RAD51 Is Required for the Occupancy of Replication Licensing Factors on the Myeloma Genome

Genomic instability and extended replicative potential arise early during oncogenic transformation and contribute to the development and progression of cancer. Although chromosomal instability and replication are distinct pathways, they share certain proteins and activities. DNA replication starts by sequential loading of replication licensing factors at replication origins. We have observed that RAD51 - a keystone protein in stabilizing the genome through homologous recombination (HR) and a dependency in multiple myeloma (MM) - interacts with the MCM complex. In fact, RAD51 knockdown inhibits DNA replication in MM cells. Because RAD51 organizes the initial steps of homologous recombination, we investigated whether RAD51 plays a similar setup role in replication - namely, that RAD51 is required for the occupancy or proper assembly of replication licensing factors (ORC1-ORC6, CDC6, CDT1, MCM2-7) on the MM genome.

To investigate this, we either inhibited (by knockdown or inhibitor) or overexpressed RAD51 in MM cells and monitored the occupancy of the replication complex on genomic DNA using ChIP with an antibody against MCM4. Subsequently, the proteins in the complex were released from DNA and identified by Western blotting. Although RAD51-KD (or inhibition) did not reduce the expression or stability of licensing factors, the occupancy of all these factors (MCM4, MCM2, MCM5, MCM6, MCM7, CDC6) on the MM genome was inhibited. Consistently, RAD51-overexpression led to an increase in the genomic occupancy of most of the licensing factors (ORC1, ORC2, MCM4, MCM2, MCM5, MCM6, MCM7, CDC6) tested. This was validated using antibodies against other members of the complex i.e., ORC1 and CDC6.

Overall, we demonstrate that RAD51 in MM cells interacts with components of the replicative protein complexes (ORC1-ORC6, CDC6, CDT1, MCM2-7) that license and ensure DNA replication once per cell cycle. Our data also demonstrate that RAD51 is required for the occupancy of most of these factors on genomic DNA in MM cells, which may explain its ability to impact DNA replication in MM cells. We are currently trying to understand if RAD51 brings one or more of these factors to the origin of replication and if some of its activities (such as ATPase, DNA binding) are involved in loading these factors onto DNA. Based on its impact both on replication and HR, RAD51 is an attractive target in myeloma. Our inhibitor studies provide further rationale to study therapeutic strategy to target RAD51 in MM.