2552 - ZDHHC15 Mediated <em>S</em>-Palmitoylation of ENO1 Reprograms Glycometabolism to Confer Non-Small Cell Lung Cancer Radioresistance through Upregulating Ku70 and Ku80

Purpose/Objective(s): Protein S-palmitoylation has been reported to participate in various aspects of cancer biology. However, its role in cancer radiosensitivity remains to be elucidated.

Materials/Methods: Using non-small cell lung cancer model, we constructed radioresistant NCI-H1299 cell lines by continuous X-ray irradiation. RNA-seq, proteomic, and metabolomic analyses were performed on radioresistant and parental cells. IP-MS/MS was used to identify interacting proteins of the palmitoyltransferase ZDHHC15. Acyl-biotinyl exchange (ABE) and click chemistry were employed to confirm the palmitoylation of ENO1. ENO1 cysteine (C) mutants were constructed to find the specific modification sites. ENO1 enzymatic activity assays and Seahorse assays were conducted to evaluate the impact of ENO1 palmitoylation on glycolysis. In vivo and in vitro radiobiological experiments were performed to assess the effect of ENO1 palmitoylation on the radiosensitivity of lung cancer cells. We also developed cell-penetrating peptides (CPPs) to specifically block ENO1 palmitoylation and tested its radiosensitizing effect in a spontaneous lung cancer mouse model.

Results: RNA-seq and proteomic analyses showed that palmitoyltransferases were widely overexpressed in radioresistant H1299 cells, with ZDHHC15 being the most prominent. The palmitoylation inhibitor 2-BP and the knockdown of ZDHHC15 both exhibit radiosensitizing effects in lung cancer cells. Metabolomic analysis showed that the glycolytic pathway and lactate levels were upregulated in radioresistant H1299 cells. IP-MS/MS identified that ENO1 could interact with ZDHHC15. The palmitoylation of ENO1 was further confirmed by ABE and click chemistry assays. By constructing ENO1 mutants, it was identified that palmitoylation of ENO1 occurs at its C399 site. It was further discovered that palmitoylation of ENO1 at the C399 site enhanced its enzymatic activity, upregulating glycolysis and lactate production. The upregulated lactate level, in turn increased the H3K9la levels in the promoter regions of XRCC6 and XRCC5. This leaded to the upregulation of Ku70 and Ku80 transcription, enhancing the NHEJ pathway and promoting radioresistance in lung cancer cells. A CPP targeting the C399 site of ENO1 was designed to specifically block the palmitoylation of ENO1 by ZDHHC15. This approach demonstrated a strong radiosensitizing effect in a spontaneous lung cancer mouse model (LSL-Kras^G12D/+; LSL-Trp53^R172H/+).

Conclusion: For the first time, we revealed the role of protein S-palmitoylation in cancer radioresistance. In lung cancer model, ZDHHC15 mediated the palmitoylation of ENO1 at the C399 site, promoting glycometabolic reprogramming and leading to increased lactate production. Through H3K9la, it enhanced the expression of key NHEJ components Ku70 and Ku80, thereby promoting radioresistance. We also designed a peptide specifically targeting this biological process, effectively enhancing the sensitivity of lung cancer to radiotherapy.