In vitro activity: Previous study found that Wdr5 could be detected readily in C/EBPα immunoprecipitates from lysates of Cebpap30/p30 cells by the treatment of OICR-9429, indicating that the Wdr5-MLL interaction could not influence p30 binding. Moreover, the gene expression profiling of OICR-9429-treated Cebpap30/p30 cells showed that Wdr5 antagonism could result in the upregulation of myeloid-specific transcripts. In addition, the gene set enrichment analyses demonstrated a close correlation between OICR-9429–induced genes and genes that were upregulated after Wdr5 knockdown. Furthermore, the gene profile of Cebpap30/p30 LICs6 was downregulated due to the Wdr5 antagonism caused by OICR-9429. Further treatment of OICR-9429 to Cebpap30/p30 cells was found to be associated with myeloid differentiation and loss of progenitor morphology.

Kinase Assay: OICR-9429 was evaluated at concentrations up to 50 μM for potential inhibition of the following human protein methyltransferases using a radioactivity based catalytic assay as previously reported: SMYD2, G9a, EHMT1, SUV39H2, SETDB1, SETD7, SETD8, SUV420H1, SUV420H2, PRMT1, PRMT3, PRMT5-MEP50 complex, PRMT6, PRMT8, PRMD9, EZH1, EZH2, NSD1, NSD2, NSD3, SETD2, DOT1L, DNMT1. The following chromatin histone binding ‘reader domains’ were tested for binding to OICR-9429 (200 μM) using differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) as previously described: TDRD3, SND1, L3MBTL1, L3MBTL3, UHRF1, 53BP1, STRAP, EED. Additional selectivity screening of>200 enzymes, receptors and transporters was performed by Cerep.

Cell Assay: 20,000 viable, actively proliferating primary human AML cells per well were seeded in 96-well plates in triplicates and treated with 0.05% DMSO or OICR-9429. Cell viability was measured using the Cell Titer-Glo luminescent cell viability assay on a VICTOR X4 luminometer after 72 h.