Hurren, Rose

Abstract Proteasome inhibitors are used in the treatment of myeloma and are under evaluation for the treatment of other malignancies including acute myeloid leukemia (AML). Via high throughput screening for inhibitors of cyclin D2 transactivation, we identified the off-patent antimicrobial, Clioquinol (CQ) that we subsequently determined acted by inhibiting the proteasome. CQ is a copper-binding halogenated 8-hydroxyquinoline that was used in the 1950’s-1970’s as an oral anti-parasitic agent. We determined that CQ decreased expression of cyclin D2, arrested cells in the G1 phase, and increased expression of p21, p27 and the abundance of ubiquitinated proteins. This constellation of changes can be associated with inhibition of the proteasome, so we evaluated the effects of CQ on proteasomal function. AML and myeloma cell lines as well as primary AML and normal hematopoietic cells were incubated with increasing concentrations of CQ. After a 24 hour incubation, cell lysates were prepared and the chymotrypsin-like activity of the proteasome was measured by monitoring the rate of cleavage of the fluorescent substrate Suc-LLVY-AMC. In cell lines and primary AML samples, CQ inhibited chymotrypsin-like activity at low micromolar concentrations and at concentrations associated with reductions in cyclin D2, G1 arrest, and increased p21 and p27 expression. Conversely, CQ did not inhibit the proteasome in normal hematopoietic cells up to 100 μM. As CQ is a copper-binding molecule, we tested the requirement of copper for its effects on the proteasome. Supplementing CQ with copper increased CQ’s potency as a proteasome inhibitor, but also negated its preferential inhibition of the proteasome in malignant cells over normal hematopoietic cells. Of note, copper alone had no effect on proteasomal function when added to intact cells. Since inhibiting the proteasome induces cell death, we tested the effects of CQ on the viability of AML (n=6), myeloma (n=14), and solid tumor (n=5) cell lines as well as primary AML samples (n=6) and primary normal hematopoietic cells (n=3). Forty-eight hours after incubation, CQ induced cell death in 6/6 AML, 12/14 myeloma, 0/5 solid tumor, 6/7 primary AML patient samples and 0/3 normal hematopoietic cells with an IC50 <20 μM. Of note, after oral administration of CQ, trough serum concentrations of 20 μM can be achieved in patients. CQ-mediated reductions in cell viability were associated with its ability to inhibit the proteosome, as no proteasomal inhibition was detected in CQ-resistant cell lines. CQ-induced cell death was copper-dependent as supplementing copper in the medium enhanced the toxicity of CQ, while adding the strong copper chelator, tetrathiomoylbdate, abrogated CQ-induced cell death. Given the effects of CQ on AML cell lines and primary patient samples, we evaluated CQ in a mouse model of leukemia. Twice daily oral gavage with CQ prevented the growth of MDAY-D2 leukemia cells in DBA2 mice without evidence of toxicity. Thus, we have identified the off-patent anti-microbial agent CQ that inhibits the proteasome and induces apoptosis preferentially in malignant cells at clinically achievable concentrations. It acts through a copper-dependent mechanism and leverages the higher copper levels present in malignant cell. As such, CQ could be rapidly advanced into clinical trial as an oral proteasome inhibitor for the treatment of AML and myeloma.

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.

Abstract The oncogene c-maf is frequently over-expressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. Therefore, small molecules that inhibit c-maf and its targets could be useful chemical probes to better understand the role and regulation of this protein. We developed a high throughput chemical screen in NIH 3T3 cells stably over-expressing the promoter of the c-maf target cyclin D2 driving firefly luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified 32 compounds that preferentially reduced cyclin D2 transactivation. Of these, 24 of the 32 hits belonged to the corticosteroid family of drugs. Indeed, the screen identified 24 of the 26 corticosteroids in the library. The most potent inhibitors were glucocorticoids such as dexamethasone. Mineralocorticoids such as fludrocortisone were weak hits, reflecting their weak glucocorticoid activity. The 24 glucocorticoids identified in this screen preferentially reduced cyclin D2 transactivation in the presence of c-maf. For example, the IC50 of dexamethasone was 11 ± 0.7 nM and >50 uM in NIH 3T3 cell with and without c-maf, respectively. Given the effects of glucocorticoids on c-maf in NIH 3T3 cells, we extended our investigation to multiple myeloma cell lines and demonstrated that nanomolar concentrations of the glucocorticoid dexamethasone reduced levels of c-maf protein and its target cyclin D2 within 6 hours of treatment. C-Maf was down regulated in isogenic MM1.S but not MM1.R cells respectively sensitive and resistant to dexamethasone. We also observed reductions in another c-maf target, beta integrin. Compared to cell lines RPMI 8226 and OCIMY5 that harbor the t(14;16) c-maf translocation, the concentration of dexamethasone required to reduce c-maf was approximately 50-fold lower in cell lines such as LP1 and OPM1 that lack the translocation but over-express c-maf. While dexamethasone reduced c-maf protein, no changes in levels of c-maf mRNA were detected. In both multiple myeloma and NIH3T3 cells, dexamethasone increased the ubiquitin-dependent destruction of c-maf. Finally, we linked glucocorticoids to c-maf ubiquitination by demonstrating that dexamethasone upregulated ubiquitin C mRNA at concentrations associated with the ubiquitination of c-maf. Moreover, ectopic expression of ubiquitin cDNA recapitulated the effects of dexamethasone and reduced levels of c-maf, suggesting that increased expression of ubiquitin C by dexamethasone is functionally important for dexamethasone’s effects on c-maf levels. Conclusion: a chemical biology screen identified glucocorticoids as c-maf dependent inhibitors of cyclin D2 transactivation. Glucocorticoids reduce c-maf by promoting its ubiquitination via the upregulation of ubiquitin C mRNA. This work provides new insights into the regulation of c-maf and has identified a novel mechanism by which glucocorticoids exert an anti-myeloma effect.

Key Points HERC4 is the first identified ubiquitin ligase that mediates c-Maf ubiquitination and degradation. HERC4 suppresses MM cell proliferation and delays MM tumor growth.

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.

AbstractThe oncogene c-maf is frequently overexpressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. To identify regulators of c-maf, we developed a chemical screen in NIH3T3 cells stably overexpressing c-maf and the cyclin D2 promoter driving luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified glucocorticoids as c-maf–dependent inhibitors of cyclin D2 transactivation. In multiple myeloma cell lines, glucocorticoids reduced levels of c-maf protein without influencing corresponding mRNA levels. Subsequent studies demonstrated that glucocorticoids increased ubiquitination-dependent degradation of c-maf and up-regulated ubiquitin C mRNA. Moreover, ectopic expression of ubiquitin C recapitulated the effects of glucocorticoids, demonstrating regulation of c-maf protein through the abundance of the ubiquitin substrate. Thus, using a chemical biology approach, we identified a novel mechanism of action of glucocorticoids and a novel mechanism by which levels of c-maf protein are regulated by the abundance of the ubiquitin substrate.