ALBERT

Description: Off-patent drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication given their prior testing for safety and toxicity. To identify such compounds, we developed, automated and conducted a cell-based chemical screen of 4800 off-patent drugs and chemicals. From this chemical screen, we identified the off-patent antimicrobial, ciclopirox olamine (CPX) that is currently used for the topical treatment of cutaneous fungal infections, but has not been previously evaluated as a systemic agent for the treatment of malignancy. As an anti-fungal agent, the mechanism of action of CPX is not well understood, but appears related to binding intracellular iron and inhibiting iron containing enzymes. To explore its efficacy and mechanism of action as an anticancer agent, leukemia, myeloma, and solid tumor cell lines were treated with increasing concentrations of CPX. 72 hours after incubation, cell viability was measured by the MTS assay. CPX decreased cell viability in 5/9 leukemia, 3/6 myeloma, and 3/5 solid tumor cells with an LD50 〈 5 uM, a concentration that is pharmacologically achievable based on prior animal studies investigating CPX as an anti-fungal. Cell death was confirmed by the presence of a subG1 peak by flow cytometry after staining cells with propidium iodide. In contrast, CPX was less toxic to MRC 5, LF1, and GMO 5757 non malignant fibroblasts with an LD50 〉 20 uM. Next, we evaluated CPX in combination with cytarabine and daunorubicin, standard chemotherapeutic agents used in the treatment of AML. In AML cell lines, CPX synergistically enhanced the cytotoxicity of cytarabine as determined by the median effect isobologram analysis. Specifically, the combination indices (CI) at the EC50, 75 and 90 were 0.18, 0.19, and 0.24, respectively, where a CI 〈 1 denotes synergy. In contrast, the addition of CPX to daunorubicin produced only additive effects. Given the effects in leukemia cells lines, we evaluated the effects of oral CPX in 3 mouse models of leukemia. Sublethally irradiated NOD-SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells or intraperitoneally with MDAY-D2 murine leukemia cells. After tumor implantation, mice were treated with CPX (25mg/kg) in water or water alone by oral gavage. Oral CPX decreased tumor weight and volume in all 3 mouse models by up to 65% compared to control without evidence of weight loss or gross organ toxicity. Mechanistically, CPX arrested cells in the G1/S phase of the cell cycle and downregulated the expression of survivin, Cyclin D1, and the transcription factors YY1 and FTII-D prior to the onset of cell death. Consistent with effects as an anti-fungal, CPX bound intracellular iron in the malignant cells and its ability to bind intracellular iron was functionally important for its cytotoxicity. In contrast to CPX, deferoxamine, a more avid extracellular iron chelator, was not significantly cytotoxic with an IC25 〉 10uM. The highest demand for intracellular iron occurs during the late G1 and S phases due, in part, to the activity of the iron-requiring enzyme ribonucleotide reductase. Therefore, we examined the effects of CPX on the activity of ribonucleotide reductase. By electron paramagnetic resonance (EPR), CPX inhibited ribonucleotide reductase at concentrations associated with cell death. Cell lines resistant to CPX-mediated inhibition of ribonucleotide reductase were also resistant to CPX-induced cell death, supporting a mechanism of action linked to ribonucleotide reductase. Thus, in summary, the off-patent anti-fungal agent CPX induces cell death through its ability to bind intracellular iron. Its ability to inhibit the iron-containing enzyme ribonucleotide reductase appears functionally important for its mechanism of action. CPX displays previously unrecognized anti-cancer activity at concentrations that are pharmacologically achievable. Thus, CPX could be rapidly repurposed for the treatment of malignancies including leukemia and myeloma.

