PMID- 34373753 OWN - NLM STAT- MEDLINE DCOM- 20220128 LR - 20220128 IS - 1838-7640 (Electronic) IS - 1838-7640 (Linking) VI - 11 IP - 17 DP - 2021 TI - Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition. PG - 8464-8479 LID - 10.7150/thno.60028 [doi] AB - As glutamine plays a central role in cancer metabolism, inhibition of glutaminolysis has become an ideal anticancer therapeutic target. However, glutaminolysis inhibition leads to activation of autophagy, which compromises its antitumor effect. Hence, we investigated the mechanism underlying glutaminolysis inhibition-induced pro-survival autophagy. Methods: High-throughput sequencing was performed on colorectal cancer (CRC) cells before and after glutaminolysis inhibition to identify differentially expressed genes. Activating transcription factor 4 (ATF4) pathway enrichment in glutaminolysis inhibited cells was identified through gene set enrichment analysis. ATF4 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. The function of ATF4 on mechanistic target of rapamycin (mTOR) regulation was assessed by western blotting. Luciferase reporter assays and chromatin immunoprecipitation were used to confirm the regulation of DNA damage inducible transcript 4 (DDIT4) by ATF4. mRNA half-life assays, RNA immunoprecipitation, qRT-PCR and western blotting were performed to determine the relationship between FTO alpha-ketoglutarate dependent dioxygenase (FTO), YTH N(6)-methyladenosine RNA binding protein 2 (YTHDF2), and ATF4. ATF4 regulation of pro-survival autophagy was measured by tandem monomeric red fluorescent protein-green fluorescent protein fluorescence microscopy. Finally, the synergistic effect of autophagy and glutaminolysis inhibition was analyzed in an azoxymethane/dextran sodium sulfate mouse model. Results: The ATF4 pathway was activated in CRC cells upon glutaminolysis inhibition. Functionally, ATF4 transcriptionally upregulated DDIT4 to suppress mTOR, which induced pro-survival autophagy during glutaminolysis inhibition. Interestingly, glutaminolysis inhibition promoted ATF4 mRNA expression by abrogating N(6)-methyladenosine (m(6)A) modification and YTHDF2-mediated RNA decay. Finally, inhibition of ATF4-induced autophagy enhanced the antitumor efficacy of glutaminolysis inhibition. Conclusion: Glutaminolysis inhibition upregulated ATF4 expression in an m(6)A-dependent manner to activate pro-survival autophagy through transcriptional activation of the mTOR inhibitor DDIT4. Targeting ATF4-induced autophagy is a new strategy to synergize glutaminolysis-targeting therapies for cancer treatment. CI - (c) The author(s). FAU - Han, Shuting AU - Han S AD - Department of Medical Oncology, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Zhu, Liyuan AU - Zhu L AD - Laboratory of Cancer Biology, Key Lab of Biotherapy, Cancer Center of Zhejiang University, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Zhu, Yiran AU - Zhu Y AD - Laboratory of Cancer Biology, Key Lab of Biotherapy, Cancer Center of Zhejiang University, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Meng, Yuan AU - Meng Y AD - Laboratory of Cancer Biology, Key Lab of Biotherapy, Cancer Center of Zhejiang University, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Li, Jiaqiu AU - Li J AD - Department of Medical Oncology, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Song, Ping AU - Song P AD - Department of Medical Oncology, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Yousafzai, Neelum Aziz AU - Yousafzai NA AD - Department of Allied Health Sciences, University of Poonch Rawalakot, AJK, Pakistan. FAU - Feng, Lifeng AU - Feng L AD - Laboratory of Cancer Biology, Key Lab of Biotherapy, Cancer Center of Zhejiang University, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Chen, Miaoqin AU - Chen M AD - Laboratory of Cancer Biology, Key Lab of Biotherapy, Cancer Center of Zhejiang University, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Wang, Yanmei AU - Wang Y AD - Department of Medical Oncology, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Jin, Hongchuan AU - Jin H AD - Laboratory of Cancer Biology, Key Lab of Biotherapy, Cancer Center of Zhejiang University, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. FAU - Wang, Xian AU - Wang X AD - Department of Medical Oncology, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20210725 PL - Australia TA - Theranostics JT - Theranostics JID - 101552395 RN - 0 (ATF4 protein, human) RN - 0 (DDIT4 protein, human) RN - 0 (RNA, Messenger) RN - 0 (RNA-Binding Proteins) RN - 0 (Transcription Factors) RN - 0RH81L854J (Glutamine) RN - 145891-90-3 (Activating Transcription Factor 4) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) SB - IM MH - Activating Transcription Factor 4/*metabolism/physiology MH - Animals MH - Autophagy/*physiology MH - Cell Line, Tumor MH - Colorectal Neoplasms/metabolism MH - Glutamine/*metabolism MH - HCT116 Cells MH - Humans MH - Male MH - Mice MH - Mice, Inbred C57BL MH - RNA Stability MH - RNA, Messenger/genetics MH - RNA-Binding Proteins/metabolism MH - TOR Serine-Threonine Kinases/metabolism MH - Transcription Factors/metabolism PMC - PMC8343999 OTO - NOTNLM OT - ATF4 OT - DDIT4 OT - Glutaminolysis inhibition OT - N6-methyladenosine OT - metabolic synthetic lethality COIS- Competing Interests: The authors have declared that no competing interest exists. EDAT- 2021/08/11 06:00 MHDA- 2022/01/29 06:00 PMCR- 2021/01/01 CRDT- 2021/08/10 06:38 PHST- 2021/03/03 00:00 [received] PHST- 2021/07/11 00:00 [accepted] PHST- 2021/08/10 06:38 [entrez] PHST- 2021/08/11 06:00 [pubmed] PHST- 2022/01/29 06:00 [medline] PHST- 2021/01/01 00:00 [pmc-release] AID - thnov11p8464 [pii] AID - 10.7150/thno.60028 [doi] PST - epublish SO - Theranostics. 2021 Jul 25;11(17):8464-8479. doi: 10.7150/thno.60028. eCollection 2021.