Hedley, David W.

Abstract The importance of aberrant signal transduction in the development and progression of cancers including multiple myeloma (MM) is well recognized, although the detailed regulation of signaling networks in relation to oncogenic mutations remains incompletely understood. However, this subject is becoming increasing relevant to clinical oncology due the rapid development of biological-based therapies that can target signaling pathways. Analytical methods that can be applied to clinical samples to measure complex populations of cells and phenotype them for multiple activation states are now feasible due to the recent advances in intracellular signal techniques, flow cytometry and phospho-antibodies. Using multi-parameter flow cytometry, we monitored phospho-protein responses to activators/cytokines and signal transduction inhibitors in a panel of 14 extensively characterized human MM cell lines with heterogeneous molecular abnormalities. A panel of 4-phospho-specific antibodies: anti-pS6, anti-ERK1/2, anti-pAKT and anti-pSTAT3 that represent downstream target genes of signaling pathways known to play central roles in myelomagenesis were used in the initial analysis. Sixteen conditions (basal, inhibitor, activator alone, activator and inhibitor) were studied to generated 64 readouts designed to survey altered signal transduction in the 14 cell lines. We collected data on unstimulated cells and cells stimulated for 7–10 min with IL-6, IGF-1 or FGF or cells inhibited with the signal transduction inhibitors U0126 (MEK), rapamycin (mTOR) or LY294002 (PI3-K). Repeat measurements were collected and the technique and monoclonal antibodies displayed a high level of reproducibly. In contrast, the basal, cytokine and inhibitor responses between cell lines varied considerably reflecting the molecular heterogeneity at the level of signaling responses. The protocols that have been refined and validated in myeloma cell lines are now being applied to primary bone marrow samples from MM patients. Although the same size thus far is small, phospho-protein responses among primary CD138 positive myeloma cells show considerable induction and variance of AKT, MAPK, STAT3 and pS6 phosphorylation. In some MM tumor samples treatment with inhibitors suggest constitutive activation of these signaling pathways while in others the nodes remain activable with phosphorylation above the basal state following stimulation. The data further suggests that MAPK phosphorylation following aFGF stimulation displays significant variance among MM samples and correlates with the detection of t(4;14) translocation by FISH analysis. Additional samples are being assessed and correlation of phospho-protein responses with clinical parameters and cytogenetics will be reported. The data demonstrate that multi-parameter flow cytometry can be applied to myeloma tumor samples to study the phospho-proteome and that considerable heterogeneity exists at the level of signaling responses.

Abstract 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.

AbstractD-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G0/G1 phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.