Chemical Dissection of the Super Elongation Complex

The proposal Chemical Dissection of the Super Elongation Complex is a cross- disciplinary approach geared to use pharmacologic and chemical-genetics approaches to probe and understand the super elongation complex (SEC). The SEC is a dynamic multi- protein assembly that orchestrates transcription elongation via differential P-TEFb recruitment to enable target-gene specific release of paused Pol II. Different lines of evidence point to a functional relevance of the SEC in human cancer by establishing aberrant transcription elongation to drive transcriptional programs that sustain cellular growth, counteract differentiation or that permit adaptions to the tumor microenvironment. However, a precise understanding of the regulatory architecture of how individual SEC subunits control and permit target-specific elongation control is lacking. This is due to the limited kinetic resolution provided by common genetic perturbation strategies as well as a lack of well-defined small-molecules that can interfere with SEC subunits. To overcome these limitations, we will devise target protein degradation as a means to acute SEC disruption, and couple acute perturbations with holistic measurements of gene expression, complex stoichiometry and chromatin structure. This will allow us to derive a systems-level understanding of how different subunits contribute to the overall role of the SEC in gene activity. Moreover, we will deploy innovative screening technology that allows us to test close to 100000 small- molecule probes for their capacity to interfere with the SEC component ENL in cancer cells. This will be coupled with downstream target-identification strategies and in-depth mechanistic validation of the molecular mechanism of action of identified small-molecule hits. We expect this research to result in well characterized chemical probes, which are expected to serve as a scalable vector to the study of transcription elongation and especially the SEC in cancer, development and homeostasis.

The controlled regulation of gene activity and the determination which genes are transcribed in a given point in time is of fundamental importance for all aspects of life. Among different transcriptional checkpoint, transcription elongation has been very actively investigated over the last couple of years. Transcription elongation has been shown to be mis regulated in various diseases, including cancer. This led to the development of small-molecule inhibitors that function by blocking a component of CDK9, a kinase that participates in a multi protein assembly called the super elongation complex (SEC). Several CDK9 inhibitors are currently in human clinical investigation for cancer treatment, but typically only have a relatively narrow therapeutic index since CDK9 inhibition blocks the transcription of most genes, thus causing cell death also in many healthy cells. In this project, we wanted to develop cell-based tools that allow us to better understand the role of the SEC in controlling transcription regulation. Moreover, we wanted to investigate the feasibility of disrupting transcription in a more specific manner to find better treatment options. To this goal, we engineered cells in order to allow us pharmacologic control over the stability of several SEC components as well as other proteins that participate in transcription, such has the human Mediator complex. By coupling acute protein degradation with transcriptional profiling, we identified that the SEC, in particular CDK9, provides a feedback loop that allows cells to safeguard cells from transcriptional insults, such as Mediator degradation. We learned that at most genes, this feedback mechanism is sufficient to maintain a given baseline transcriptional output. Interestingly, this feedback mechanism was not active at genes that determine cell specificity and are often overexpressed in cancer, such as MYC or MYB. This opens up the exciting possibility to achieve gene-selective transcriptional defects by targeting components of the Mediator complex with small-molecule drugs, including small-molecule degraders, which we are currently following up with additional experiments.