Poster Presentation 46th Lorne Genome Conference 2025

Multi-point targeting of RNA polymerase II transcription cycles and epigenetic networks to investigate their coordinated control of gene-expression (#230)

Shenali A. Ranasinghe 1 2 , Jennifer R. Devlin 1 2 , Ricky W. Johnstone 1 2
  1. Division of Laboratory Research, Peter MacCallum Cancer Centre, Parkville, VIC, Australia
  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia

Gene-expression is tightly controlled by a network of transcriptional and epigenetic regulators, including transcriptional-cyclin-dependent kinases (tCDKs) that control RNA polymerase II (Pol II) advancement through different checkpoints in its transcription cycle, and epigenetic regulators comprising writers, readers, and erasers, that promote or suppress transcription through modification of histone proteins and DNA. However, the causal or consequential relationship between the epigenetic landscape and Pol II transcription cycle remains obscure and is an active area of research. Hence, I will combine inhibitors of transcriptional and epigenetic regulators and explore their combined effect on gene expression, epigenetic signatures, and cancer cell phenotypes.

Cell viability-based combination screens using 5 tCDK and 280 epigenetic inhibitors were performed using mixed-lineage-leukemia-rearranged acute-myeloid-leukemia (MLLr-AML) cells (THP1). Putative synergistic and antagonistic drug interactions were observed, spanning a range of epigenetic regulators (e.g., histone deacetylases, bromodomain and extra-terminal domain proteins), with multiple potential synergistic combinations identified for CDK7 (Pol II initiation), CDK9 (Pol II pause-release) and CDK12/13 (Pol II elongation) inhibitors. The molecular impacts of these candidate combinations will be further investigated using Multiplexed-analysis-of-cells-sequencing (MAC-seq), a high-throughput RNA-sequencing-based method to simultaneously profile differential gene expression across hundreds-to-thousands of treatment conditions. Combinations exhibiting synergistic effects on MLLr-AML cell viability will be assessed across an expanded cohort of human haematological and solid cancer cells using CellTraceVioletTM (cell proliferation) and propidium-iodide (cell death) assays, and on-target efficacy will be confirmed using CRISPR/Cas9-knockout approaches. Chromatin-immunoprecipitation (ChIP) and transient-transcriptome (TT) sequencing assays will assess genome-wide impacts of inhibitor combinations on the epigenetic landscape, Pol II chromatin occupancy, and Pol II catalytic activity. Finally, in vitro validated combinations will be assessed in vivo using mouse models of aggressive cancers.

This research will provide new knowledge about how tCDKs and epigenetic regulators control gene-expression, and expand opportunities to more effectively utilise transcriptional regulator inhibitors for cancer treatment.

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