Mechanisms of ruxolitinib combined with adavosertib. (A) Downregulation of DDR/replication stress gene sets after JAK1/2 inhibition. Various GSEA (gene set enrichment analysis) gene sets regulating cell cycle, DDR and replication were significant downregulated (FDR q value < 0.001) after 24-hour ruxolitinib treatment in the NKL cell line (left). (B) Cell cycle analyses indication G0/G1, S, and G2/M phases in after 24 hours of treatment with DMSO, 500 nM adavosertib, 5 μM ruxolitinib, or the combination of adavosertib and ruxolitinib. (C) Multiple genes from the Repstress gene set (Takahashi et al³⁴) were significant downregulated (P < .05) after treatment (right). (D) Assessment of EdU negative S phase cells through adavosertib, ruxolitinib and combination treatment. Illustrated are mean values of 3 independent experiments. (E) Immunoblotting shows protein expression changes of proteins involved in JAK/STAT signaling, DNA damage repair, replication stress and cell cycle regulation. (F) Accumulation of cell death during cell cycle progression through combined WEE1/JAK inhibition. Significances were assessed with a 2-way ANOVA after Dunnett correction. Bar graphs illustrate mean values of 3 independent experiments. ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. FDR, false discovery rate; NES, normalized enrichment score.