PROJECT 1 – TARGETING ONCOGENIC TRANSCRIPTION FACTORS PROJECT SUMMARY DNA-binding transcription factors have exceptional specificity as cancer drivers and dependencies, but these targets lack deep hydrophobic cavities that allow for drug discovery. Hence, there is striking lack of pharmacology for modulating such targets in our arsenal of cancer therapies. The main objective of Project 1 is to reveal therapeutically-relevant mechanisms involving the oncogenic transcription factors KLF5 and TEAD:YAP/TAZ, which are strong dependencies in carcinomas of the breast and pancreas while being dispensable for tissue homeostasis. This Project has applied the latest innovations in genetic screening technology to discover novel interactions involving these onco-transcription factors. Our objectives will now be to elucidate these mechanisms with maximum precision and explore how such mechanisms can be leveraged to develop novel pharmacology. Aim 1 will focus on exploring the link between TEAD:YAP/TAZ and the poorly understood kinases MARK2/3. Using paralog co-targeting `double knockout' screening, we have discovered that MARK2/3 catalytic function is selectively essential in YAP/TAZON cancer cells. Our epigenomic and epistasis experiments indicate that the essential function of MARK2/3 in cancer is to sustain TEAD:YAP/TAZ function. In this aim, we will employ an organoid biobank to rigorously evaluate the YAP/TAZ activity as a predictive biomarker of sensitivity to catalytic MARK2/3 inhibition. In addition, we will use `bump-and-hole' chemical-genetic tools, peptide-based inhibitors, and epistasis experiments to reveal the critical substrates of MARK2/3 in the Hippo-YAP/TAZ pathway that account for its cancer maintenance function. The mechanistic research outlined in Aim 1 will test the hypothesis that chemical modulation of MARK2/3 can exploit the addiction of carcinoma cells to the TEAD:YAP/TAZ complex. Aim 2 focuses on the zinc finger protein KLF5, which is major driver and dependency in pancreatic and breast cancer. Using an innovative marker-based genetic screening strategy, we have identified several novel cofactor ligands of KLF5, including both transcriptional coactivators as well as RNA splicing factors. We will perform rigorous biochemical and genetic experiments to definitively demonstrate whether KLF5 requires direct binding these cofactors to execute its essential function in cancer. Such interactions might serve as opportunities for developing orthosteric inhibitors of KLF5 function. In addition, we will collaborate with the other Projects to identify vulnerabilities of the KLF5 mRNA using antisense oligonucleotides (ASOs). Organoid models will be employed to evaluate the therapeutic potential of several ASO-based targeting strategies that modulate KLF5 expression or splicing. Collectively, this Project features a unique ensemble of technologies and hypotheses in pursuit of a major objective in molecular oncology: to overcome `undruggable' targe...