Abstract
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.
A Small-Molecule Inhibitor of WEE1, AZD1775, Synergizes with Olaparib by Impairing Homologous Recombination and Enhancing DNA Damage and Apoptosis in Acute Leukemia
