Instructor Dana-Farber Cancer Institute, United States
Introduction: Multiple myeloma (MM) is associated with significant DNA damage and genomic instability, enabling them to acquire new characteristics for growth and disease progression. We previously identified apurinic/apyrimidinic nuclease 1 (APEX1) as part of a gene signature that correlated with genomic instability in MM cells. APEX1 is best known for its critical role in the base-excision repair pathway. However, APEX1 is also known to participate in transcriptional regulation. In multiple myeloma cells, APEX1 binds to P73 at the RAD51 promotor to regulate the expression of RAD51, a key factor in homologous DNA repair (HR).
Methods: To find APEX1-interaction sites throughout the myeloma genome, we did Chromatin Immuno-precipitation (IP) with APEX1 antibody followed by sequencing (ChIP-seq). To find APEX1 and RAD51 proteomes in myeloma cells, we performed IP using APEX1, RAD51 or IgG followed by mass spectrometry.
Results: Using ChIP-seq, we found that ~60% of the APEX1 interaction sites are in the expressed regions of the genome i.e., in promoters, exons, introns or UTRs. APEX1 was predominantly bound to genes involved in immune function, growth, and cancer-related pathways. Interestingly, treatment with camptothecin (CPT), a chemical that induces double-strand DNA breaks, redirected APEX1 to genes involved in the cell cycle, DNA repair, mTOR signaling and cancer-related pathways. Some of the genes where APEX1 peaks appeared were TP53 (involved in DNA repair and apoptosis), GADD 45B (involved in DNA repair, cell cycle and the ability of the cell to cope with genotoxic stress), LMNA (involved in the maintenance of chromatin structure, telomeres, DNA repair and gene expression), IKBKB (involved in immune response, growth control) and MAP2K1 (involved in growth, adhesion, survival and differentiation). Since coordination among cell cycle, DNA repair and apoptosis pathways is required for regulating growth and genome stability, our data suggest a possible role of APEX1 in the coordination of these vital processes. We previously demonstrated that elevated APEX1 impacts genome stability through dysregulation of base excision repair as well as HR. To further study the mechanisms and protein networks involved in APEX-related dysregulation of HR and genomic instability, we investigated proteins interacting with APEX1 and RAD51. Thirteen proteins were common in APEX1 and RAD51 proteomes. For example, PARP1, which contributes to both single and double stranded DNA break repair (through recruitment of BRCA1), is part of both APEX1 and RAD51 proteomes. This further confirms the functional link between APEX1 (base excision repair) and RAD51 (HR) related pathways.
Conclusions: Our data suggest that APEX1, in addition to its canonical DNA repair function, is also involved in the regulation of the cell cycle, apoptosis and multiple DNA repair pathways and may assist in coordination of these vital processes. Thus, inhibitors of APEX1 have the potential to target genomic instability and growth of MM cells.
