Figure 6. Depletion of NKAP Decreases NF-κB Activation and PDHK1 Expression and Induces Synthetic Lethality Specifically upon PTEN Protein Phosphatase Loss.

(A and B) Effects of stable NKAP knockdown in PTEN-deficient cancer cell lines on NF-κB activity (A) or PDHK1 promoter activation (B) by luciferase reporter assays (STAR Methods) are shown. shA and shB, shRNAs to NKAP; SC, scrambled control shRNA. Data are shown as mean ± SEM (n = 3 replicates). **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with “scrambled control shRNA expressing PTEN-deficient cells” by Tukey’s multiple-comparisons one-way ANOVA.

(C) Western blots showing NKAP, PDHK1, and phospho-AKT expression in PTEN-deficient cancer cell lines with or without stable NKAP knockdown. shA and shB, shRNAs to NKAP; SC, scrambled control shRNA.

(D) Western blots showing HA-NKAP and PDHK1 expression in PTEN-deficient A2058 cancer cells stably expressing empty vector (EV) or wild-type NKAP (HA-NKAP^WT) or de-phosphorylation-deficient mutant NKAP (HA-NKAP^S9A-S149A).

(E and F) Effects of stable NKAP knockdown in PTEN-deficient cancer cell lines without (E) or with stable PTEN^WT or PTEN^G129E or PTEN^Y138L or GFP expression (F) on cell growth by crystal violet staining assay are shown, with quantification of cell viability under each condition relative to cells expressing the scrambled control shRNA. shA and shB, shRNAs to NKAP; SC, scrambled control shRNA.

(G) Model of cellular survival and energy metabolism regulation specifically by the protein-phosphatase activity of PTEN via a NKAP-NF-κB-PDHK1-driven signaling axis. Loss of the PTEN protein-phosphatase activity promotes NKAP phosphorylation, NF-κB activation, and PDHK1 upregulation, thereby enhancing aerobic glycolysis and rendering PTEN protein-phosphatase deficient cells dependent on NKAP and PDHK1 for survival.

See also Figure S10.