Wednesday, April 2, 2025
4/2/2025
Targeted degradation of RNAs by using small molecules - PROJECT SUMMARY We propose a transformative approach, inspired by the mechanism of action of antisense oligonucleotides (ASOs), to cleave RNAs with small molecules in cells and animals. As an alternative to ASOs, we will continue to develop a class of small molecules that selectively cleave an RNA target, with supporting, validating data. Our approach, dubbed ribonuclease targeting chimeras (RiboTACs), engineers small molecules to recruit endogenous RNase L, expressed in a latent (hence RNase L) form. The chimeras, comprising an RNA-binding module and an RNase L-recruiting small molecule, activate RNase L locally at the site of the desired target. Indeed, the goals of our previously funded grant were successfully achieved, showing that the RiboTAC approach cleaves oncogenic microRNA-21 sub-stoichiometrically and catalytically. Importantly, recent studies showed that RiboTACs can induce cleavage of RNA targets even when the RNA binder is biologically inert.1 Further, we designed two RiboTACs that degrade oncogenic mRNAs that encode difficult-to-target (generally considered “undruggable”), intrinsically disordered proteins. One of these RiboTACs binds the internal ribosomal entry site (IRES) in MYC mRNA, reducing MYC mRNA and protein levels in cellular models of triple-negative breast cancer (TNBC), cervical cancer, Burkitt’s lymphoma, and multiple myeloma (MM).1 This c-Myc-RiboTAC will be fully characterized in myeloma cell lines and patient tumor cells and then lead optimized. Collectively, we will establish a method to program small molecules to cleave specific, oncogenic transcripts that encode “undruggable” proteins in cells and in vivo. In support of these goals, we propose in Aim 1 to comprehensively characterize c-Myc-RiboTAC against MYC mRNA in vitro and in situ, a benchmark for lead optimization. Our preliminary studies show that c-Myc- RiboTAC reduces MYC mRNA and protein levels in various MM cell lines, patient tumor cells, and in vivo. Moreover, the extent of cleavage correlates with RNase L expression. In Aim 2, c-Myc-RiboTAC will be lead optimized to deliver a proof-of-concept compound with properties amenable for in vivo testing. These drug metabolism and pharmacokinetics (DMPK)-driven studies will optimize all three components of the RiboTAC, the RNA-binding module, the linker, and the RNase L-recruiting module. We will rigorously assess top RiboTACs in MM cell lines, including measuring selectivity transcriptome- and proteome-wide. Finally, in Aim 3 we will study optimized RiboTACs for activity against a panel of MM patient tumor cells and in vivo. After confirming MYC knockdown, we will assess their effects on: (i) cell proliferation; (ii) cell survival; and (iii) expression of epithelial- mesenchymal transition (EMT) and MM cancer stem cell markers. The RiboTAC with the broadest activity against MM cells will be evaluated in vivo.
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