Gain of even a single chromosome leads to changes in human cell physiology and uniform perturbations of specific cellular processes, including downregulation of DNA replication pathway, upregulation of autophagy and lysosomal degradation, and constitutive activation of the type I interferon response. Little is known about the molecular mechanisms underlying these changes. We show that the constitutive nuclear localization of TFEB, a transcription factor that activates the expression of autophagy and lysosomal genes, is characteristic of human trisomic cells. Constitutive nuclear localization of TFEB in trisomic cells is independent of mTORC1 signaling, but depends on the cGAS-STING activation. Trisomic cells accumulate cytoplasmic dsDNA, which activates the cGAS-STING signaling cascade, thereby triggering nuclear accumulation of the transcription factor IRF3 and, consequently, upregulation of interferon-stimulated genes. cGAS depletion interferes with TFEB-dependent upregulation of autophagy in model trisomic cells. Importantly, activation of both the innate immune response and autophagy occurs also in primary trisomic embryonic fibroblasts, independent of the identity of the additional chromosome. Our research identifies the cGAS-STING pathway as an upstream regulator responsible for activation of autophagy and inflammatory response in human cells with extra chromosomes, such as in Down syndrome or other aneuploidy-associated pathologies.

Microhomology-mediated end-joining (MMEJ) is an intrinsically mutagenic pathway of DNA double strand break repair essential for proliferation of homologous recombination (HR) deficient tumors. While targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as a novel MMEJ effector. We show that loss of APE2 blocks the fusion of deprotected telomeres by MMEJ and inhibits MMEJ in DNA repair reporter assays to levels comparable to Pol Theta suppression. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease domain and further identify uncharacterized domains required for recruitment to damaged DNA. We conclude that HR-deficient cells are addicted to APE2 due to a previously unappreciated role in MMEJ, which could be exploited in the treatment of cancer.

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