KRAS allelic imbalance reshapes tumor evolution through selective clonal outgrowth and chromosomal instability in NSCLC

KRAS -mutant non-small cell lung cancer (NSCLC), which accounts ~25% of all NSCLC cases, remains one of the most refractory solid tumors due to its pronounced evolutionary adaptability and propensity to develop resistance. KRAS allelic imbalance, including loss of heterozygosity (LOH), is recurrently observed in patient tumors, its functional role in shaping clonal selection and tumor evolution remains unclear.We hypothesize that distinct subpopulations harbors LOH of KRAS driven by competitive fitness and acquire unique genomic events. To investigate how KRAS allelic imbalance influences tumor evolutionary trajectories, we developed a somatic mosaic genetically engineered mouse model (smGEMM) that integrates a 200-gene CRISPR-Cas9-GFP perturbation library with an allele-resolving fluorescent reporter (Tdtomato) embedded on murine chromosome 6, ~30 Mb from Kras ^WT and in trans with Kras ^LSL-G12D. Each perturbation is encoded by an sgRNA coupled to a unique capture sequence, allowing the combined sgRNA-capture element to serve as a recoverable molecular barcode for clonal tracing. This platform allows simultaneous readout of allelic configuration and competitive fitness, facilitating in vivo tracking of both Kras -heterozygous (TdT⁺/GFP⁺) and Kras -LOH (TdT⁻/GFP⁺) subclones.Preliminary lineage-resolved analyses revealed a consistent and reproducible selective expansion of Kras -LOH subclones across biological replicates. LOH subclones consistently formed larger tumors and overtook heterozygous populations, demonstrating that allelic imbalance confers a potential competitive advantage rather than representing neutral drift. We performed whole-genome profiling of reporter-mapped subclones showed that Kras -LOH tumors undergo extensive genomic rearrangements, including broad copy-number alterations, structural variations, and chromothripsis events. Our findings indicate that genome-scale instability emerges as a positively selected trait during the competitive expansion of LOH subclones.Our study identifies Kras allelic imbalance as an active driver of clonal evolution, linking selective advantage, clonal expansion, and increasing genomic complexity. By coupling allele-specific fluorescent tracing with a focused, CRISPR perturbation library, we uncover mechanistic insights into Kras -mutant NSCLC evolution and reveal evolutionary vulnerabilities that may be leveraged to prevent or overcome resistance in KRAS -driven lung cancer.