Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem‐like cells

While previous studies have identified cancer stem‐like cells (CSCs) as a crucial driver for chemoresistance and tumor recurrence, the underlying mechanisms for populating the CSC pool remain unclear. Here, we identify hypermitophagy as a feature of human lung CSCs, promoting metabolic adaption via the Notch1‐AMPK axis to drive CSC expansion. Specifically, mitophagy is highly active in CSCs, resulting in increased mitochondrial DNA (mtDNA) content in the lysosome. Lysosomal mtDNA acts as an endogenous ligand for Toll‐like receptor 9 (TLR9) that promotes Notch1 activity. Notch1 interacts with AMPK to drive lysosomal AMPK activation by inducing metabolic stress and LKB1 phosphorylation. This TLR9‐Notch1‐AMPK axis supports mitochondrial metabolism to fuel CSC expansion. In patient‐derived xenograft chimeras, targeting mitophagy and TLR9‐dependent Notch1‐AMPK pathway restricts tumor growth and CSC expansion. Taken together, mitochondrial hemostasis is interlinked with innate immune sensing and Notch1‐AMPK activity to increase the CSC pool of human lung cancer. Synopsis Lung cancer stem‐like cells show high levels of mitophagy, resulting in lysosomal mtDNA accumulation that activates TLR9 and Notch1‐AMPK signaling. The TLR9‐Notch1‐AMPK pathway promotes CSC expansion and can be therapeutically targeted. CSCs show mitophagic hyperactivity, resulting in the accumulation of mtDNA in lysosomes. Lysosomal mtDNA activates TLR9 and drives the expansion of lung CSCs. TLR9 interacts with Notch1, triggering Notch1 activation and subsequent AMPK phosphorylation. Blocking the TLR9‐dependent Notch1‐AMPK pathway inhibits tumor outgrowth and reduces lung CSCs. Graphical Abstract Lung cancer stem‐like cells show high levels of mitophagy, resulting in lysosomal mtDNA accumulation that activates TLR9 and Notch1‐AMPK signaling. The TLR9‐Notch1‐AMPK pathway promotes CSC expansion and can be therapeutically targeted.