The Novel Pan-HDAC Inhibitor CR2408 Inhibits Multiple Myeloma Cell Growth and Proliferation

Myeloma (MM) is still an incurable disease, and new cytotoxic drugs are urgently needed. CR2408 is a novel pan-histon deacetylases inhibitor with promising properties and effects in MM cells.

The effect of CR2408 in MM cells was characterized by multiple assays. HDAC inhibition was shown by western blotting as well as HDAC enzyme inhibition assays. Cell growth and viability was shown by the common WST-1 assay. Induction of apoptosis was detected using flow cytometry after annexin-V-FITC staining as well as caspase cleavage detected by western blotting. Basal and cytokine stimulated cell growth rates of myeloma cells were measured by the WST-1 assay. Myeloma cell proliferation was determined by the BrdU assay. Alterations of the cell cycle were determined by flow cytometry after staining with propidium iodide. Modulation of intracellular signalling was shown by western blotting.

We have found CR2408 to induce profound hyperacetylation of histone H4 in MM cells. Our experiments revealed that nanomolar concentrations of CR2408 abrogate HDAC activity in 11 HDAC enzymes. Comparison with SAHA shows lower IC50 values for CR2408 (HDAC1 27nM, HDAC2 76nM, HDAC3 28nM, HDAC4 151nM, HDAC5 51nM, HDAC6 13nM, HDAC7 360nM, HDAC8 522nM, HDAC9 92nM, HDAC10 77nM and HDAC11 56nM). CR2408 abrogated myeloma cell growth at nanomolar concentrations (250nM: NCI-H929: −93%; OPM-2: −85%; U266: −87%; RPMI-8226: −86%) and induced apoptosis in multiple myeloma cell lines and primary cells, as shown by the annexin V assay (500nM: NCI-H929: 75%, OPM-2: 65%, RPMI-8226: 80%, U266: 18%, primary cells: 50%). Induction of apoptosis was confirmed by showing cleavage of caspase 3, 8 and 9. Furthermore, increased cell growth induced by conditioned medium obtained from bone marrow stromal cells was abrogated by CR2408. The BrdU assay revealed that inhibition of cell growth was due to inhibition of myeloma cell proliferation (500nM: OPM-2: −50%; RPMI-8226: −58%, U266: −55%). Furthermore, we analysed cell cycle distribution and found that in contrast to other HDAC inhibitors, CD2408 does not provoke a G0/G1 cell cycle arrest but leads to immediate DNA and cell fragmentation, resulting in an accumulation of cell fragments in the subG1 phase. Inhibition of cell proliferation was accompanied by a strong downregulation of the proteins cdc25A, cdk4 and hypophosphorylation of RB. Incubation of myeloma cells with CR2408 did not alter the phosphorylation of 4E-BP-1, P70S6k, but the mitochondrial proteins Bad and Bcl-Xl were downregulated and Bim and pJNK were upregulated. Finally, CR2408 shows significant synergistic effects when combined with doxorubicin and bortezomib.

The HDAC inhibitor CR2408 inhibits MM cell proliferation and induces apoptosis. This is accompanied by a strong perturbation of mitochondrial proteins. Since CR2408 inhibit myeloma growth and proliferation as low nanomolar levels, this study provides the rationale for the further in vivo evaluation of CR2408 in order to find a more efficient and less toxic member of this group of compounds.

Stefan:4SC AG: Employment.