Phosphorylation of RAB7 by TBK1/IKKε Regulates Innate Immune Signaling in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TBK1/IKKe on the same serine-72 site. An unbiased search for novel TBK1/IKKe substrates using stable isotope labeling with amino acids in cell culture (SILAC) phosphoproteomic analysis identified Rab7 serine-72 (S72) as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated STING degradation. Thus, convergence of PTEN loss and TBK1/IKKe activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFN-b. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyper-responsive to STING agonists. Together these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKe in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic.