The Hybrid Molecule, Edo-S101, Impairs Double Strand Breaks Repair in Multiple Myeloma and Synergizes with Bortezomib and Dexamethasone
Authors: López-Iglesias Ana Alicia , Herrero Ana Belen , San-Segundo Laura , Hernández-García Susana , González-Méndez Lorena , Martín-Sánchez Montserrat , Algarín Esperanza M , Paino Teresa , Garayoa Mercedes , Mehrling Thomas , Gutierrez Norma C , San-Miguel Jesús F. , Mateos Maria-Victoria , Ocio Enrique M. 
Published: October 2019

Abstract
Introduction. EDO-S101 is a hybrid molecule of bendamustine plus vorinostat, new in its class. Our group has previously demonstrated that EDO-S101 is effective in vitro in MM cell lines independently of p53 state, and also in a murine plasmacytoma model where it decreases tumor growth and prolongs survival with respect to bendamustine and/or vorinostat treatment. The objective of this work was to gain further insights into the efficacy of EDO-S101, its mechanism of action and its combination with other drugs used in MM.
Methods. The mechanism of action was assessed by western blot, comet assay, immunohistochemistry, and flow cytometry. Homologous recombination (HR) efficiency was calculated using chromosomally integrated green fluorescent protein reporter construct-based assay. The efficacy of different combinations was studied in vitro (HMCLs), in vivo (murine plasmacytoma model CB-17 SCID mice) and ex vivo (cells from patients).
Results. In addition to the activity of EDO-S101 in MM cell lines we demonstrated that it was active ex vivo in cells isolated from 7 MM patients, with median IC50 of 5 µM (ranging from 1,8 to 8 µM), some of them previously exposed and resistant to alkylators such as melphalan. Interestingly, EDO-S101 could also overcome alkylators-resistance in vitro, as it was active in melphalan resistant cells (U266-LR7 and RPMI8226-LR5). EDO-S01 was also effective in the presence of factors that confer proliferative advantage to plasma cells, like IL-6, IGF or co-culture with mesenchimal cells hMSC-TERT.
Regarding its mechanism of action, we found that the apoptosis induced by EDO-S101 was caspase-independent but calpain-dependent, since PD150606, an inhibitor of this protein could overcome EDO-S101-induced apoptosis, whereas the caspase inhibitor Z-VAD -FMK did not. This data was consistent with the finding that under treatment with EDO-S101, MM1S cells showed AIF (apoptotic inducing factor) translocation from the mitochondria into the nucleus. Interestingly, the release of this pro-apoptotic protein from the mitochondria could be mediated by calpains, as it has been described in literature.
We subsequently demonstrated that EDO-S101 causes DNA damage, as revealed by the phosphorylation and subsequent activation of several components of the DNA Damage Response (DDR) such as ATM, H2AX, chk1, chk2 or p53, and the induction of DNA fragmentation, that was detected by the comet assay. EDO-S101 was also found to induce cell cycle arrest in different phases depending on the dose and cell line.
It has previously been suggested that DACi may impair DNA repair by inhibiting homologous recombination (HR), a pathway related with genomic instability and progression, very active in MM. Therefore we next evaluated the efficiency of HR using a reported construct that was chromosomally integrated in two MM cell lines, JJN3 and U266. Treatment with EDO-S101 significantly reduced the efficiency of HR in both cell lines, by 50% and 20% of untreated controls respectively.
Finally, we tested potential combinations with other antimyeloma agents like lenalidomide and thalidomide; and also with proteasome inhibitors (bortezomib, carfilzomib and oprozomib). EDO-S101 potentiated the activity of all these agents, but the most synergistic combination was that including Bortezomib + Dexamethasone (CI 0,4). This combination was also evaluated in vivo, where it significantly decreased tumor growth and prolonged survival compared to agents in monotherapy and in double combinations. We are currently deepening into the mechanism of action of this combination.
Conclusions. EDO S101 is active ex vivo in cells isolated from patients and is able to overcome resistance to alkylators. It induces caspase-independent apoptosis, and cell cycle arrest in MM cell lines. These effects are due to the potent DNA damage which is enhanced by HR impairment induced by the hybrid molecule. Moreover, the combination with bortezomib and dexamethasone is especially attractive to be taken into the clinical setting.

Disclosures
Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Mateos:Takeda: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy.