Fig. 6. Two novel point mutations on FOXK (FOXK1 N128K and FOXK2 N59K) are important for FOXK function in DNA damage–induced autophagy and response to chemotherapy.

(A and B) HEK293T cells transfected with indicated FOXK1 (A) or FOXK2 (B) constructs were treated with vehicle or 20 μM cisplatin for 24 hours and purified using anti–HA-agarose beads. The immunoprecipitates were then blotted with the indicated antibodies. (C) A549 cells depleted of endogenous FOXK and stably expressing HA-FOXK WT or HA-FOXK NK were treated with 20 μM cisplatin and/or CQ as indicated. Western blot was performed with the indicated antibodies. (D) Cells from (C) were used to perform colony formation as indicated. (E and F) Tumor growth assay for FOXK WT or FOXK NK cells treated with vehicle, CQ, cisplatin, or cisplatin together with CQ. Tumor images were acquired as shown in (E), and weights (F) were measured. n = 5; data points in (F) represent mean tumor weight ± SD. **P < 0.01. Statistical analyses were performed using Student’s t test. (G) Under normal conditions, FOXK localizes to the nucleus as a transcriptional repressor to control the expression of ATGs. (H) Upon DNA damage conditions (caused by agents such as cisplatin and etoposide), ATM activates CHK2, which further directly phosphorylates FOXK (FOXK1 at S130 and FOXK2 at S61) and creates a 14-3-3γ binding site, which, in turn, traps FOXK proteins in the cytoplasm. Because FOXK functions as the transcriptional suppressor of ATGs, DNA damage–mediated FOXKs’ cytoplasmic trapping induces expression of ATGs and facilitates autophagy in response to DNA damage. Moreover, DNA damage–induced autophagy results in chemoresistance.