The Alkylating Histone Deacetylase Inhibitor Fusion Molecule Edo-S101 Displays Full Bi-Functional Properties in Preclinical Models of Hematological Malignancies
Authors: López-Iglesias Ana Alicia , San-Segundo Laura , González-Méndez Lorena , Hernández-García Susana , Primo Daniel , Garayoa Mercedes , Hernández Ana Belén , Paíno Teresa , Mateos Maria-Victoria , Chen Yi , Ocio Enrique M. , Mehrling Thomas 
Published: October 2019

Abstract
Background
Alkylating histone deacetylase inhibitors (HDACi) enhance the anticancer efficacy of alkylators by increasing chromatin accessibility and also down regulating DNA repair. EDO-S101 is a first-in-class fusion molecule that combines DNA damaging effect of bendamustine with the pan-HDACi vorinostat.
Objectives
To study the bi-functional properties of EDO-S101 as an alkylating agent and a pan-HDACi in various in vitro and in vivo xenograft models of hematological malignancies.
Methods
In vitro inhibition of HDAC Class I and II enzymes by EDO-S101 and vorinostat was tested using an recombinant human enzymatic assay (BPS Bioscience, Enzo Life Science) and in vivo in rat peripheral blood mononuclear cells (PBMCs). The degree of inhibition was measured 1 hour following a single dose of 10–50 mg/kg i.v. and duration of inhibition over 24 hours after a single i.v. dose of EDO-S101 of 25 mg/kg. HDAC inhibition, alkylation and apoptotic activity were evaluated in vitro in myeloid (HL60 AML cell line) and lymphoid cell lines, including Daudi Burkitt’s lymphoma (BL) and a panel of 6 MM cell lines (MM1S, MM1R, RPMI-8226, RPMI-LR5, U266, U266-LR7). In vivo intra-tumor effects were analyzed after short courses of treatment with EDO-S101 in MM1S human plasmacytoma (PC) and BL xenograft models. Changes in pathway activation, protein expression and activities influencing the cell cycle were measured by Western blot and immunohistochemistry. Anti-tumor activity in vitro was measured by MTT and in vivo using a caliper to assess tumor size at regular intervals.
Results
In vitro, EDO-S101’s pan-HDACi activity, at nanomolar concentrations in Class I and II recombinant enzymes, was similar to vorinostat. In vivo, in intact rat PBMCs, HDAC inhibition was maximal at 1 hour after a single dose of 10 mg/kg i.v.–the dose where antitumor activity starts. HDAC inhibition did not increase with doses up to 50 mg/kg, recovery began within 3 hours and was nearly complete at 16 hours.
In the AML HL60 cell line in vitro, hyperacetylation of lysine residues K9, K14, K23 and K56 on histone 3 was found after exposure to 2–4 µM of EDO-S101. Histone 3 and 4 hyperacetylation was also demonstrated in MM cell lines at 1–5 µM concentrations. In xenograft models of human plasmacytoma and BL, EDO-S101 induced histone 3 hyperacetylation, indicating an HDACi effect in vivo.
Alkylating activity was demonstrated in vitro in HL60 and MM cell lines by DNA cross-linking and double strand break formation in the comet assay by immunofluorescence. In vivo, in xenograft models of human plasmacytoma (60 mg/kg d 1, 8, 15) and BL (40 and 80mg/kg d1) exposure to EDO-S101 caused a strong DNA-repair response shown by activation of pH2AX and p53 (PC and BL) followed by an increase of DNA damage check point proteins pCHK1 (PC) and even more prominent pCHK2 (PC and BL). The kinetics of this effect, studied in vivo in BL tumors, showed that the pH2AX response fell at Day 8 after dosing while the p53 response lasted, particularly in the group treated with 80mg/kg. In Daudi-bearing mice tumors, p-ATR was completely suppressed at Day 8 after treatment, which was not clear in the PC tumors.
EDO-S101 triggered apoptosis in vitro and in vivo, resulting in strong antitumor activity in HL60, Daudi and the panel of six MM cell lines. Initial in vitro experiments in HL60 cells showed an activation of the intrinsic pathway of apoptosis with cleavage of caspases 3, 9 and PARP and a marked reduction of anti-apoptotic proteins XIAP and Mcl-1. In the MM cell line, MM1S activation of the intrinsic and extrinsic pathways of apoptosis (C 8, 9, 3, 7 and PARP cleavage) was seen with a loss of mitochondrial membrane potential by DiOC6.
Tumors of human plasmacytoma and BL in vivo were rapidly shrinking or completely eradicated after i.v. administration of EDO-S101. A decrease in proliferation (Ki67) and slight PARP cleavage was found in the tumor tissue (PC), and evidence of activation of apoptosis by cleavage of caspases 7 and 9 at Day 4 and caspase 8 and PARP at Day 8 after treatment in BL tumors. The level of caspase 3, different to MM, remained unchanged. Importantly, EDO-S101 induced a rapid and dose-dependent strong decrease of XIAP and Mcl-1 which lasted until Day 8.
Conclusions
This study demonstrates the bi-functional mechanism of ED0-S101 in both myeloid and lymphoid hematological malignancies. The data support the clinical investigation of EDO-S101 in treating hematological malignancies.

Disclosures
Ocio: Mundipharma: Honoraria, Research Funding. Mehrling:Mundipharma: Employment.