Description: Abstract 61 To identify new therapeutic strategies for AML, we compiled and screened an in-house library of on-patent and off-patent drugs to identify agents cytotoxic to leukemia cells. From this screen, we identified mefloquine, an off-patent drug indicated for the treatment and prophylaxis of malaria. In secondary assays, mefloquine decreased the viability of 9/10 human and murine leukemia cell lines (EC50 3.25–8.0 μM). Moreover, it reduced the viability of 4/5 primary AML samples, but was not cytotoxic to normal hematopoietic cells (EC50〉31 μM). Importantly, mefloquine reduced the clonogenic growth of primary AML samples, but not normal hematopoietic cells, and completely inhibited engraftment of primary AML cells into immune deficient mice. Finally, systemic treatment with oral mefloquine (50 mg/kg/day) decreased leukemic burden without evidence of toxicity in 4 mouse models of leukemia, including mice engrafted with primary AML cells. Thus, mefloquine effectively targets leukemic cells, including leukemia stem cells, at concentrations that appear pharmacologically achievable and are not toxic to normal hematopoietic cells. To identify the mechanisms of mefloquine-mediated cell death in AML cells, we performed a binary drug combination screen, hypothesizing that drugs that synergized with mefloquine may share overlapping mechanism of action. From this combination screen of 550 drugs, we identified 18 that reproducibly synergized with mefloquine as measured by the Excess over Bliss additivism score, including 3 members of the artemisinin class of anti-malarials: artemisinin, artesunate and artenimol. Strikingly, 10/18 synergistic compounds, including the artemisinins, were known generators of reactive oxygen species (ROS). Therefore we tested mefloquine's ability to increase ROS in leukemic cells. Mefloquine increased ROS production in leukemia cells in a dose- and time-dependent manner. Co-treatment with ROS scavengers α-tocopherol and N-acetyl-cysteine abrogated mefloquine-induced ROS production and cell death, indicating that ROS production was functionally important for mefloquine-mediated cell death. Moreover, the artemisinins induced ROS as single agents, and synergistically increased ROS when combined with mefloquine. To identify cellular target(s) of mefloquine's anti-leukemic effects, we performed a yeast genome-wide functional screen to identify heterozygous gene deletions that rendered yeast more sensitive to mefloquine. 21/37 genes whose depletion conferred 〉4-fold sensitivity to mefloquine were associated with function of the yeast vacuole, equivalent to the mammalian lysosome. Consistent with these data, fluorescent confocal microscopy demonstrated that mefloquine and artesunate disrupted lysosomes. Cell death after mefloquine and artesunate treatment was caspase-independent and associated with increased incorporation of monodancylcadaverin in autophagosomes, consistent with the effect of these drugs on the lysosomes. To further explore the anti-leukemic activity of lysosomal disruption, we evaluated the anti-leukemic effects of the known lysosomal disrupter L-leucine-leucine methyl ether (LeuLeuOMe). Similar to mefloquine and artesunate, LeuLeuOMe induced cell death in leukemia cells, increased ROS production, and disrupted the lysosomes. Highlighting the potential clinical utility of lysosomal disrupters for the treatment of leukemia, a patient with relapsed/refractory juvenile myelomonocytic leukemia self-administered artemisinin. The artemisinin cleared the circulating blasts from the circulating blasts and the patient proceeded to allotransplant. Finally, to investigate the basis of leukemic cell hypersensitivity to lysosomal disruption, we assessed lysosomal characteristics of primary AML and normal hematopoietic cells. By gene expression analysis, AML patient samples had higher mRNA levels of the lysosomal cathepsins A, B, C, D, H, L, S and Z, compared to CD34+ normal hematopoietic cells, and cathepsins C, D and Z were significantly over-expressed in the LSC compartment, compared to normal HSCs. In summary, our data demonstrate that lysosomal disruption preferentially targets AML cells and AML stem cells through a mechanism related to increased ROS production. Thus, this work highlights lysosomal disruption as a novel therapeutic strategy for AML. Disclosures: Off Label Use: This study includes a case report of off-label use of the anti-malarial artemisinin in the treatment of a case of juvenile myelomonocytic leukemia.

Description: The 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.

