Figure 3. Activation of the GCN2 pathway enhances the response to WEE1 kinase inhibition in vivo.

(A) Left: in vivo competition assay with GCN2^−/− (mCherry⁺) and WT (GFP⁺) NCI-H82 AZD1775-naïve cells. Right: percent of GCN2^−/− cells between the treated group and untreated group. t test, *p = 0.05–0.01, **p = 0.01–0.001, ***p < 0.001. Mouse numbers per condition are indicated in the plot (independent replicates in Figures S3C and S3D).

(B) Targeted in vivo CRISPR-Cas9 screening strategy. Cas9-expressing cells were infected with lentiviral particles containing the targeted library (focusing on sensitizing hits in culture) and subsequently injected into the flanks of mice. After tumors reached ~1 cm³ in size, the tumors were collected for analysis.

(C) Volcano plots from targeted in vivo CRISPR-Cas9 screens as in (B). Each screen was performed in replicates (n = 2). Mice (n = 12, 3 per group) were treated for 21 days. PPP1CC (PP1c) is bolded. Fold change (log2) < −1 or > 1, ANOVA: p < 0.05 (MEMcrispR package).

(D) Proposed model: inhibition of the WEE1 kinase by the ATP-competitive inhibitor AZD1775 not only inhibits WEE1 in SCLC cells but also activates GCN2 and the integrated stress response (ISR) pathway, which contributes to enhanced cell death. Further activation of the ISR pathway by inhibiting a phosphatase in the GCN2 pathway can lead to greater cancer cell death.