Enhancing Chemotherapy Efficacy by Targeting the Mutagenic Translesion Synthesis Pathway - PROJECT SUMMARY Chemoresistance remains a major barrier to effective non-small cell lung cancer (NSCLC) treatment, often leading to relapse and treatment failure despite initial therapeutic response. Platinum-based chemotherapy and radiotherapy, the current standard-of-care, lack selectivity and induce broad DNA damage, contributing to severe side effects such as neurotoxicity, nephrotoxicity, and an increased risk of secondary malignancies. A key mechanism driving both intrinsic and acquired resistance is the mutagenic translesion synthesis (TLS) pathway, which enables cancer cells to bypass chemotherapy-induced DNA lesions while accumulating mutations that accelerate disease progression. Despite its critical role in promoting chemoresistance, no FDAapproved therapies currently target TLS. This project aims to develop a first-in-class synthetic RNA-based therapy that selectively inhibits TLS to enhance chemotherapy efficacy in NSCLC. Our approach utilizes a nucleic acid construct encoding a dominant-negative REV1 C-terminal domain (REV1-CTDDN) to block TLS polymerase recruitment, delivered via lung cancer-targeted lipid nanoparticles (LNPs). By preventing mutagenic lesion bypass, this strategy is expected to increase chemotherapy-induced DNA damage accumulation, enhance tumor cell death, and reduce adaptive resistance. Our preliminary studies demonstrate that this construct-encoded REV1-CTDDN sensitizes ovarian (OVCAR5, OVCAR8), melanoma (A375), lung (KP), fibrosarcoma (HT1080) and leukemia (K562) cancer cells to cisplatin, reinforcing the therapeutic potential of TLS inhibition across multiple cancer types. To establish proof-of-concept, we will first evaluate TLS inhibition and chemo-sensitization in a panel of NSCLC cell lines, including KRAS-mutant (A549, H460), TP53-mutant (H1299), and EGFR-mutant (H1975) models, to determine its therapeutic impact across different genomic backgrounds. We will then conduct in vivo validation using syngeneic lung cancer models (KP mouse models) to assess tumor regression, survival benefit, and molecular markers of TLS inhibition. The KP NSCLC model is a robust syngeneic mouse model that is extensively used because it recapitulates the progression of the human disease. Additionally, we will develop a single-agent therapy co-encapsulating a cisplatin prodrug with the TLS-inhibitory construct, synchronizing DNA damage with TLS inhibition to maximize therapeutic synergy while reducing systemic toxicity. Vertebrate animals are essential to this project because the efficacy, survival benefit, and in vivo safety of the LNP-delivered TLS-inhibitory therapy cannot be recapitulated in vitro and require an immunocompetent host with an intact tumor microenvironment; the KP syngeneic mouse model provides this physiologically faithful context for evaluating therapeutic response and toxicity prior to clinical translation. Successful completion of this project will establish TLS inhibition as a novel strategy to overcome NSCLC chemoresistance, providing essential preclinical data for future IND-enabling studies and clinical translation. This work aligns with the NIH mission to advance transformative cancer therapeutics and improve treatment outcomes for lung cancer patients, a population with limited therapeutic options and high mortality rates
