Targeting Non-Canonical NFκB2 to Determine the Effects on Radiation Sensitization

Abstract

<h3>Purpose/Objective(s)</h3> Homologous recombination (HR) provides repair of DNA double stranded breaks and lesion that block DNA replication. While non-homologous end joining (NHEJ) proficiency is commonly attributed to tumor cell resistance to radiation, HR also plays a role. This is supported by the observation that simultaneous disruption of both pathways makes cells more sensitive to radiation than either pathway alone. Thus, HR inhibition may improve radiation sensitization. Since NF-κB is known to influence HR gene expression, we hypothesize that targeting NF-κB signaling pathways, specifically non-canonical signaling, may cause radiation sensitization. <h3>Materials/Methods</h3> The Cancer Cell Line Encyclopedia (CCLE) was used to evaluated <b>NFKB2</b> gene expression levels in various cell lines. Immunofluorescence, using an NFKB2 antibody (Santa Cruz sc-7386), was performed in several cell lines (including NHI-H650, A-172, MCF-7, and SK-N-BE2) to compare total NFKB2 protein expression level and nuclear protein expression level to CCLE data. Cells were irradiated using an irradiator at radiation doses between 0.0 Gy and 8.0 Gy. Cells were fixed 24 hours after irradiation and immunofluorescence was performed using NFKB2 antibody. We performed CRISPR-Cas mutagenesis to generate U2OS clones, which carry null mutations in either <b>NFKB2</b> (encodes p100/p52) or <b>Relb</b>. We used U2OS cells with a safe harbor control locus for a control cell line. Surviving cell fraction was determined after a single fraction of radiation at 2.0 Gy by using a fixable viability staining kit. <h3>Results</h3> <b>NFKB2</b> gene expression corresponds with total non-canonical NF-κB factor p52 protein expression and nuclear p52 protein expression at baseline when results from the Cancer Cell Line Encyclopedia (CCLE) are compared to p52 immunofluorescent protein expression levels. We find nuclear protein expression of p52 increases with increasing doses of radiation from 0.0 Gy-8.0 Gy. We compared a CRISPR knockout of <b>NFKB2</b> and <b>Relb</b> in a U2OS cell line to a U2OS cell line with a safe harbor control. We found that the surviving fraction of cells is decreased in the <b>NFKB2</b> CRISPR knockout compared to the safe harbor control when given 2.0 Gy of radiation and examined 24 hours later (<i>P</i> = 0.034), but is not decreased in the <b>Relb</b> CRISPR knockout (<i>P</i> = 0.558). <h3>Conclusion</h3> NFKB2 total and nuclear protein expression increases as radiation dose increases. Knocking out <b>NFKB2</b> causes radiation sensitization, suggesting that NFKB2 may be a useful target for future cancer therapies.