AZI2 mediates TBK1 activation at unresolved selective autophagy cargo receptor complexes with implications for CD8 T-cell infiltration in breast cancer

ABSTRACT

Most breast cancers do not respond to immune checkpoint inhibitors and there is an urgent need to identify novel sensitization strategies. Herein, we uncovered that activation of the TBK-IFN pathway that is mediated by the TBK1 adapter protein AZI2 is a potent strategy for this purpose. Our initial observations showed that RB1CC1 depletion leads to accumulation of AZI2, in puncta along with selective macroautophagy/autophagy cargo receptors, which are both required for TBK1 activation. Specifically, disrupting the selective autophagy function of RB1CC1 was sufficient to sustain AZI2 puncta accumulation and TBK1 activation. AZI2 then mediates downstream activation of DDX3X, increasing its interaction with IRF3 for transcription of pro-inflammatory chemokines. Consequently, we performed a screen to identify inhibitors that can induce the AZI2-TBK1 pathway, and this revealed Lys05 as a pharmacological agent that induced pro-inflammatory chemokine expression and CD8⁺ T cell infiltration into tumors. Overall, we have identified a distinct AZI2-TBK1-IFN signaling pathway that is responsive to selective autophagy blockade and can be activated to make breast cancers more immunogenic.

Abbreviations: AZI2/NAP1: 5-azacytidine induced 2; CALCOCO2: calcium binding and coiled-coil domain 2; DDX3X: DEAD-box helicase 3 X-linked; FCCP: carbonyl cyanide p-triflouromethoxyphenylhydrazone; a protonophore that depolarizes the mitochondrial inner membrane; ICI: immune checkpoint inhibitor; IFN: interferon; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1

KEYWORDS:

• AZI2
• breast cancer
• RB1CC1
• immune checkpoint inhibitor
• TBK1

Acknowledgements

We would like to thank Dr. Kefeng Lu [63] from Sichuan University, Chengdu for the pLV-ORF3a plasmid and Dr. Richard Youle (NIH) for the HeLa Penta KO cells [42]. We are grateful to Dr. Ravi Amaravadi (University of Pennsylvania) for providing Lys05. We appreciate the assistance from Ken Greis and Michael Wyder from the UC Proteomics Laboratory core for mass spectrometry analysis and the University of Cincinnati LAMS staff for their support with regards to mouse colony maintenance and husbandry. We are grateful to members of the Guan lab for critical appraisal and suggestions in the preparation of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

All relevant data to evaluate the conclusions in the paper are within the paper and/or the Supplementary Materials.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2259775

Additional information

Funding

This research was supported by NIH Grants R01CA163493, R01NS094144, and R01HL073394 to J.-L. Guan.