[Show abstract][Hide abstract] ABSTRACT: Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA-mini linear covalently closed DNA vectors-that are devoid of unwanted bacterial sequences and encode only the gene(s) of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
[Show abstract][Hide abstract] ABSTRACT: To identify novel anti-cancer agents, we created and screened a unique nutraceutical library for activity against acute myeloid leukemia (AML) cells. From this screen, we determined that glucopsychosine was selectively toxic toward AML cell lines and primary AML patient samples with no effect toward normal hematopoietic cells. It delayed tumor growth and reduced tumor weights in mouse xenografts models without imparting toxicity. Glucopsychosine increased cytosolic calcium and induced apoptosis through calpain enzymes. Extracellular calcium was functionally important for glucopsychosine-induced AML cell death and surface calcium channel expression is altered in AML cells highlighting a unique mechanism of glucopsychosine's selectivity.
[Show abstract][Hide abstract] ABSTRACT: The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes. In particular, Stat3 protein has received particular attention as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Multiple myeloma (MM) is an incurable haematologic malignancy that often exhibits abnormally high levels of Stat3 activity. Although current treatment strategies can improve the clinical management of MM, it remains uniformly incurable with a dismal median survival time post-treatment of 3-4 years. Thus, novel targeted therapeutics are critically needed to improve MM patient outcomes. We herein report the development of a series of small molecule Stat3 inhibitors with potent anti-MM activity in vitro. These compounds showed high-affinity binding to Stat3's SH2 domain, inhibited intracellular Stat3 phosphorylation, and induced apoptosis in MM cell lines at low micromolar concentrations.
Journal of Medicinal Chemistry 08/2013; · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC50's <5μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold's history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.
[Show abstract][Hide abstract] ABSTRACT: Parthenolide is selectively toxic to leukemia cells; however, it also activates cell protective responses which may limit its clinical application. Therefore, we sought to identify agents that synergistically enhance parthenolide's cytotoxicity. Using a high-throughput combination drug screen, we identified the hypoglycemic, vildagliptin, which synergized with parthenolide to induce death of the leukemia stem cell line, TEX (combination index (CI) = 0.36 and 0.16, at EC 50 and 80, respectively; where CI < 1 denotes statistical synergy). The combination of parthenolide and vildagliptin reduced the viability and clonogenic growth of cells from acute myeloid leukemia patients and had limited effects on the viability of normal human peripheral blood stem cells. The basis for synergy was independent of vildagliptin's primary action as an inhibitor of dipeptidyl peptidase (DPP) IV. Rather, using chemical and genetic approaches we demonstrated that the synergy was due to inhibition of the related enzymes DPP8 and 9. In summary, these results highlight DPP8 and 9 inhibition as a novel chemosensitizing strategy in leukemia cells. Moreover, these results suggest that the combination of vildagliptin and parthenolide could be useful for the treatment of leukemia.Leukemia accepted article preview online, 15 January 2013; doi:10.1038/leu.2013.9.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 01/2013; · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.
The Journal of clinical investigation 12/2012; · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein-protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein-protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (K(a) ∼ 10(7) M(-1)) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines.
[Show abstract][Hide abstract] ABSTRACT: To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.
Cancer cell 11/2011; 20(5):674-88. · 25.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advancing novel therapeutic agents for the treatment of malignancy into the marketplace is an increasingly costly and lengthy process. As such, new strategies for drug discovery are needed. Drug repurposing represents an opportunity to rapidly advance new therapeutic strategies into clinical trials at a relatively low cost. Known on-patent or off-patent drugs with unrecognized anticancer activity can be rapidly advanced into clinical testing for this new indication by leveraging their known pharmacology, pharmacokinetics, and toxicology. Using this approach, academic groups can participate in the drug discovery field and smaller biotechnology companies can "de-risk" early-stage drug discovery projects. Here, several scientific approaches used to identify drug repurposing opportunities are highlighted, with a focus on hematologic malignancies. In addition, a discussion of the regulatory issues that are unique to drug repurposing and how they impact developing old drugs for new indications is included. Finally, the mechanisms to enhance drug repurposing through increased collaborations between academia, industry, and nonprofit charitable organizations are discussed.
[Show abstract][Hide abstract] ABSTRACT: 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 G(0)/G(1) 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.
[Show abstract][Hide abstract] ABSTRACT: On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.