Abstract P42: Beyond the Canonical: uORF-encoded Micro Peptides as Novel Kinase Inhibitors in Cancer Therapy

Abstract About 40% of human mRNAs contain upstream open reading frames (uORFs) in their 5’-untranslated regions. Some of these uORF sequences thought to attenuate scanning ribosomes or lead to mRNA degradation were recently shown to be translated, although the function of most encoded peptides remains unknown. Our recent study was the first to reveal a uORF-encoded peptide exhibiting kinase inhibitory activity (Jayaram DR, et al. PNAS (2021)). This uORF (uORF2), upstream of a protein kinase C (PKC) family member (PKCeta) main ORF, encodes for a peptide (uPEP2) containing the typical PKC pseudosubstrate (PS) motif present in all PKCs that auto-inhibits their kinase activity. We show direct binding and selective inhibition of the catalytic activity of novel PKCs by uPEP2 (but not of classical or atypical PKCs). Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 triple-negative breast cancer in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. The endogenous deletion of uORF2 by CRISPR/Cas9 in the non-tumorigenic MCF-7 cells showed that it impedes cell proliferation, acting as a growth suppressor. Its role in tumor formation and metastasis is presently studied in breast cancer models with metastasis to the lungs. Together, we point to a new layer of protein regulation by a uORF-encoded kinase inhibitor, which could present a forerunner for other uORFs, possessing kinase inhibitory function. Notably, we have recently identified an additional uORF with a PS motif upstream of another PKC. As kinase inhibitors, uORF-encoded peptides may play a role in signaling and stress, acting in cis and/or in trans, forming diverse regulatory networks, thereby opening new views into eukaryotic protein regulation and cancer biology. Citation Format: Divya Ram Jayaram, Sigal Frost, Chanan Argov, Vijayasteltar Belsamma Liju, Nikhil Ponnoor Anto, Amitha Muraleedharan, Assaf Ben-Ari, Rose Sinay, Ilan Smoly, Ofra Novoplansky, Noah Isakov, Debra Toiber, Chen Keasar, Moshe Elkabets, Esti Yeger-Lotem, Etta Livneh. Beyond the Canonical: uORF-encoded Micro Peptides as Novel Kinase Inhibitors in Cancer Therapy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P42.