Novel strategies for achieving synthetic lethality in KRAS-dependent cancers

For many years KRAS has been considered an undruggable target. Recent advancements have enabled the development of therapeutic strategies aimed at modulating KRAS activity at both transcriptional and post-transcriptional levels. Among these, the emergence of KRAS-specific enzymatic inhibitors has represented a significant milestone in oncology, offering selective efficacy against KRAS-mutant malignancies and progressing to clinical evaluation. Nevertheless, the clinical utility of these inhibitors is frequently compromised by the onset of acquired resistance, underscoring the urgent need for innovative approaches to circumvent the adaptive responses displayed by neoplastic cells. This study seeks to elucidate the regulatory mechanisms governing KRAS expression, with the ultimate objective of identifying novel molecular targets for therapeutic intervention in tumors harboring gain-of-function KRAS mutations. Building upon prior findings from our laboratory, which demonstrated the presence of G-quadruplex (G4) structures within the 5’ untranslated region (5’UTR) of KRAS mRNA, and considering the growing body of evidence supporting the regulatory roles of both DNA and RNA G4 motifs, the first section of this thesis investigates the functional implications of KRAS 5’UTR G4s in transcript stability and translational control. Experimental data revealed that these RNA-G4 (rG4) structures are specifically recognized by long non-coding RNAs (lncRNAs) transcribed from the LINC01750 locus. The interaction between KRAS rG4s and LINC01750-derived lncRNAs facilitates the formation of double-stranded RNA hybrids, which are subsequently degraded via RNase III-mediated cleavage. This process is modulated by the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which acts to unwind the G4 structures, thereby promoting hybridization between KRAS mRNA and the corresponding lncRNAs. Notably, the presence of rG4s within the KRAS 5’UTR intrinsically enhances transcript stability by protecting the RNA from degradation, suggesting that pharmacological disruption of these structures using G4-remodeling agents may represent a viable strategy to suppress KRAS expression. In parallel, we investigated the compensatory mechanisms employed by cancer cells that exhibit resistance to KRAS enzymatic inhibitors. Our findings identified peptidyl arginine deiminases (PADIs), specifically PADI1 and 3, as critical mediators of chemoresistance. Pharmacological inhibition of PADIs using the pan-PADI inhibitor BB-Cl-amidine (BB-Cl) sensitized pancreatic ductal adenocarcinoma (PDAC) cells that were refractory to KRAS-targeted degradation. Mechanistically, this synergistic cytotoxicity was associated with perturbation of glutathione metabolism and downregulation of resistance-associated transcriptional programs, including those involved in cytoskeletal remodeling and nuclear expulsion. Importantly, BB-Cl synergized with several KRAS(OFF) and KRAS(ON) inhibitors in different pancreatic and colorectal cancer models, showing adaptability across different KRAS mutations (G12C and G12D) and minimal off-target effects in healthy cells. Although the precise molecular role of PADIs in modulating resistance to KRAS inhibition remains to be fully delineated, the combinatorial approach of dual KRAS and PADI inhibition emerges as a promising avenue for the treatment of KRAS-driven malignancies. Furthermore, our findings pave the way for the development of G4-targeting compounds directed at the KRAS 5’UTR, which may be employed in conjunction with existing KRAS inhibitors and BB-Cl-based co-treatment regimens to enhance therapeutic efficacy.