Cyclin Ds are key regulators of the cell cycle that are frequently over-expressed in multiple myeloma and leukemia and act to promote the phosphorylation of Rb, thereby facilitating the transition from G1 to S phase. Over-expression of cyclin Ds increases cell proliferation and chemoresistance. In contrast, reducing cyclin Ds levels directly or indirectly through G1 arrest can decrease cellular proliferation and induce apoptosis. To identify novel pharmaceutical inhibitors of cyclin D transactivation, we screened the LOPAC and Prestwick libraries of drugs and natural compounds (n = 2400) using NIH 3T3 cells stably expressing the Cyclin D2 promoter-driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Cyproheptadine as a novel inhibitor of Cyclin D2 transactivation. Cyproheptadine has been used previously for the treatment of atopic dermatitis and anorexia, but its ability to inhibit cyclin D expression has not previously been reported. By immunoblotting, Cyproheptadine decreased expression of cyclin D1, D2 and D3 proteins in human myeloma and leukemia cell lines at low micromolar concentrations. Consistent with effects on the cyclin Ds, Cyproheptadine arrested cells in the G1 phase at concentrations associated with reduction in cyclin D expression. Decreased cyclin D expression and G1 arrest can induce apoptosis, so we tested the effects of Cyproheptadine on cell viability. Myeloma and leukemia cell lines were treated with increasing concentrations of Cyproheptadine and viability was measured by the MTS assay. Cyproheptadine reduced the viability of 7/10 myeloma and 7/8 AML cells lines with an IC50 ranging from 10–25μM. In contrast, it was less toxic to HeLa or NIH3T3 cells with IC20 > 50 μM. Cyproheptadine also reduced the viability of primary myeloma (8/8) and AML patient samples (7/9) with an IC50 <25 μM, but was less toxic to normal hematopoietic cells (IC20 > 50 μM). In a MDAY-D2 mouse model of leukemia, treatment with Cyproheptadine (50mg/kg/d) abolished formation of malignant leukemic ascites without untoward toxicity. Cyproheptadine-induced cell death was associated with reductions in mitochondrial membrane potential. Furthermore, reductions of pro-caspases -3 and -9 were observed prior to the reduction in pro-caspase-8, indicating that Cyproheptadine activates the mitochondrial pathway of caspase activation. Cyproheptadine is a known H1 histamine and serotonin receptor inhibitor, but pre-incubation with histamine, serotonin, or a combination of histamine and serotonin did not abrogate Cyproheptadine-induced cell death. Moreover, the structurally related H1 receptor inhibitor loratadine did not decrease cyclin D expression or reduce cell viability. Therefore, the pro-apoptotic activity of Cyproheptadine is not due to a competitive inhibition of the H1 and/or serotonin receptors, suggesting that Cyproheptadine has additional targets. In summary, Cyproheptadine arrests cells in G1, reduces cyclin D expression, and induces apoptosis via the mitochondrial pathway of caspase activation. Given the prior safety and toxicity record of Cyproheptadine, this drug could be rapidly advanced into clinical trial for the treatment of hematologic malignancies.
Disclosure:Research Funding: Multiple Myeloma Research Foundation.