Site-Specific Mutagenesis Screening in KRAS(G12D) Mutant Library to Uncover Resistance Mechanisms to KRAS(G12D) Inhibitors

KRAS, a pivotal member of the RAS GTPase family, plays a crucial role in regulating cell survival and proliferation and is one of the most commonly mutated oncogenes in human cancers. The novel KRAS(G12D) inhibitor, MRTX1133, demonstrates promising antitumor efficacy in vitro and in vivo. However, the development of acquired resistance in treated patients presents a considerable challenge to sustained therapeutic effectiveness. In response to this challenge, we conducted comprehensive site saturation mutagenesis screening to identify potential secondary mutations that could induce resistance to the KRAS(G12D) inhibitor MRTX1133. Using our approach, we screened a range of KRAS(G12D) variants harboring potential secondary mutations. From this screening, 44 representative variants were selected for in-depth validation of the pooled screening outcomes. We identified eight variants (G12D combined with the mutations V9E, V9W, V9Q, G13P, T58Y, R68G, Y96W, and Q99L) that exhibited substantial resistance. Further investigations into these variants, particularly focusing on their interaction with MRTX1133, were conducted using downstream signaling analyses. Our results revealed the diminished efficacy of MRTX1133 in these specific contexts. Particularly, the V9W variant exhibited pronounced resistance. Structural modeling of this variant provides insights into how its unique conformation may hinder the effective binding of MRTX1133. Our findings offer valuable perspectives for predicting and understanding secondary mutations that lead to acquired resistance to KRAS(G12D) inhibitors. This evidence is critical for devising new strategies to counteract resistance mechanisms and, ultimately, enhance treatment outcomes in patients with KRAS(G12D)-mutant cancers