Abstract 3279: Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations
Authors: Mensah Afua Adjeiwaa , Valente Sergio , Matkovic Milos , Sartori Giulio , Falzarano Chiara , Tarantelli Chiara , Cascione Luciano , Pileri Stefano A. , Cavalli Andrea , Mai Antonello , Bertoni Francesco 
Published: June 2022

Abstract
Background: Lymphomas are characterized by aberrations in epigenetic proteins that contribute to establishing and maintaining the malignant phenotype. Gain of function mutations in EZH2 histone methyltransferase and inactivating mutations in CREBBP acetyltransferase occur in up to 30% of diffuse large B cell lymphomas (DLBCLs). Half of DLBCLs with mutated EZH2 have also mutated CREBBP. In vitro, mutated EZH2 DLBCLs have a lower sensitivity to HDAC inhibitors, HDACi (Mensah et al, 2021). Here, we explored the dual pharmacological inhibition of EZH2 and HDAC for an improved anti-lymphoma activity.
Methods: Biochemical evaluation and characterization of target engagement were done using fluorescence polarization, thermal shift, surface plasmon resonance, isothermal titration calorimetry and microscale thermophoresis. Computer modelling was performed using available EZH2 and HDACs crystal structures. Anti-proliferative activity was assessed after 7 days (d) using MTT and live imaging in DLBCL cells (n = 5). For cell cycle analysis by flow cytometry, cells were treated, fixed then stained with 7-AAD.
Results: We designed and synthesized 2 dual EZH2/HDAC inhibitors, MC4343 and MC4355, starting from the structures of EZH2 inhibitor tazemetostat (taz) and HDACi vorinostat. In biochemical assays, MC4343 and MC4355 had equal potencies towards EZH2 (0.032 nM) but different specificities towards class I and class II HDACs: MC4355 showed 7.5-fold greater inhibition of HDAC3 compared to MC4343 (0.38 µM and 2.85 µM, respectively) and more potently inhibited HDACs 6 and 8 (0.016, 0.17 µM and no activity, respectively). Computational modelling showed that the coordinative functional group of MC4343, but not of MC4355, caused steric clashes with several HDACs in increasing order: HDAC1 = HDAC3 &gt; HDAC8 &gt;&gt; HDAC4 = HDAC6. These results closely mirrored those obtained from the biochemical analysis. Both compounds inhibited proliferation in DLBCL cell lines irrespective of EZH2 or CREBBP mutational status but EZH2 and/or CREBBP mutants were more sensitive. MC4355 (IC50 range = 0.17 - 1.68 µM; median = 0.2 µM) was more potent than MC4343 (IC50 range = 0.17-2.72 µM; median = 1.78 µM). This was confirmed by live imaging analyses. SUDHL4, with EZH2 Y666N, showed poorer sensitivity to both inhibitors compared to DLBCLs with EZH2 Y646N/S. MC4343 and MC4355 induced cell death and G1 arrest in a dose-dependent manner. Pfeiffer, KARPAS422, WSUDLCL2, most sensitive to taz alone (IC50 = 4, 16, 77 nM), were most sensitive to MC4355 (IC50 = 200, 174, 171 nM). Notably, dual inhibitor treatment of Toledo and SUDHL4 with low sensitivity to taz (IC50 = 9, 14 µM), partially rescued sensitivity (IC50 = 1.7, 1.6 µM).
Conclusions: We designed and synthesized 2 novel dual EZH2/HDAC inhibitors, MC4343 and MC4355, with robust anti-proliferative effects in DLBCL. Our data show the efficacy of this novel class of epigenetic agents in lymphomas.
Citation Format: Afua Adjeiwaa Mensah, Sergio Valente, Milos Matkovic, Giulio Sartori, Chiara Falzarano, Chiara Tarantelli, Luciano Cascione, Stefano A. Pileri, Andrea Cavalli, Antonello Mai, Francesco Bertoni. Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3279.