We have made some exciting observations during this interim period that hopefully will have translational applications for the targeted therapy of cyclin D1 and D3 (+) myeloma. Our previous results funded by the IMF used gene targeting to ablate cyclin D1 expression in multiple myeloma cells (U266). The complete elimination of cyclin D1 expression in myeloma and mantle cell lymphoma cell lines was associated with a phenotype of increased growth in vitro and tumorigenicity in vivo. Western analysis revealed a dramatic increase in cyclin D3 protein in cyclin D1(-) cells. These results demonstrate ablation of cyclin D1 expression has little or no effect on the in vitro growth of MM and MCL cells. This is apparently due to the compensatory upregulation of cyclin D3 expression, primarily at the translational or posttranslational level. These results suggested that potential therapeutic agents directed at cyclin D1 may not be effective if compensatory upregulation of cyclin D3 occurs. We tested several classes of agents for their ability to inhibit cell growth in vitro and cyclin D1 and D3 expression. A recent report in Blood suggested that iron chelating agents may be capable of inhibiting cyclin D1 and D3 expression. The effects of desferroxamine (desferal) was tested and at potentially achievable drug concentrations cell growth of cyclin D1 (+) and cyclin D3 (+) multiple myeloma and mantle cell lymphoma cell lines was inhibited. Interestingly, the molecular mechanism by which this drug causes growth arrest on these cell lines may be different in cyclin D1 vs. D3 (+) cell lines. Flow cytometry shows that desferal causes a G1 and G2 arrest in cyclin D1 (+) cell lines and apoptosis in cyclin D3 (+) cell lines. A Burkitt’s lymphoma line overexpressing c-myc (Manca) and a bcr-abl overexpressing human leukemia cell line (K562) were not growth inhibited by desferal, suggesting that this effect may be specific to cyclin D1 or D3 dependent cell lines. Further experiments to define the mechanism of action of desferal are under way including RT-PCR and Western blotting for cyclins D1 and D3 as well as p21 and p52. We are also initiating experiments in SCID mice to test the effects of desferal on in vivo tumorigenicity in cyclin D1 and D3 (+) myeloma cell lines.
2. Structural studies of histone acetylation and DNA methylation around the cyclin D1 locus.
We have continuing to investigate the chromatin structure and patterns of DNA methylation in cell lines derived from patients with the (11:14) translocation as well as controls. Using restriction enzyme analysis with HpaII/Msp followed by Southern blotting, the DNA in this region appears fully unmethylated in MCL and MM (U266) cell lines. Further studies including MM lines with or without the 11:14 translocation are continuing. Using antibodies to acetylated histones H3 and H4, we have begun to define the role of histone acetylation in IgH mediated cyclin D1 deregulation. We have found that the cyclin D1 expression in B cell malignancies is correlated with DNA hypomethylation and histone hyperacetylation at the cyclin D1 promoter. The MTC region was hypoacetylated and under methylated in all cell lines regardless of expression status. These results suggest local promoter hyperacetylation and DNA hypomethylation correlate with cyclin D1 transcription. We are continuing to investigate the chromatin structure and patterns of histone acetylation in the chromosome region.
3. Gene targeting studies into multiple myeloma cell lines.
We are continuing our gene targeting studies into multiple myeloma cell line that contain deregulated expression of cyclin D1, D3, c-maf, and FGFR3. Several of these lines have proven difficult to electroporate. We have successfully used lipofection to transfect the cyclin D3 expressing KMM1 cell line and colonies are being expanded and screened currently. Other myeloma lines are being transfected using this technique.