INTRODUCTION: KRAS mutations have been recognized as undruggable for many years. 
Recently, novel KRAS G12C inhibitors, such as sotorasib and adagrasib, are being 
developed in clinical trials and have revealed promising results in metastatic 
NSCLC. Nevertheless, it is strongly anticipated that acquired resistance will 
limit their clinical use. In this study, we developed in vitro models of the 
KRAS G12C cancer, derived from resistant clones against sotorasib and adagrasib, 
and searched for secondary KRAS mutations as on-target resistance mechanisms to 
develop possible strategies to overcome such resistance.
METHODS: We chronically exposed Ba/F3 cells transduced with KRASG12C to 
sotorasib or adagrasib in the presence of N-ethyl-N-nitrosourea and searched for 
secondary KRAS mutations. Strategies to overcome resistance were also 
investigated.
RESULTS: We generated 142 Ba/F3 clones resistant to either sotorasib or 
adagrasib, of which 124 (87%) harbored secondary KRAS mutations. There were 12 
different secondary KRAS mutations. Y96D and Y96S were resistant to both 
inhibitors. A combination of novel SOS1 inhibitor, BI-3406, and trametinib had 
potent activity against this resistance. Although G13D, R68M, A59S and A59T, 
which were highly resistant to sotorasib, remained sensitive to adagrasib, Q99L 
was resistant to adagrasib but sensitive to sotorasib.
CONCLUSIONS: We identified many secondary KRAS mutations causing resistance to 
sotorasib, adagrasib, or both, in vitro. The differential activities of these 
two inhibitors depending on the secondary mutations suggest sequential use in 
some cases. In addition, switching to BI-3406 plus trametinib might be a useful 
strategy to overcome acquired resistance owing to the secondary Y96D and Y96S 
mutations.