Ku70 Serine 155 mediates Aurora B inhibition and activation of the DNA damage response.

The Ku heterodimer (Ku70/Ku80) is the central DNA binding component of the classical non-homologous end joining (NHEJ) pathway that repairs DNA double-stranded breaks (DSBs), serving as the scaffold for the formation of the NHEJ complex. Here we show that Ku70 is phosphorylated on Serine 155 in response to DNA damage. Expression of Ku70 bearing a S155 phosphomimetic substitution (Ku70 S155D) in Ku70-deficient mouse embryonic fibroblasts (MEFs) triggered cell cycle arrest at multiple checkpoints and altered expression of several cell cycle regulators in absence of DNA damage. Cells expressing Ku70 S155D exhibited a constitutive DNA damage response, including ATM activation, H2AX phosphorylation and 53BP1 foci formation. Ku70 S155D was found to interact with Aurora B and to have an inhibitory effect on Aurora B kinase activity. Lastly, we demonstrate that Ku and Aurora B interact following ionizing radiation treatment and that Aurora B inhibition in response to DNA damage is dependent upon Ku70 S155 phosphorylation. This uncovers a new pathway where Ku may relay signaling to Aurora B to enforce cell cycle arrest in response to DNA damage.

RB1 deficiency in triple-negative breast cancer induces mitochondrial protein translation.

Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low-like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration-approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.

The Ku heterodimer: function in DNA repair and beyond.

Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, that is best characterized for its central role as the initial DNA end binding factor in the "classical" non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. Ku binds double-stranded DNA ends with high affinity in a sequence-independent manner through a central ring formed by the intertwined strands of the Ku70 and Ku80 subunits. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. There is also evidence that Ku is involved in signaling to the DNA damage response (DDR) machinery to modulate the activation of cell cycle checkpoints and the activation of apoptosis. Interestingly, Ku is also associated with telomeres, where, paradoxically to its DNA end-joining functions, it protects the telomere ends from being recognized as DSBs, thereby preventing their recombination and degradation. Ku, together with the silent information regulator (Sir) complex is also required for transcriptional silencing through telomere position effect (TPE). How Ku associates with telomeres, whether it is through direct DNA binding, or through protein-protein interactions with other telomere bound factors remains to be determined. Ku is central to the protection of organisms through its participation in C-NHEJ to repair DSBs generated during V(D)J recombination, a process that is indispensable for the establishment of the immune response. Ku also functions to prevent tumorigenesis and senescence since Ku-deficient mice show increased cancer incidence and early onset of aging. Overall, Ku function is critical to the maintenance of genomic integrity and to proper cellular and organismal development.

Ku regulates signaling to DNA damage response pathways through the Ku70 von Willebrand A domain.

The Ku heterodimer (Ku70/Ku80) is a main component of the nonhomologous end-joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs). Ku binds the broken DNA end and recruits other proteins to facilitate the processing and ligation of the broken end. While Ku interacts with many proteins involved in DNA damage/repair-related functions, few interactions have been mapped to the N-terminal von Willebrand A (vWA) domain, a predicted protein interaction domain. The mutagenesis of Ku70 vWA domain S155/D156 unexpectedly increased cell survival following ionizing radiation (IR) treatment. DNA repair appeared unaffected, but defects in the activation of apoptosis and alterations in the DNA damage signaling response were identified. In particular, Ku70 S155A/D156A affected the IR-induced transcriptional response of several activating transcription factor 2 (ATF2)-regulated genes involved in apoptosis regulation. ATF2 phosphorylation and recruitment to DNA damage-induced foci was increased in Ku70-deficient cells, suggesting that Ku represses ATF2 activation. Ku70 S155A/D156A substitutions further enhanced this repression. S155A substitution alone was sufficient to confer enhanced survival, whereas alteration to a phosphomimetic residue (S155D) reversed this effect, suggesting that S155 is a phosphorylation site. Thus, these findings infer that Ku links signals from the DNA repair machinery to DNA damage signaling regulators that control apoptotic pathways.