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

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.