The histone Deacetylase Inhibitor Givinostat in Combination with Sorafenib Induces Reactive Oxygen Species (ROS) Generation and Exerts Potent Antitumor Effects in NOD/SCID Mice with Hodgkin Lymphoma Cell Line Xenografts
Authors: Locatelli Silvia L , Guidetti Anna , Cleris Loredana , Tartari Silvia , Gianni Alessandro M. , Anichini Andrea , Carlo-Stella Carmelo 
Published: October 2019

Abstract
Abstract 3711

INTRODUCTION:
Patients with refractory or relapsed classical Hodgkin Lymphoma (cHL) represent an unmet medical need and would benefit from the development of new therapies. Histone deacetylases (HDACs) and the RAF/MEK/ERK pathway are aberrantly controlled in cHL and influence a broad repertoire of tumor processes, suggesting a rationale for therapeutically targeting these pathways. We targeted these pathways using the HDAC inhibitor Givinostat (Italfarmaco S.p.A., Milan, Italy), and the RAF/MEK/ERK inhibitor Sorafenib (Nexavar, Bayer, Germany, EU) in order to investigate in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination.

METHODS:
Three cHL cell lines, including HDLM-2, L-540 and HD-MyZ, were used to investigate the effects of Givinostat and Sorafenib, used alone or in combination, by means of in vitro assays evaluating cell growth and cell survival. Additionally, live cell imaging was used to asses the production of reactive oxygen species (ROS), and Western blotting (WB) to assess modulating effects of the two-drug combination on MAPK, PI3K/AKT, HDACs as well as the apoptotic pathways. The efficacy of Givinostat/Sorafenib combination was finally confirmed in NOD/SCID mice with cHL cell line xenografts.

RESULTS:
While Givinostat and Sorafenib as single agents exerted a limited activity against cHL cells, the combined Givinostat/Sorafenib treatment was associated with potent dephosphorylation of MAPK and PI3K/Akt pathways and significantly increased H3 and H4 acetylation due to a nearly complete inhibition of class I and II HDACs. Furthermore, these events were associated with a time-dependent synergistic cell growth inhibition (70% to 90%) in all Givinostat/Sorafenib-treated cHL cells. Upon Givinostat/Sorafenib exposure, HDLM-2 and L-540 cell lines showed significantly (P ≤.0001) increased levels of apoptosis (90 ± 2% and 96 ± 1%, respectively) and mitochondrial dysfunction (up to 70%, P≤.0001), as compared with single agents. Apoptosis induced by Givinostat/Sorafenib combination failed to induce processing of caspase-8, −9, −3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, suggesting the occurrence of caspase-independent apoptosis. Besides downregulating the expression of the anti-apoptotic protein Mcl-1 and ERK1/2 phosphorylation, Givinostat/Sorafenib strongly increased expression of the BH-3 only protein Bim, compared to single treatments. These findings were dependent on a potent, early and time-dependent ROS generation (up to 60%, P≤.0001) that was synergistically induced by Givinostat/Sorafenib treatment. Additionally, pretreatment of cHL cells with the ROS inhibitor YCG063 prevented the generation of ROS as well as mitochondrial membrane depolarization along with cell death induced by the two-drug combination, suggesting that ROS generation is the triggering event in Givinostat/Sorafenib induced-cell death. In vivo Givinostat/Sorafenib treatment significantly reduced the growth of L-540 and HD-MyZ nodules, resulting in an average 35% to 65% tumor growth inhibition (P ≤.0001) compared to single treatments, in the absence of any toxicity. Interestingly, as compared to controls or treatment with single agents, the combined Givinostat/Sorafenib treatment significantly increased in vivo Bim expression (7- to 21-fold increase, P ≤.0001), resulting in a marked tumor necrosis (3- to 5-fold increase, P ≤.0001).

CONCLUSIONS:
The combined Givinostat/Sorafenib treatment demonstrates a potent preclinical in vitro and in vivo activity against cHL cell lines by targeting aberrant expression of HDACs and MAPK. Antitumor activity of this combination involves ROS generation and Bim upregulation and provides a rationale for clinical studies using this combination in refractory/relapsed cHL patients.

Disclosures:
No relevant conflicts of interest to declare.