Description: D-cyclins are universally dysregulated in multiple myeloma and frequently overexpressed in leukemia. To better understand the role and impact of dysregulated D-cyclins in hematologic malignancies, we conducted a high-throughput screen for inhibitors of cyclin D2 transactivation and identified 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161), which inhibited the expression of cyclins D1, D2, and D3 and arrested cells at the G0/G1 phase. After D-cyclin suppression, S14161 induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of leukemia, S14161 inhibited tumor growth without evidence of weight loss or gross organ toxicity. Mechanistically, S14161 inhibited the activity of phosphoinositide 3-kinase in intact cells and the activity of the phosphoinositide 3-kinases α, β, δ, and γ in a cell-free enzymatic assay. In contrast, it did not inhibit the enzymatic activities of other related kinases, including the mammalian target of rapamycin, the DNA-dependent protein kinase catalytic subunit, and phosphoinositide-dependent kinase-1. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation via the phosphoinositide 3-kinase/protein kinase B signaling pathway. Given its potent antileukemia and antimyeloma activity and minimal toxicity, S14161 could be developed as a novel agent for blood cancer therapy.

Description: Abstract 1586 Background Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by the t(11;14) translocation and concomitant over-expression of cyclin D1. MCL has a variable natural history; while some patients have prolonged survival similar to other indolent B-cell lymphomas, most follow an aggressive course with short survival. While the t(11;14) is pathognomonic of MCL, it is not necessary for disease pathogenesis, as a subset of MCL cases lack the translocation. Furthermore, in vivo models demonstrate that cyclin D1 over-expression alone is unable to bring about the disease, and that deregulation of additional cellular pathways is required for its pathogenesis. Assessment of microRNA (miR) expression in MCL may help determine mechanisms of gene deregulation and reveal pathways involved in disease pathogenesis. In this study we examined MCL in relation to both aggressive and indolent B-cell NHL to determine a miR signature that characterizes MCL. Design and Methods Total RNA from a training set of 36 B-cell NHL cases (19 indolent and 17 aggressive) and 32 MCL was applied to a high-throughput quantitative real-time PCR platform assessing the expression of 365 miRs [TaqMan Human MicroRNA Array v1.0 (Early Access) or TLDA]. miRs that were differentially expressed between MCL and aggressive NHL, and between MCL and indolent NHL were then validated using RNA from a second, independent, set of B-cell NHL cases (28 indolent and 28 aggressive) and 50 MCL cases. Validated miRs were determined and potential targets for each miR were examined. A map of targets common to the MCL miR signature was created, revealing important proteins involved in MCL pathogenesis. Results 66 miRs (11 over-expressed, 55 under-expressed) were differentially expressed between MCL and aggressive B-cell NHL and 8 miRs (7 over-expressed, 1 under-expressed) were differentially expressed between MCL and indolent B-cell NHL (false discovery rate = 0.2). 6 miRs from each group were chosen for validation in an independent set of MCL and NHL cases. Of these 12 miRs, 7 miRs validated (2 were under-expressed in MCL relative to aggressive B-cell NHL, and 5 were over-expressed in MCL relative to indolent B-cell NHL). Genes and pathways involved in disease pathogenesis are most likely targeted by multiple miRs. We thus determined a set of 123 genes predicted to be targets of this MCL miR signature, based on five miR target prediction databases from the mirDIP (microRNA data integration) portal. These genes were significantly enriched for focal adhesion and integrin signalling, proteasome-mediated degradation, and the PI3K signalling pathway. Conclusions Using the largest set of MCL cases evaluated to date, a 7-miR signature characteristic of MCL was discovered. The gene targets of these miRs are enriched for roles in proteasome-mediated protein degradation, consistent with the reported sensitivity of MCL to proteasome inhibitors. In addition, these miRs are predicted to be involved in regulation of PI3K/AKT signalling, confirming reports of the importance of this pathway in MCL pathogenesis. Enrichment of target genes involved in focal adhesion and integrin signalling indicate the importance of MCL-stromal interactions and motivates further study into the role of the tumor microenvironment in MCL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is an aggressive malignant disease characterized by poor patient outcome and suboptimal front-line chemotherapy. To identify novel anti-AML compounds, we performed a high-throughput screen of a natural products library (n=800). This screen was performed against the AML cell line (TEX), which has several properties of leukemia stem cells, the cells responsible for disease pathophysiology and patient relapse. Here, avocatin B was identified as a potent and novel anti-leukemia agent. Avocatin B, at concentrations as high as 20µM, had no effect on normal peripheral blood stem cell viability. In contrast, it induced death of primary AML cells with an EC50 of 1.5-5.0 µM. Selective toxicity towards a functionally defined subset of primitive leukemia cells was also demonstrated. Avocatin B (3µM) reduced clonogenic growth of AML progenitor cells with no effect on clonogenic growth of normal hematopoietic stem cells. Further, treatment of primary AML cells with avocatin B (3µM) diminished their ability to engraft into the bone marrow of pre-conditioned, NOD/SCID mice (t18=6.5; p80%) reduction in mitochondria were confirmed by nonyl acridine orange staining and flow cytometry and a near absence of the mitochondria specific proteins ANT and ND1, as measured by Western blotting. Avocatin B’s activity was abolished in leukemia cells lacking mitochondria. Next, using lentiviral knockdown, we generated leukemia cells lacking CPT1, the enzyme that facilitates transport of 16-20 carbon lipids into mitochondria. Avocatin B’s activity was abolished in cells with reduced CPT1 expression (〉70% as measured by qPCR analysis). To further confirm the importance of CPT1 in avocatin B-induced death, we chemically inhibited CPT1 with etomoxir. Avocatin B’s activity was blocked in the presence of etomoxir, further demonstrating that avocatin B accumulates in mitochondria. Since avocatin B is a lipid that targets mitochondria and that mitochondria can oxidize fatty acids for energy, we next assessed the impact of avocatin B on fatty acid oxidation, using the Seahorse Bioanalyzer. Avocatin B inhibited leukemia cell fatty acid oxidation (〉40% reduction in oxygen consumption at 10µM) and this occurred at a 10-fold less concentration than etomoxir, the standard experimental molecule used to probe this pathway. Further, avocatin B resulted in a 50% reduction in levels of NADPH, an important co-factor generated during fatty acid oxidation that participates in catabolic processes during cell proliferation. These results show that avocatin B accumulates in mitochondria to inhibit fatty acid oxidation and reduce NADPH to result in ROS-mediated leukemia cell apoptosis. This highlights a novel AML-therapeutic strategy by which mitochondria are targeted to impair cellular metabolism leading directly to AML cell death. Disclosures No relevant conflicts of interest to declare.

