The BET Inhibitor INCB054329 Primes AML Cells for Venetoclax-Induced Apoptosis

Abstract Bromodomain and extra-terminal (BET) inhibitors may be efficacious for treatment of acute myeloid leukemia because they attenuate the expression of critical oncogenes including MYC and BCL2. These BET inhibitors (BETi) disrupt the transcriptional elongation process by displacing BET family members BRD2,3, and 4 off of chromatin, and causing RNA polymerase promoter-proximal pausing. We used precision nuclear run-on transcription sequencing (PROseq) to directly measure the effects of INCB054329, a potent BETi, on RNA polymerase II pausing and elongation. We found dramatic reductions on the elongation of key oncogenes such as MYC and BCL2 within 15 min of adding the drug. These effects became more significant over time, eventually affecting nearly two thousand genes. By four hours after drug addition, we found a loss of ribosomal gene expression and a loss of mitochondrial gene expression that is characteristic of genes regulated by MYC, suggesting that these were secondary to turning off MYC expression. When we examined the potential of the BETi INCB054329 for therapeutic efficacy in AML using Alamar Blue assays, which measure cellular redox potential, we noted marked growth inhibition of AML cell lines. However, growth assays and measurements of apoptosis using Annexin V staining found that BETi induced minimal apoptosis and cells were largely cytostatic. BrdU incorporation assays showed that INCB054329 caused the cells to accumulate in the G0/G1 phase of the cell cycle. Metabolic studies indicated that INCB054329 treatment for 48 hours caused disruption of mitochondrial respiration rate and severely reduced glycolytic capacity. Taken together, the growth inhibition, cell cycle arrest and reduced metabolic rate suggests that INCB054329 promoted quiescence in AML cells, but that this is reversible, consistent with the clinical experience of single-agent treatment of hematologic malignancies with BETi. MLL fusion proteins enhance transcription by stimulating RNA polymerase elongation, suggesting INCB054329may provide a therapeutic option to reverse this effect. However, the cell cycle arrest suggested that a second compound may be needed to trigger cell death. We first performed in vivo studies with INCB054329 using a systemic AML xenograft model of MV4-11 cells that express MLL-AF4. Engrafted NSGS mice received INCB054329 in 3 different doses (vehicle vs 10, 30 and 75mg/kg q.d) daily. During treatment, the kinetics of MV-4-11 expansion was monitored via flow cytometry for the detection of human AML in the blood. At approximately 4 weeks after transplant, the vehicle mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Significant decreases in leukemic expansion were evident in the bone marrow (vehicle vs75mg/kg, p Disclosures Stubbs: Incyte: Employment. Liu: Incyte: Employment. Rathmell: Calithera: Research Funding. Hiebert: Incyte: Research Funding. Savona: Boehringer Ingelheim: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding.