(A) (Left) Growth of sotorasib-resistant tumors after subcutaneous implantation in immunodeficient mice treated either with vehicle (black circles) or 100 mg/kg sotorasib following a discontinuous treatment (blue circles) in which they were preexpanded in untreated immunodeficient mice. (Right) Growth of sotorasib-resistant tumors after subcutaneous implantation in immunodeficient mice treated either with vehicle (black circles) or with 100 mg/kg sotorasib following a continuous treatment (red circles) in which they were preexpanded in immunodeficient mice continuously treated with 100 mg/kg of sotorasib. Dotted lines indicate the differential time scale for tumors to become sotorasib resistant following discontinuous versus continuous exposure to sotorasib. (B) Viability of lung tumor cells treated with the indicated concentrations of sotorasib for 72 hours in 2D (left) and 3D (right) cultures. Tumor cells derived from untreated tumors expressing KrasG12V (white circles, n = 3) or KrasG12C (black circles, n = 3) as well as tumor cells obtained from sotorasib-resistant tumors either left untreated in culture (blue circles, n = 4) or cultured in the presence of 10 μM sotorasib (red circles, n = 2). Data are represented as mean ± SEM. (C) Specific GST activity (U/mg) of untreated control KrasG12C tumor cells, tumor cells obtained from sotorasib-resistant tumors untreated in vitro (R/U), and tumor cells obtained from sotorasib-resistant tumors cultured in the presence of 10 μM of sotorasib (R/T). Colors are those described in B. Data are represented as mean ± SEM. P values were calculated using an ANOVA test. **P < 0.01. (D) Representative images of interphase and metaphase FISH analyses of 2 cell lines (1 and 2) obtained from sotorasib-resistant tumors, cultured in the absence of sotorasib (sotorasib withdrawal) or in the presence of 10 μM of sotorasib (+ sotorasib). Scale bars: 5 μm. (E) Absence (white bars) or presence of Kras (black bars) amplification in cell lines 1 and 2 grown in the absence (–) or presence (+) of 10 μM of sotorasib.