Abstract 801: Cellular pharmacokinetics and molecular pharmacodynamics studies of a novel BET inhibitor NHWD870 in sensitive and resistant leukemic cell lines
Authors: Riveiro Maria E. , Huguet Samuel , Gauzan Marie-francoise , Bekradda Mohamed , Wang Nenghui , Lokiec François , Madar Olivier , Rezai Keyvan 
Published: August 2018

Background: NHWD870 (Ningbo Wenda Pharma, China) is a novel oral bromodomain and extraterminal (BET) protein family inhibitor. BET proteins are transcriptional co-activators that interact with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors disrupt transcriptional regulatory complexes and have broad anticancer activity. The relation between the cellular pharmacokinetics (PK) and targeted-gene regulation governing the biological effects of BET inhibitors is still largely not understood. We analyzed cellular PK properties and CMYC and related genes modulation in NHWD870-sensitive and resistant leukemic cell lines.

Materials and Methods. Established human cell lines from acute and chronic myeloid leukemia (HL-60, U937, K562), acute lymphoblastic leukemia (Jurkat, MOLT-3) and SET-2 a JAK2 V617F mutated-megakaryoblastic cell line were studied. Anti-proliferative effects of NHWD870 were assessed by MTT after 72h-exposure. For cellular PK studies, cells were seeded at 2.106cells/ml and exposed to 245.5 ng/ml (500nM) NHWD870 for 0, 3, 10, 30, 60, 120 and 180 min. At each time point, NHWD870 extracellular and intracellular concentrations were analyzed in cell supernatants and pellets respectively, using Ultra Performance Liquid Chromatography with tandem Mass Spectrometry (concentration range 0.1-100 ng/mL). In cell pellets, protein modulation was analyzed by Western blot using commercial antibodies.

Results: IC50 values were between 20-600 nM in HL60, U937, SET-2 and Jurkat cells and were ≥ 1,000 nM for other cell lines. Cellular uptake of NHWD870 was rapid (3 min) in both sensitive and resistant leukemic cell lines, with a mean concentration of ~5.0 ng/106 cells (3-8.5 ng/106cells) and ~3.1 ng/106 cells (2.8-3.5 ng /106cells) for HL60 and K562 cell line respectively, whereas extracellular levels of NHWD870 were stable with up to 3h exposure. In the resistant cell line, K562, we observed CMYC mRNA downregulation after 2h exposure, even though NHWD870 intracellular levels were detected after 5 min exposure. A rapid downregulation of CMYC and P21 proteins were observed in the sensitive cell lines, HL60, U937, SET-2 and Jurkat, whereas other BET-targeted genes, such as HEXIM and BCL2 levels were modulated after longer exposure time points in sensitive cell lines.

Conclusions: NHWD870 intracellular accumulation occurred to a similar extent and timing in NHWD870 sensitive and resistant leukemic cell lines. Rapid modulation of NHWD870-target genes was observed at the protein levels only in sensitive cell lines.

Citation Format: Maria E. Riveiro, Samuel Huguet, Marie-francoise Gauzan, Mohamed Bekradda, Nenghui Wang, François Lokiec, Olivier Madar, Keyvan Rezai. Cellular pharmacokinetics and molecular pharmacodynamics studies of a novel BET inhibitor NHWD870 in sensitive and resistant leukemic cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 801.