Perturbation of Genomic Instability by Wortmannin in Myeloma.

Abstract 1105

Poster Board I-127

Genetic recombination plays a critical role in telomere maintenance, chromosomal translocation, and gene amplification, and may therefore underlie the chromosomal aberrations observed with high frequency in number of malignancies. The molecular mechanism/s inducing genomic instability remains ill-defined and their elucidation may provide methods to prevent tumor progression and development of drug resistance. Our earlier work has demonstrated that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to increased rate of mutation and progressive accumulation of genetic variation over time. We have further demonstrated that the inhibition of HR activity in MM cells by siRNAs targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome; and conversely, induction of HR activity leads to significant elevation in the number of new mutations over time, and development of drug resistance in MM cells. Here we have evaluated a PI3K inhibitor Wortmaninin which has significant inhibitory activity against both HR and non-HR (nHR) pathways. Exposure of MM cells (OPM1, ARP and RPMI 8226) to wortmannin (WM) led to reduced expression of recombinase (hsRAD51) and nearly complete inhibition of HR activity, within 24 hrs as determined by a plasmid based assay in which generation of active gene product by recombination is measured. Similarly nHR was evaluated by measuring generation of intact gene product from a linearized plasmid. We evaluated effect of WM on nHR by 3 hours preincubation before transfecting the plasmid followed by cell culture for 72 hrs at 37° C. Cells were harvested and analyzed for nHR as previously described. Treatment with WM led to >40% reduction in nHR, indicating that WM affects both HR and NHR pathways. Downregulation of these pathways by wortmannin was also associated with a reduced growth rate of myeloma cells in culture by 20-25% at 48 hours. Importantly, WM treatment markedly decreased the acquisition of new genomic changes in MM as measured by genome-wide loss of heterozygosity assay as an indicator of genomic stability. To evaluate the impact of WM on in vivo tumor growth, OPM1 cells were injected subcutaneously in SCID mice and following appearance of palpable tumors, mice were treated with WM at 0.75 mg/kg, injecting daily intraperitoneally. Treatment with WM was associated with almost complete inhibition of tumor growth in vivo. Long term exposure of myeloma cells to WM was consistently associated with reduced telomere length, probably by blocking HR dependent ALT pathway. These data identifies dysregulated recombination activity as a key mediator of DNA instability and progression of MM, and WM as a potential therapeutic agent for prevention of myeloma progression and possibly drug resistance.