The First in Class, Alkylator-Histone-Deacetylase-Inhibitor Fusion Molecule Edo-S101 in Combination with Proteasome Inhibitors Induces Highly Synergistic Pro-Apoptotic Signaling through UPR Activation and Suppression of c-Myc and BCL2 in Multiple Myeloma

Background. EDO-S101 is a first-in-class alkylating, histone-deacetylase inhibitor (HDACi) fusion molecule with dual activity that is currently in Phase I. It structurally combines the strong DNA damaging effect of bendamustine with a fully functional pan-HDAC inhibitor, vorinostat. Bendamustine has substantial activity against B-cell malignancies; vorinostat sensitizes the same type of cancers against alkylators or proteasome inhibitors (PI). Bendamustine combined with the PI bortezomib (BTZ) is active against multiple myeloma (MM). Cytotoxicity of PI in MM relies on excess induction of proteotoxic stress and triggering of the unfolded protein response (UPR). Upon proteasome inhibition, HDACi synergize with PI by interfering with the a-tubulin-mediated transport of poly-ubiquitinated proteasome substrates to lysosomal destruction. Indeed, EDO-S101 has strong synergistic cytotoxicity with PI in vitro against hematological malignancies, including MM, mantle cell lymphoma and ABC type diffuse large B-cell lymphoma. The aim of this work is to characterize the molecular mechanism of action of the synergy of EDO-S101 with PI in comparison to its established structurally related drugs, bendamustine and vorinostat.

Methods. The cytotoxic and molecular activity of EDO-S101 in combination with BTZ and other types of PI was assessed in vitro using the RPMI-8226 and several other MM cell lines. HDAC-inhibiting activity, accumulation of poly-ubiquitinated proteins and induction of ER stress, apoptotic signaling and autophagy induction were assessed by quantitative PCR and western blotting. Proteasome activity was measured with activity based probes (ABP). Apoptosis was assessed by AnnexinV/FITC staining with flow cytometry. Cell viability was evaluated by MTS assay.

Results. EDO-S101 showed substantially stronger cytotoxicity in combination with PI than melphalan, bendamustine, cyclophosphamide or PI combined with equimolar vorinostat. EDO-S101 had higher HDACi-type of activity, compared to vorinostat, as demonstrated judged in particular by increased a-tubulin acetylation, providing a potential mechanistic basis for its superior synergy with PI. Consistent with this, EDO-S101 alone induced moderate cellular accumulation of poly-ubiquitinated proteins already in the absence of proteasome inhibition, which was potentiated when EDO-S101 was combined with BTZ. EDO-S101 induced activation of the UPR-regulators XBP1 and IRE1 known to control BTZ sensitivity of MM, in contrast to vorinostat or bendamustine alone. Co-treatment with BTZ and EDO-S101 or vorinostat resulted in highly synergistic triggering of the UPR (ATF4, CHOP, BIP). Interestingly, EDO-S101 in addition induced the pro-apoptotic machinery via upregulation of NOXA, downregulation of BCL2 and an increase of the BAX/BCL2 ratio, and also activated autophagy, as evidenced by upregulation of LC3A and LC3B. While this pro-apoptotic signaling of EDO-S101 was highly synergistic with BTZ-induced apoptotic signals, co-treatment with BTZ and vorinostat reduced apoptotic signaling compared to BTZ alone. EDO-S101 reduced c-Myc expression by 60%, while vorinostat had no effect on c-Myc levels. The combination BTZ+EDO-S101 decreased c-Myc levels by approx. 90%, while these levels remained unchanged during treatment with BTZ+vorinostat.

Conclusion. EDO-S101 is a first-in-class, dual-mechanism, alkylator-HDAC-inhibitor fusion molecule that combines key structural features of bendamustine and vorinostat. The molecular mode of action of EDO-S101 differs from that of its structurally related drugs by a more effective interaction with a-tubulin, which may in part explain superior synergy with PI. Most importantly, EDO-S101 has a direct pro-apoptotic activity via downregulation of c-Myc and BCL2 while upregulating NOXA, features not observed with vorinostat. This results in highly synergistic signaling with the PI-induced pro-apoptotic effects. EDO-S101 is a promising advancement of bendamustine with molecular features clearly different from and superior to a combination of bendamustine with vorinostat. EDO-S101 should be explored in combination with proteasome inhibitors in particular in poor risk B cell neoplasms with c-Myc overexpression such as aggressive MM, Burkitt lymphoma or "double hit" aggressive B cell lymphoma.