Validation of Anticorrelated TGFß Signaling and Alternative End-Joining DNA Repair Signatures that Predict Response to Genotoxic Cancer Therapy.

PURPOSE: Loss of TGFß signaling increases error-prone alternative end-joining (alt-EJ) DNA repair. We previously translated this mechanistic relationship as TGFß and alt-EJ gene expression signatures, which we showed are anticorrelated across cancer types. A score representing anticorrelation, ßAlt, predicts patient outcome in response to genotoxic therapy. Here we sought to verify this biology in live specimens and additional datasets. EXPERIMENTAL DESIGN: Human head and neck squamous carcinoma (HNSC) explants were treated in vitro to test whether the signatures report TGFß signaling, indicated by SMAD2 phosphorylation, and unrepaired DNA damage, indicated by persistent 53BP1 foci after irradiation or olaparib. A custom NanoString assay was implemented to analyze the signatures' expression in explants. Each signature gene was then weighted by its association with functional responses to define a modified score, ßAltw, that was retested for association with response to genotoxic therapies in independent datasets. RESULTS: Most genes in each signature were positively correlated with the expected biological response in tumor explants. Anticorrelation of TGFß and alt-EJ signatures measured by NanoString was confirmed in explants. ßAltw was significantly (P < 0.001) better than ßAlt in predicting overall survival in response to genotoxic therapy in The Cancer Genome Atlas (TCGA) pancancer patients and in independent HNSC and ovarian cancer patient datasets. CONCLUSIONS: Association of the TGFß and alt-EJ signatures with their biological response validates TGFß competency as a key mediator of DNA repair that can be readily assayed by gene expression. The predictive value of ßAltw supports its development to assist in clinical decision making.

Loss of TGFß signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types.

Among the pleotropic roles of transforming growth factor-ß (TGFß) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFß signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFß broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFß and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFß and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFß competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFß and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFß signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFß biology.