Squamous cell carcinomas (SCCs) arising from aerodigestive or anogenital epithelium that are associated with the human papillomavirus (HPV) are far more readily cured with radiation therapy than HPV-negative SCCs. The mechanism behind this increased radiosensitivity has been proposed to be secondary to defects in DNA repair, although the specific repair pathways that are disrupted have not been elucidated. To gain insight into this important biomarker of radiosensitivity, we first examined genomic patterns reflective of defects in DNA double-strand break repair, comparing HPV-associated and HPV-negative head and neck cancers (HNSCC). Compared to HPV-negative HNSCC genomes, HPV+ cases demonstrated a marked increase in the proportion of deletions with flanking microhomology, a signature associated with a backup, error-prone double-strand break repair pathway known as microhomology-mediated end-joining (MMEJ). Then, using 3 different methodologies to comprehensively profile double-strand break repair pathways in isogenic paired cell lines, we demonstrate that the HPV16 E7 oncoprotein suppresses canonical nonhomologous end-joining (NHEJ) and promotes error-prone MMEJ, providing a mechanistic rationale for the clinical radiosensitivity of these cancers.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

During aging, wear, and tear of intervertebral discs, human discs undergo a series of morphological and biochemical changes. Degradation of extracellular matrix proteins, e.g., collagen, arises as an important contributor and accelerator in this process. Existing methods to detect collagen degradation at the tissue level include histology and immunohistochemistry. Unfortunately, most of these methods only depict overall collagen content without the ability to specifically discern degraded collagen and to assess the severity of degeneration. To fill this technological gap, we developed a robust and simple approach to detect and assess early disc degeneration with a collagen hybridizing peptide (CHP) that hybridizes with the flawed triple

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Cancer cells defective in homologous recombination (HR) are responsive to DNA crosslinking chemotherapies, PARP inhibitors, and inhibitors of polymerase theta, a key mediator of the backup pathway alternative end-joining. Such cancers include those with pathogenic bi-allelic alterations in core HR genes and another cohort of cases that exhibit sensitivity to the same agents and harbor genomic hallmarks of HR deficiency (HRD). These HRD signatures include a single base substitution pattern, large rearrangements, characteristic tandem duplications, and small deletions. Here, we utilized what is now known about the backup pathway alternative end-joining (Alt-EJ) through the key factor polymerase theta to design and test novel signatures of polymerase theta mediated (TMEJ) repair. We generated two novel signatures; a signature composed of small deletions with microhomology and another consisting of small, templated insertions. We find that templated insertions (TINS) consistent with TMEJ repair are highly specific to tumors with pathogenic bi-allelic mutations in BRCA2 and that high TINS genomic signature content in advanced ovarian cancers associate with overall survival following treatment with platinum agents. Additionally, the combination of TINS with other HRD metrics significantly improves the association of platinum sensitivity with survival compared to current state-of-the-art signatures. Implications: Small, templated insertions indicative of theta-mediated end-joining likely can be used in conjunction with other HRD mutational signatures as a prognostic tool for patient response to therapies targeting HR deficiency.