Generation of Human Haematopoietic Model Cell Lines Revealed Distinct Replication Stress Tolerance Between Two...

KRAS is one of the most frequently mutated genes in all human cancers, and its oncogenic mutation hotspots are glycine 12 (G12), glycine 13 (G13), glutamine 61 (Q61) and alanine 146 (A146). Among these hotspot mutations, A146 substitution mutations (A146X) occur relatively infrequently, except for haematopoietic and lymphoid cancers, suggesting that A146X causes intrinsically distinct KRAS signalling compared to other KRAS oncogenic alleles. However, due to the absence of model A146X cell lines derived from haematopoietic sources, the cellular mechanisms that cause the differences between KRAS.A146X and other common KRAS mutants, such as KRAS.G12X, remain largely unexplored. In this study, we developed a set of isogenic model haematopoietic cell lines expressing KRAS.A146T, KRAS.G12V and KRAS.G12G (non-mutated) from the endogenous locus by genetically modifying the human lymphoblastoid TK6 cell line. We found that TK6 cells carrying KRAS^A146T/+ or KRAS^G12V/+ exhibited increased replication stress compared to KRAS wild-type cells. Strikingly, KRAS^A146T/+ cells strongly rely on PrimPol for maintaining cellular survival upon replication stress. In contrast, KRAS^G12V/+ cells exhibited hypersensitivity to inhibitors for the ATR-Chk1 checkpoint signalling axis and to nucleoside analogues commonly used to treat cancers and viral infections. Our findings demonstrate that the endogenously expressed oncogenic KRAS mutations exacerbate the replication stress and reveal KRAS allele-specific replication phenotypes, facilitating the development of effective chemotherapies tailored to specific oncogenic KRAS mutation alleles and types of cancer. Moreover, our study offers valuable model cell lines for investigating mechanisms underlying replication vulnerability in cancers harbouring oncogenic KRAS mutations.