DDDR-31. Targeting transcriptional elongation by BRD4 and CDK9 inhibition for the treatment of diffuse midline glioma

Abstract Diffuse midline glioma (DMG) K27-altered is one of the most aggressive pediatric cancers. The identification of effective therapies has been extremely challenging. Over 250 clinical trials involving different combinations of chemotherapy and radiation have been unsuccessful in DMG patients, highlighting the need to develop new therapeutic strategies based on molecular characteristics to improve treatment outcomes. We recently showed that targeting BET domain protein 4 (BRD4) activity using small-molecule inhibitors results in delayed tumor progression and extended survival of mice bearing K27M DMG patient-derived xenografts (PDX). However, tumors that initially respond to small-molecule inhibitors, such as those targeting BRD4 activity, eventually show resistance to monotherapy treatments. To find new therapeutic targets and discover novel combinatorial approaches to prevent or delay acquired resistance to BRD4 monotherapy, we performed an unbiased genome-wide CRISPR/Cas9-based genetic screening of patient-derived DMG cells. We found that DMG cell maintenance depends on a set of downstream effectors including POLR2I, which encodes a subunit of RNA Polymerase II (Pol II), in addition to cyclin-dependent kinase 9 (CDK9), a key component of the super elongation complex involved in transcriptional elongation. We observed that targeting POLR2I and CDK9 activity using short-hairpin RNA knockdown and small-molecule inhibitors blocks Pol II transcriptional elongation and inhibits the growth of DMG in vitro and in vivo. We hypothesized that dual inhibition of BRD4 and CDK9 activity will further suppress gene transcription at levels of both transcriptional initiation and elongation and will either delay or prevent DMG from acquiring resistance to monotherapies. DMG cells treated with combined BRD4 and CDK9 inhibitors showed time-dependent growth inhibition and increased apoptosis, outperforming each monotherapy. Combination therapy in mice bearing DMG PDX also inhibited tumor growth and extended survival. This study has shown promising results demonstrating that this combined therapy is a potentially effective strategy for treating DMG.