Description: Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC–enriched CD34+CD38− AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti–L-IC compounds for human leukemias.

Description: D-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.

Description: Abstract 2036 Poster Board II-13 D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in acute leukemia. To better understand the regulation of D-cyclins and the effects of targeting their expression in myeloma and leukemia cells, we conducted a chemical screen of the 56,000 compound Maybridge chemical library for inhibitors of the human cyclin D2 promoter using NIH 3T3 cells stably expressing the Cyclin D2promoter-driving a luciferase reporter gene. From this screen, 11 compounds that reproducibly inhibited transactivation of the cyclin D2 promoter, but did not inhibit transactivation of an unrelated RSV promoter driving luciferase, and did not reduce the growth and viability of NIH3T3 in an MTS assay. Among these 11 compounds, the most active was 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161). Given the identification of S14161 as a potential inhibitor of cyclin D2 transactivation, we evaluated its effects on D-cyclin expression in myeloma and leukemia cell lines. S14161 decreased D-cyclin protein and mRNA levels across a range of events that dysregulated D-cyclins including cyclin D1 translocation (KMS12), c-maf over-expression (RPMI-8226, KMS11, LP1, JJN3), and FGFR3 translocation (KMS11) at low micromolar concentrations. Consistent with its effects on D-cyclin expression, S14161 arrested cells in the G0/G1 phase of the cell cycle at concentrations associated with its ability to decrease D-cyclins. Next, we tested the effects of S14161 on the viability of myeloma and leukemia cells. S14161 induced cell death in 6/7 myeloma and 6/7 leukemia cell lines with an IC50

Description: 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.