Abstract Lymphoma cells have frequent deregulation of their epigenome. The Bromodomain (BRD) and Extra-Terminal domain (BET) proteins are key regulators of the transcription process. The acetyltransferases cyclic AMP response element binding protein (CREB)-binding protein (CBP) and the E1A interacting protein of 300 kDa (EP300) are highly homologous BRD-containing transcriptional co-activators and are often mutated in diffuse large B cell lymphoma (DLBCL). Targeting the individual classes of proteins is a new therapeutic approach, with BET inhibitors having both preclinical and early clinical anti-lymphoma activity. NEO2734 and NEO1132 are novel chemically distinct dual inhibitors of both BET and CREBBP/EP300 proteins with different affinity for their targets. Here, we present initial data exploring their anti-tumor activity in DLBCL models. Lymphoma cell lines were exposed to increasing doses of compounds for 72h followed by MTT assay. Twenty-seven DLBCL cell lines were exposed to NEO2734 and NEO1132. NEO2734 showed anti-tumor activity with a median IC50 of 157 nM (95% C.I., 135-214). While, NEO1132 showed a lower activity compared to NEO2734, with a median IC50 of 400nM (95% C.I., 316-622). For both compounds, cell lines derived from activated B-cell-like (ABC) DLBCL (n.=8) were more sensitive than those derived from germinal center B-cell (GCB) DLBCL (n.=19) (P=0.009, P=0.03 respectively). No differences were observed based on double hit MYC/BCL2 (yes, n.=6; no, n.=14), MYC (translocation: yes, n=8; no, n.=13), BCL2 (translocation: yes, n=12; no, n.=6), TP53 (inactive: yes, n=14; no, n.=6), CREBBP (mutated, n.=10; wild type, n=16), or EP300 (mutated, n.=5; wild type, n.=20) gene status. All the cell lines were also exposed to a BET inhibitor (birabresib, OTX015) (Boi et al, Clinical Cancer Res 2015) and to a CREBBP/EP300 inhibitor (CBP30) (Hammitzsch et al, PNAS 2015). The median IC50 values of the two molecules were 237 nM (95% C.I., 171-344) and 5.5 μM (95% C.I., 4.2-8.3 μM) respectively. The four compounds presented a similar pattern of anti-proliferative activity across all the cell lines (NEO2734 vs NEO1132 R2=0.98, P < 0.001; NEO2734 vs birabresib: R2=0.84, P < 0.001; NEO2734 vs CBP30, R2 = 0.73, P < 0.001; birabresib vs CBP30, R2 = 0.73, P < 0.001) but with different degrees of IC50. NEO2734 was significantly more potent than birabresib (P=0.0182), CBP30 (P<0.001) and NEO1132 (P<0.001). The higher activity of NEO2734 compared to NEO1132 may be attributable to its superior potency in binding CREBBP and EP300, confirming the importance of the BET/CREBBP/EP300 simultaneous inhibition. The novel dual BET and CREBBP/EP300 inhibitors NEO2734 and NEO1132 showed potent in vitro anti-tumor activity across a large panel of DLBCL cell lines and their activity appear to be proportional to the binding affinity for both BET and CREBBP/EP300 proteins. Citation Format: Filippo Spriano, Eugenio Gaudio, Chiara Tarantelli, Gaetanina Golino, Luciano Cascione, Emanuele Zucca, Anastasios Stathis, Francis Giles, Francesco Bertoni. NEO1132 and NEO2734, novel dual bromodomain inhibitors of both BET and CREBBP/EP300, compared to single BET or CREBB/EP300 inhibitors in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3829.