Targeting of Non-canonical NF-κB Factor p100/p52 Inhibits Homologous Recombination and Sensitizes Cancer Cells to DNA-damaging Therapy

Abstract

Homologous recombination (HR) repairs DNA double strand breaks (DSBs) and promotes tolerance of replication-blocking lesions. Cells deficient in HR are hypersensitive to radiation and DNA-damaging chemotherapies. The central recombinase protein, RAD51, is frequently overexpressed in human malignancies, thereby elevating HR efficiency and promoting resistance to DNA-damaging therapies. Our preliminary data demonstrate that non-canonical NF-κB factors control RAD51 expression, suggesting that the non-canonical NF-κB pathway may represent a therapeutic target to reverse RAD51 overexpression in tumors, rendering them susceptible to DNA-damaging treatments including radiotherapy. NFKB1, which encodes the central non-canonical NF-κB factor proteins p100/p52, was transcriptionally targeted using pools of siRNAs. Parallel experiments were performed using various human cells lines derived from a wide range of malignancies, including prostate (PC-3), osteosarcoma (U2OS), kidney (HEK-293), and colorectal carcinomas (DLD-1). Two such cell lines (U2OS and HEK-293) carry integrated reporter constructs that quantify the proficiency of HR, as well as related DSB repair pathways including single strand annealing (SSA), micro-homology mediated end joining (MMEJ), and non-homologous end joining (NHEJ). Transcriptional silencing of p100/p52 reduces RAD51 protein levels in multiple human cancer cells lines. Whole transcriptome sequencing of U2OS cells following treatment with siNFKB1 demonstrates that RAD51 is the most differentially expressed gene (4.5-fold downregulated, p<10-46) out of 40 select genes with known relevance to HR. The functional impact of p52-dependent signaling on HR proficiency was measured using the DR-GFP assay after treatment with siNFKB1. We find that siNFKB2 reduces HR proficiency by 60.6% in U2OS cells and by 58.3% in HEK-293 cells. By contrast, the proficiency of SSA (p = 0.32), MMEJ (p = 0.62), and NHEJ (p = 0.29) are unaffected by siNFKB2. Clonogenic survival assays were performed using CHO or DLD-1 cell lines that differ only in their expression of a key HR protein (XRCC3 or BRCA2, respectively). In both cell pairs, targeted disruption of NFKB2 sensitizes the HR-competent cells to either camptothecin or radiation. However, sensitization is absent in HR-incompetent control cells. The non-canonical NF-κB factor p100/p52 is a key transcriptional regulator of DNA repair proteins, most notably, the RAD51 recombinase that promotes HR. Inhibition of p100/p52-dependent signaling leads to a significant reduction HR activity in cancer cells, thereby promoting sensitization to DNA-damaging oncologic treatment.