Abstract We investigated the in vivo function of NONO, a protein that has been shown to promote a distinct sub-pathway of nonhomologous end joining (NHEJ) repair in vitro and in cultured cells. We used a gene trap strategy to create a null mutant for Nono, the mouse homolog of the human NONO gene. We investigated hematopoietic stem cells (HSCs), which are known to be sensitive to deficiencies in other DNA repair proteins. Nono-deficient mice showed reduced bone marrow and spleen cellularity. HSCs from Nono-deficient mice showed severe impairment in competitive repopulation assays in primary and secondary recipients. HSCs from Nono-deficient mice also displayed significantly higher levels of reactive oxygen species, bromodeoxyuridine incorporation, and Ki-67 positivity, consistent with hyperproliferation in response to chronic genotoxic stress. To investigate DNA repair competence more directly, we exposed HSCs ex vivo to the DNA crosslinking agent, mitomycin C (MMC), or to ionizing radiation. HSCs from Nono-deficient and wild type mice showed increased sensitivity to both of these agents in colony forming cell assays, that NONO protein is induced after X-ray treatment, and that Nono-deficient HSCs show delayed resolution of γ-H2AX foci and increased apoptosis. Separately, we evaluated changes in the testes, another organ that is frequently affected by DNA repair gene mutations. Testes of Nono-deficient mice showed growth retardation, which became apparent between 18 and 26 days of postnatal development, as well as reduced sperm count. X-irradiation of Nono-deficient mice at 4 Gy led to increased apoptosis in the seminiferous tubules after 24 h, as determined in a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. There was also nearly complete depletion of germ cells at 48 h. Significantly less effects were seen at the same time points in wild type control mice. Similar to the results in HSCs, NONO was up regulated in the testes following X-ray exposure. Taken together, results are consistent with an in vivo defect in DNA repair. Notably, the co-occurrence of hematopoietic and germ cell phenotypes is reminiscent of other DNA repair gene mutations, including Fanconi anemia. Although DNA repair contributes to genomic stability in normal tissues, heightened repair activity can be a cause of treatment resistance in cancer. In this respect, it is interesting that NONO has been reported to be highly expressed in melanoma specimens and cell lines. We confirmed that NONO is expressed in melanoma cell lines and showed that it is additionally upregulated in melanoma cell lines following irradiation. Moreover, siRNA-mediated attenuation of NONO expression led to inhibition of melanoma cell proliferation. We are currently investigating NONO expression patterns of human melanoma specimens (stage I to IV) in order to explore NONO potential role as a useful prognostic biomarker for melanoma. Citation Format: Shuyi Li, Fengjue Shu, Mohammad K. Khan, Brian P. Pollack, Zhentian Li, Morgan McLemore, William S. Dynan. Investigating the function of NONO, a novel double strand break repair factor, and exploring its potential role as a biomarker for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2015-3027