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Cellular and Molecular Biology

Disruption of DNA-PKcs-mediated cGAS retention on damaged chromatin potentiates DNA damage-inducing agent-induced anti-multiple myeloma activity

Abstract

Background

Targeting DNA damage repair factors, such as DNA-dependent protein kinase catalytic subunit (DNA-PKcs), may offer an opportunity for effective treatment of multiple myeloma (MM). In combination with DNA damage-inducing agents, this strategy has been shown to improve chemotherapies partially via activation of cGAS-STING pathway by an elevated level of cytosolic DNA. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated into the cytoplasm upon DNA damage, leading to cGAS-STING activation.

Methods

We examined the role of DNA-PKcs inhibition on cGAS-STING-mediated MM chemosensitivity by performing mass spectrometry and mechanism study.

Results

Here, we found DNA-PKcs inhibition potentiated DNA damage-inducing agent doxorubicin-induced anti-MM effect by activating cGAS-STING signaling. The cGAS-STING activation in MM cells caused cell death partly via IRF3-NOXA-BAK axis and induced M1 polarization of macrophages. Moreover, this activation was not caused by defective classical non-homologous end joining (c-NHEJ). Instead, upon DNA damage induced by doxorubicin, inhibition of DNA-PKcs promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of cytosolic cGAS and DNA, activating cGAS-STING.

Conclusions

Inhibition of DNA-PKcs could improve the efficacy of doxorubicin in treatment of MM by de-sequestrating cGAS in damaged chromatin.

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Fig. 1: DNA-PKcs inhibition synergistically enhanced doxorubicin-induced cell death of MMCs.
Fig. 2: DNA-PKcs inhibition potentiated doxorubicin-induced cGAS-STING-mediated response of type I IFNs in MMCs.
Fig. 3: NU7441/doxorubicin promoted cGAS-STING-dependent but type I IFNs-independent NOXA-mediated apoptosis of MMCs.
Fig. 4: Activation of the cGAS-STING signaling by NU7441/doxorubicin in MMCs induced M1 polarization of macrophages (Mφs).
Fig. 5: DNA-PKcs inhibition but not c-NHEJ deficiency potentiated doxorubicin-induced cGAS activation in MMCs by dissociating cGAS from cytoplasmic nucleosomes.
Fig. 6: Disruption of cytoplasmic cGAS-nucleosome binding abolished the effect of NU7441 on doxorubicin-induced activation of the cGAS-STING signaling in MMCs.

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Data availability

The raw RNA-seq datasets for this study are available from the NCBI Sequence Read Archive (SRA) with the accession number code PRJNA984948. Flow cytometry datasets are publicly accessible at the repository Zenodo (https://doi.org/10.5281/zenodo.8042604). Source data for the figures and supplementary figures are provided as a Source Data file. Source Data are provided with this paper.

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Acknowledgements

We thank members of the Cai lab and the Xie lab for helpful discussions. We also thank Bi Chao and Hong Xiaoli from the Core Facilities, Zhejiang University School of Medicine for their technical support.

Funding

This work was supported by the National Natural Science Foundation of China (82100212 to MMD, U22A20291 to ZC, No. 31870806 to AYX and 32071439 to YLF), the Department of Science and Technology of Hangzhou (202204A05 to AYX), the Zhejiang Provincial Natural Science Foundation of China (LQ21H160015 to EFZ and LZ22H160009 to JSH) and the Key Research and Development Plan Project of Zhejiang Province (2020C03014 to ZC).

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Contributions

ZC, AYX, MMD and JNZ initiated and designed the study. JNZ, MMD and WC performed experiments. JNZ, MMD, YLF and AYX analyzed experimental results. AYX and JNZ wrote the paper. HGC performed the bioinformatics analyses. HYG, EFZ, YLF and SCL provided assistance and valuable advice on the manuscript. JSH, AYX and ZC supervised the experiments.

Corresponding authors

Correspondence to An-Yong Xie or Zhen Cai.

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All experimental methods were carried out in accordance with relevant guidelines such as the Declaration of Helsinki. All procedures involving animals were in accordance with the Animal Experimental Ethical Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (Reference Number: 2022-1578). Human samples-based study was approved by the Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China (Reference Number: 2022-930).

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Zhang, JN., Dong, MM., Cao, W. et al. Disruption of DNA-PKcs-mediated cGAS retention on damaged chromatin potentiates DNA damage-inducing agent-induced anti-multiple myeloma activity. Br J Cancer (2024). https://doi.org/10.1038/s41416-024-02742-3

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