Targeting Both BET and Crebbp/EP300 Proteins with the Novel Dual Inhibitor NEO2734 Leads to More Preclinical Anti-Tumor Activity in Diffuse Large B Cell Lymphomathan with Single BET or Crebbp/EP300 Inhibitors
Authors: Spriano Filippo , Gaudio Eugenio , Tarantelli Chiara , Golino Gaetanina , Cascione Luciano , Zucca Emanuele , Stathis Anastasios , Giles Francis J. , Bertoni Francesco 
Published: October 2019

Abstract
Background: Lymphoma cells have frequent deregulation of their epigenome. The Bromodomain (BRD) and Extra-Terminal domain (BET) proteins are key regulators of the transcription process (Stathis & Bertoni, Cancer Discovery 2018). The acetyltransferases cyclic AMP response element binding protein (CREB)-binding protein (CBP) and the E1A interacting protein of 300 kDa (EP300 or P300) are highly homologous BRD-containing transcriptional co-activators and their genes are often mutated in diffuse large B cell lymphoma (DLBCL) (Pasqualucci et al, Nature 2011; Morin et al, Nature 2011). Targeting the individual classes of proteins is a new therapeutic approach, as shown especially by BET inhibitors with both preclinical and early clinical anti-lymphoma activity (Stathis & Bertoni, Cancer Discovery 2018). NEO2734 (Epigene Therapeutics Inc) is a novel oral dual inhibitor of BET and CREBBP/EP300 proteins with pre-clinical activity in a spectrum of solid tumors (Giles et al, ESMO 2018). Here, we present the first data exploring its anti-tumor activity in DLBCL models.
Methods: Lymphoma cell lines were exposed to increasing doses of compounds for 72h. Cell proliferation was measured by using the MTT assay.
Results: Twenty-seven DLBCL cell lines were exposed to NEO2734. The compound showed anti-tumor activity with a median IC50 of 157 nM (95% C.I., 135-214). Cell lines derived from activated B-cell-like DLBCL (ABC DLBCL) (n.=7) were more sensitive than the ones derived from germinal center B-cell (GCB) DLBCL (n.=20) (P = 0.04). No difference were observed based on MYC gene status (translocation: yes, n=8; no, n.=13), BCL2 gene status (translocation: yes, n=12; no, n.=6), TP53 gene status (inactive: yes, n=14; no, n.=6), double hit MYC/BCL2 (yes, n.=6; no, n.=14), CREBBP gene status (mutated, n.=10; wild type, n.=16), or EP300 gene status (mutated, n.=5; wild type, n.=20).
As comparison, 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 data obtained for birabresib were in agreement with what we had previously reported (Boi et al, Clinical Cancer Res 2015). The three compounds presented a similar pattern of anti-proliferative activity across all the cell lines (NEO2734 and birabresib: R2 =0.84, P < 0.001; NEO2734 and CBP30, R2 = 0.73, P < 0.001; birabresib and CBP30, R2 = 0.73, P < 0.001) but with different degrees of IC50. Both NEO2734 and birabresib were more potent than CBP30 (P <0.0001). The novel dual inhibitor was more potent than birabresib (P=0.0182) and the difference was even bigger considering the compounds' IC90 (P = 0.0025): median values were 1.1 μM (95% C.I., 735 nM - 2.7 μM) and 20 μM (95% C.I., 2 - 30 μM) for the dual inhibitor and for the BET inhibitor, respectively.
Conclusions: The novel dual BET and CREBBP/EP300 inhibitor NEO2734 showed strong in vitro anti-tumor activity across a large panel of DLBCL cell lines and it appeared more potent than single BET or CREBBP/EP300 inhibitors.

Disclosures
Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Stathis:Oncology Therapeutic Development: Research Funding. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership.