Multiple myeloma (MM) cells demonstrate significant genomic instability with acquisition of new genomic events over time. In an effort to decipher the pathways utilized by MM cells to genomically evolve and to acquire proliferative advantage as well as develop drug resistance, we have investigated role of various pathway intermediates. Based on our prior results showing elevated homologous recombination (HR) contributing to genomic instability and development of drug resistance, we have further studied RAD51, the key HR gene. Also based on the dysfunctional base excision repair (BER) that can increase DNA damage and dysregulate genome stability, we have investigated apurinic/apyrimidinic endonuclease (APEX)1/2 in MM.
We first observed that both APEX1 and APEX2 interact with multiple HR genes in MM. APEX1 interacts with P73, a known transcriptional regulator of RAD51 at RAD51 promoter, and contributes to its transcriptional upregulation and increase in HR activity. To further extend these observations, we conducted sequential chromatin immunoprecipitation (ChIP-reChIP) assays, using MM.1S cells. For the first ChIP, anti-APEX1 and for the reChIP anti-p73 antibody was used. DNA recovered was used for real-time Q-PCR assays, using promoter-specific primers. Colocalization of APEX1 and p73 was thus confirmed on the promoters of three HR related genes (RAD51, BRCA1 and BLM), whereas interaction was not observed for CDK2 promoter. These data suggested that APEX1 may be involved in the overall regulation of HR machinery. To investigate the role of APEX1 in genomewide transcriptional regulation in the context of HR, we cultured MM (H929 and MM.1S) cells in the presence or absence of camptothecin (CPT; a topoisomerase I inhibitor that induces DSBs which are mainly repaired by HR), and conducted ChIP assays using anti-APEX1 antibody followed by DNA sequencing. We have confirmed the interaction of APEX1 with RAD51 promoter and the impact of CPT, using Q-PCR and observe that the occupancy of RAD51 promoter by APEX1 increases following treatment with CPT. The sequencing data to be presented will identify genomic regions occupied by APEX1 under spontaneous as well as modulated HR conditions.
We have further confirmed interrelationship between these two key pathway intermediates in MM. We observe that overexpression of APEX1 increases abasic sites in MM as well as another cancer (esophageal adenocarcinoma) cells, while RAD51-knockdown in these cells prevents the increase in abasic sites by APEX1. Similarly, APEX1-overexpression increased HR activity which was prevented by RAD51 inhibition, indicating that APEX-induced RAD51 contributes to dysregulation of both the HR and base excision repair.We also demonstrated thatAPEX-overexpression in normal hematopoietic cells increased HR and genomic instability (as assessed by micronuclei), which was reversed by RAD51 inhibitor. The treatment of control plasmid transfected CD138+ normal plasma cells with RAD51 inhibitor reduced micronuclei by 53.5%. Importantly, APEX1-overexpression in these cells increased micronuclei by 34.7%, whereas treatment of these cells with RAD51 inhibitor completely reversed this increase. Similarly, the treatment of bone marrow/stromal (HS5) cells with RAD51 inhibitor reduced micronuclei in APEX1-overexpressing cells by 55%.
This unique functional interaction between APEX nuclease and RAD51 also provides rationale for combining APEX inhibitors with chemotherapeutic agents. Consistently, we observe thatAPEX inhibition synergistically increases the efficacy of chemotherapeutic agents in MM cells. We also investigated the impact of simultaneous suppression of APEX1 and RAD51. Cancer cells (FLO-1) were transduced with control shRNAs or those targeting APEX1, RAD51 or both, and following puromycin selection evaluated for HR and evidence of apoptosis. Combined RAD51/APEX1 knockdown led to a significantly increased apoptosis (P=0.0003), relative to RAD51 or APEX1 inhibition alone.
In summary, we report an interesting interaction/crosstalk between 2 major DNA repair pathway intermediates, APEX1 and RAD51 recombinase, driving genomic evolution in MM. These results provide insight into processes driving genomic progression and suggest possible avenues to control evolution of the disease and its clinical consequences in MM.
Munshi:Janssen: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.
Asterisk with author names denotes non-ASH members.