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Background <p>Neoadjuvant chemoradiotherapy is a standard treatment for locally advanced rectal cancer, yet acquired resistance limits durable tumor control. Whether stress-adaptive acetyl-group metabolism contributes to this resistance remains unclear.</p> Methods <p>We performed a genome-wide CRISPR–Cas9 loss-of-function screen under fractionated irradiation and integrated the results with transcriptomic and proteomic profiling of radioresistant colorectal cancer models established by stepwise irradiation. CRAT function was interrogated using genetic perturbation, catalytic-dead reconstitution, acetylation-mimetic p65 rescue, RELA depletion, stable-isotope tracing, free coenzyme A quantification, NF-κB reporter assays, chromatin immunoprecipitation-qPCR, and xenograft and syngeneic tumor models.</p> Results <p>CRAT was prioritized through cross-platform convergence of the CRISPR screen with transcriptomic and proteomic signatures of radioresistant cells, and post-treatment tumors with poorer pathological regression showed higher CRAT and nuclear p65 immunohistochemical scores. CRAT manipulation had limited, model-dependent effects on basal proliferation but more pronounced effects after irradiation, where CRAT loss reduced clonogenic survival and was associated with increased oxidative stress and persistent DNA damage. Stable-isotope tracing showed that irradiation increased acetate-to-acetylcarnitine flux and expanded the free coenzyme A pool in control cells, whereas CRAT loss disrupted this acetyl-group buffering. Mechanistically, CRAT catalytic activity helped maintain irradiation-induced p65 Lys310 acetylation, H3K27ac enrichment at NF-κB-responsive loci, and NF-κB-associated transcriptional output through a p300/CBP-sensitive acetylation axis. p65-K310Q rescue and RELA depletion supported p65 acetylation and RELA-dependent transcription as downstream contributors to, rather than sole mediators of, CRAT-dependent radioprotection. In syngeneic MC38 tumors, Crat deletion enhanced the response to fractionated radiotherapy.</p> Conclusions <p>These findings support CRAT-dependent acetyl-group metabolism as a contributor to an NF-κB-associated radioprotective program and nominate this metabolic dependency as a candidate vulnerability for radiosensitization in colorectal cancer.</p>

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CRAT-mediated acetylcarnitine shuttling sustains p65 acetylation and NF-κB-associated radioresistance in colorectal cancer

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月31日

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  • Yixuan Liu,
  • Yu Xiao,
  • Yiran Chen,
  • Chaoxiong Huang,
  • Feifei Lin,
  • Qingyang Zhuang,
  • Hui Li,
  • Peicheng Lin,
  • Lirui Tang,
  • Jinluan Li

摘要

Background

Neoadjuvant chemoradiotherapy is a standard treatment for locally advanced rectal cancer, yet acquired resistance limits durable tumor control. Whether stress-adaptive acetyl-group metabolism contributes to this resistance remains unclear.

Methods

We performed a genome-wide CRISPR–Cas9 loss-of-function screen under fractionated irradiation and integrated the results with transcriptomic and proteomic profiling of radioresistant colorectal cancer models established by stepwise irradiation. CRAT function was interrogated using genetic perturbation, catalytic-dead reconstitution, acetylation-mimetic p65 rescue, RELA depletion, stable-isotope tracing, free coenzyme A quantification, NF-κB reporter assays, chromatin immunoprecipitation-qPCR, and xenograft and syngeneic tumor models.

Results

CRAT was prioritized through cross-platform convergence of the CRISPR screen with transcriptomic and proteomic signatures of radioresistant cells, and post-treatment tumors with poorer pathological regression showed higher CRAT and nuclear p65 immunohistochemical scores. CRAT manipulation had limited, model-dependent effects on basal proliferation but more pronounced effects after irradiation, where CRAT loss reduced clonogenic survival and was associated with increased oxidative stress and persistent DNA damage. Stable-isotope tracing showed that irradiation increased acetate-to-acetylcarnitine flux and expanded the free coenzyme A pool in control cells, whereas CRAT loss disrupted this acetyl-group buffering. Mechanistically, CRAT catalytic activity helped maintain irradiation-induced p65 Lys310 acetylation, H3K27ac enrichment at NF-κB-responsive loci, and NF-κB-associated transcriptional output through a p300/CBP-sensitive acetylation axis. p65-K310Q rescue and RELA depletion supported p65 acetylation and RELA-dependent transcription as downstream contributors to, rather than sole mediators of, CRAT-dependent radioprotection. In syngeneic MC38 tumors, Crat deletion enhanced the response to fractionated radiotherapy.

Conclusions

These findings support CRAT-dependent acetyl-group metabolism as a contributor to an NF-κB-associated radioprotective program and nominate this metabolic dependency as a candidate vulnerability for radiosensitization in colorectal cancer.