Multiple myeloma (MM) is an incurable cancer of antibody-producing plasma cells. A prominent feature of MM cells is chromosomal instability, often leading to chemotolerance, disease progression and poor prognosis. Although the mechanisms underlying genomic instability are not well understood, deranged DNA damage repair pathways seem to play a significant role, as myeloma cells display pervasive DNA double-strand breaks (DSBs), whose unfaithful repair may contribute to chromosomal instability. Of the two major DSB repair pathways, RAD51-mediated homologous recombination (HR) is elevated in MM cells and is commensurate with genomic instability and disease progression; whereas impaired nonhomologous end-joining has been found. MM cells may thus rely heavily on RAD51-dependent HR for survival, compared with normal cells. In this dissertation, I hypothesized that targeting RAD51-mediated HR will enhance the efficacy of DNA-damaging chemotherapy agents in MM.
I first targeted transcription of the RAD51 gene with a peptide nucleic acid, and then used a RAD51-specific small-molecule inhibitor, B02, to block the RAD51 recombinase activity. Since cyclin-dependent kinases (CDKs) are deregulated in MM and they can promote RAD51-mediated HR, I also used a CDK inhibitor, dinaciclib, to block these CDKs. I used qRT-PCR and immunoblotting to examine the effects of these treatments on the expression HR repair factors. Immunofluorescence microscopy and a chromosomal DSB repair reporter system for HR, the I-SceI/DR-GFP construct, were used to assess their effects on the recruitment of HR repair factors to DSB sites and HR repair of DSBs, respectively. I used in vitro cell culture systems and in vivo mouse models of MM to study their anti-myeloma effects.
These strategies effectively reduced DNA damage repair by HR, as indicated by reduced expression of HR repair factors including RAD51 and/or their impaired focus formation at DSB sites, and reduced HR repair of chromosomal DSBs. Consequently, the efficacy of anti-myeloma agents including melphalan, doxorubicin and the poly-ADP-ribose-polymerase inhibitor ABT-888, was enhanced by these strategies, respectively.
These studies provide proof-of-principle that RAD51 is a valid therapeutic target in MM and that inhibition of CDKs is an effective means to extend the use of PARP inhibitors to HR-proficient cancers such as MM.
|Advisor:||Shmookler Reis, Robert J.|
|Commitee:||Barger, Steven W., Haun, Randy S., Suva, Larry J., Yaccoby, Shmuel|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 76/09(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Oncology|
|Keywords:||Chemoresistance, Chemotherapy, Dna repair, Homologous recombination, Multiple myeloma, Rad51|
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