9 (LEHD-fmk) inhibitors. Caspase-8 inhibition decreased the cytotoxicity of the panobinostat + marizomib combination compared to control, whereas no difference was observed on cells treated with panobinostat + bortezomib. Western blotting for cleaved caspase-8 corroborated these data. Caspase-9 inhibitors did not significantly protect against DNA fragmentation in any of the combinations. These AML results were consistent with our prior published work using ALL cells, highlighting a role for caspase-8 in sensitivity to synergistic combinations of HDACi and PI.
Bortezomib resistance is an emerging problem in the treatment of hematological malignancies. Therefore, RPMI-8226vr10 (bortezomib-resistant multiple myeloma) cells were treated with equimolar doses of either of the HDACi; panobinostat was able to induce higher DNA fragmentation than vorinostat. When panobinostat was combined with either PI, the combination with marizomib caused higher DNA fragmentation than the bortezomib combination. Because caspase-2 has been implicated in panobinostat cytotoxicity in other systems, caspase-2 cleavage was assessed by western blotting assays. At 12 hours of treatment, panobinostat + marizomib caused stronger caspase-2 cleavage than the panobinostat + bortezomib combination, suggesting that marizomib may uniquely augment the caspase-2 activating capacity of panobinostat, providing insight into a potential mechanism of bortezomib resistance.
Further experiments will focus on the molecular mechanisms of panobinostat and marizomib combinations and how efficacy could be further optimized. Overall, these data support the use of these novel anticancer agents in hematological malignancies.

Disclosures:
Orlowski: Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Resverlogix: Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity’s Board of Directors or advisory committees.