PROJECT SUMMARY Transcription factors (TFs) are master regulators of gene expression and have been implicated in many disease states, including in cancer. Of these, the basic helix-loop-helix (bHLH) MYC TF family members are notorious drivers of oncogenic expression programs and are implicated in approximately 70% of all cancers. TFs influence gene expression by binding to their cognate DNA sequences and recruiting the appropriate transcriptional machinery to activate or inhibit gene expression. Although TFs are well-validated targets for cancer therapeutics, the featureless nature of the protein-protein and protein-DNA interactions required for their function is resistant to traditional drug development pipelines. Indeed, some small molecule inhibitors of bHLH-TF proteins have been reported, but their low potency and unclear mechanism of action have stalled their translation into clinical use. To address this challenge, we have developed a platform of fully synthetic, modular TF mimetics. Our approach employs strategic chemical stabilization of peptide secondary, tertiary and quaternary structure to yield synthetic transcriptional repressors (STRs) capable of binding target DNA sequences with high affinity and specificity. The initial class of STRs, derived from the bHLH protein MAX, inhibit MYC/MAX-DNA binding and block MYC-driven oncogenic phenotypes in cells. Building upon these preliminary data, this proposal aims to explore and expand into novel STR architectures and validate lead STRs capable of opposing oncogenic gene expression programs and phenotypes in animal models of MYC-driven cancers. Our expertise in synthetic chemistry and biochemical profiling of TF function, as well as our established collaborations with leaders in the fields of epigenetic and transcriptomic profiling and in vivo imaging, will be leveraged in the service of the following specific aims: 1) structural and biochemical optimization of hyperstable and ultrapotent STRs targeting MYC, 2) enhancing cellular and pharmacologic delivery of STRs coupled with quantitative mapping of STR- reprogramming of epigenetic and transcriptomic landscapes in MYC-dependent cancer cells, and 3) evaluation of lead STRs in in vivo models of both MYC-dependent solid tumors (neuroblastoma) and liquid cancers (lymphoma). Successful completion of these aims will generate potent, specific, and pharmacologically tenable STRs and prioritize them for further study as MYC-targeted therapeutics. Beyond this direct goal, this work will provide new insight into MYC-mediated gene regulation in cancer and establish a blueprint for the development of STRs targeting other TF-dependent gene expression networks in the future.