Base Excision Repair and Homologous Recombination Pathway Intermediates Drive Genomic Instability and Evolution in Myeloma

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. Disclosures 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.

Elevated APE1 Mediates Dysregulation of Homologous Recombination in Myeloma: Mechanisms and Translational Significance

We have previously shown that elevated homologous recombination (HR) activity mediates genomic instability and progression in myeloma. Moreover, elevated HR also plays critical role in tumor growth by contributing to telomere maintenance and other survival mechanisms. We have now investigated molecular mechanisms driving dysregulated HR in MM. We observe that elevated apurinic apyrimidic endonuclease 1 (APE1) significantly contributes to dysregulation of HR, directly through transcriptional control of RAD51 as well as indirectly through its ability to induce DNA breaks. The transgenic suppression using APE1-specifc shRNA inhibits RAD51 expression, HR activity, and genomic instability as measured by SNP array profile in MM cells; whereas its induction leads to increased RAD51 expression, HR activity, genomic instability and oncogenic transformation in normal human cells. We have further investigated how APE1, a base excision repair protein, regulates RAD51, the key component of HR in myeloma and evaluated a novel small molecule inhibitor of APE1 for its impact on HR and associated genomic instability. Using an antibody array we observed that APE1 physically interacts with p73, a known transcriptional regulator of RAD51. To demonstrate that APE1 and P73 interact with RAD51 promoter in MM cells, we conducted chromatin immunoprecipitation (chip) assays and observed both P73 and APE1 binding to adjacent loci on RAD51 promoter. Taken together, these data suggest that elevated APE1 induces RAD51 expression through its interaction with P73. We next evaluated effect of a small molecule inhibitor specifically targeting nuclease function of APE1 in MM cells, and observed that it inhibits RAD51 expression, RAD51 foci, HR activity and reduces DNA breaks as assessed by g-H2AX levels on western blotting. The suppression of APE1 by this small molecule was associated with significant loss of RAD51 promoter activity, as assessed by a RAD51-promoter driven luciferase construct, as well as reduced RAD51 transcript levels. As APE1 is required for DNA repair which plays a critical part in development of drug resistance, we evaluated if APE1 inhibitor can help sensitize MM cells to DNA damaging agents. To investigate this we pretreated RPMI8226 and LR5 MM cells with the small molecule inhibitor of APE1 and then exposed them to various concentrations of melphalan for 48 hrs and cell viability and growth assessed. Pretreatment with APE1 inhibitor not only sensitized RPMI8226 cells to melphalan but also resistant LR5 cell line. These observations suggest that elevated APE1 is a critical target to induce DNA damage or overcome certain type of resistance possibly driven by repair mechanisms. In summary, we conclude that elevated APE1 is a critical intermediate for dysregulated HR and associated genomic instability, and small molecule inhibitor of APE1 has potential to reduce genomic instability, prevent/delay progression and improve clinical outcome in MM. Disclosures No relevant conflicts of interest to declare.

Critical Role of Recombinase (HsRAD51) in Genetic Instability in Multiple Myeloma.

A number of cancers, including multiple myeloma (MM), show a significant genetic instability that evolves with the progression of disease. The molecular basis for the generation of genetic diversity in cancer cells has therefore emerged as an important focus of investigation. We have previously shown that homologous recombination (HR), required for a variety of normal cellular processes, is dysregulated in multiple myeloma and leads to a progressive accumulation of genetic variation over time. Moreover the inhibition or stimulation of HR activity in MM cells, by altering components of the HR pathway, concordantly affects the acquisition of new genetic changes. Consistent with this, an elevated frequency of cancer is observed in patients whose cells have elevated rate of recombination. HsRAD51 recombinase, is a key molecule in HR pathway and its dysregulated expression can increase in HR activity, leading to increased rate of mutations and development or progression of cancer. Based on these data, we hypothesized that hsRAD51 promoter activity may be a target for carcinogenic agents and ability to measure the hsRAD51 promoter activity may provide a simpler method to assess oncogenic potential of test agents. To this end we constructed a mammalian expression vector containing firefly luciferase gene downstream of hsRAD51 promoter. This plasmid was transfected into normal human plasma cells, multiple myeloma cell lines (ARP, OPM1, U266), and normal human diploid fibroblasts. The cells were allowed to recover for 24 hrs and then treated with agents with carcinogenic or anti-oxidant activities, or control vehicles for 2 hrs; lysed and luciferase activity assessed. RAD51 promoter activity was elevated 7- to 1000-fold in MM cells relative to normal plasma cells. Exposure of MM cells to a carcinogen (nickel chloride) was able to further induce hsRAD51 promoter activity (2-fold) in MM cells with already over-expression of hsRAD51. However, the anti-oxidant tocopherol and cannabinol were able to reduce hsRAD51 promoter activity by 3- and 8-fold respectively, while vitamin C and Selenium had minimal effects. In normal diploid fibroblasts, although the exposure to a non-carcinogen, Sodium azide did not induce hsRAD51 promoter activity, the exposure to agents with known transforming ability, Mitomycin C and Nickel chloride, led to a significant induction of hsRAD51 promoter activity (2.5 and 3.5-fold respectively). Moreover, the ability of these chemicals to induce RAD51 promoter activity covaried with their ability to induce homologous recombination activity as measured by a novel plasmid based assay (r = 0.9). We also demonstrate that Induction of RAD51 and HR or its inhibition using siRNAs targeting hsRAD51 can inhibit acquisition of new mutations in MM cells as measured by change in genome-wide loss of heterozygosity (LOH) using the 100K single nucleotide polymorphism arrays. These preliminary results provides us with a simple and powerful method to measure oncogenic potential of known carcinogens and demonstrate a critical role for RAD51 in ongoing genomic instability in MM and a rationale to target RAD51 to prevent progression.