Abstract The mitotic kinase polo-like kinase 1 (PLK1) is over-expressed in neoplastic cells, including non-Hodgkin’s lymphoma (NHL) cells, but not in normal cells, prompting development of PLK1 inhibitors e.g., volasertib (Boehringer-Ingelheim). PLK1 plays key roles in cell cycle progression, mitosis, cytokinesis, centriole duplication, and the DNA damage response (DDR). Notably, volasertib demonstrated preliminary signs of activity in NHL patients in a phase I trial. HDAC inhibitors (HDACIs) modify chromatin structure and gene expression, but also induce multiple mitotic abnormalities including mitotic slippage and DNA damage checkpoint disruption. Given these complementary mechanisms, we sought to examine volasertib interactions with the pan-HDACI belinostat (Spectrum Pharmaceuticals) in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells in vitro and in vivo. Exposure of DLBCL cells to extremely low (e.g., 5-30 nM), minimally toxic concentrations of volasertib and low (e.g., 100-400 nM), marginally toxic belinostat concentrations for 48 hr synergistically increased apoptosis in SU-DHL16, SU-DHL4, SU-DHL8 (GC subtype), HBL-1, U2932 (ABC-subtype), and OCI-LY18, Carnaval (double hit) DLBCL cells. Similar interactions occurred in primary lymphoma cells, Granta-519 and Rec-1 (MCL), SU-DHL4/BR and SU-DHL16/BR (bortezomib-resistant cells exhibiting up-regulation of the proteasome sub-unit PSMB5 and bortezomib binding site mutation (Ala49Val). In contrast, identical regimens were minimally toxic to normal hematopoietic progenitors. Volasertib/belinostat induced massive M-phase arrest, p-histone H3 up-regulation, a marked increase in mitotic errors, and pronounced M-phase cell death associated with striking DNA damage (γH2A.X formation). Belinostat diminished c-Myc mRNA and protein in DLBCL cells, an effect significantly enhanced by volasertib. Notably, PLK1 knock-down in DLBCL cells significantly increased belinostat-induced M-phase accumulation, phospho-histone H3 accumulation, γH2AX generation, and apoptosis. Analogously, c-Myc knock-down increased DNA damage and apoptosis in response to volasertib, arguing that c-Myc down-regulation plays a functional role in lethality. Volasertib/belinostat co-administration dramatically reduced tumor growth in an ABC-DLBCL flank model (U2932) and significantly increased survival (56.2 vs 39.7 days; p < 0.001) without weight loss or other toxicities. The regimen also enhanced survival and reduced tumor cell bone marrow engraftment in a systemic double-hit lymphoma model (OCI-Ly18). Together, these findings indicate that PLK1/HDAC inhibition warrants attention as a therapeutic strategy in NHL. Accordingly, based on these findings, a phase I volasertib/belinostat trial in patients with aggressive NHL is planned (NCT02875002). Citation Format: Tri Nguyen, Rebecca Parker, Elisa Hawkins, Victor Yazbeck, Akhil Kolluri, Maciej Kmieciak, Mohamed Rahmani, Steven Grant. Synergistic interactions between PLK1 and HDAC inhibitors in non-Hodgkin’s lymphoma cells occur in vitro and in vivo and proceed through multiple mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2018. doi:10.1158/1538-7445.AM2017-2018