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Rida Mourtada,
Sonali B Fonseca,
Simon P Wisnovsky,
Mark P Pereira, Xiaoming Wang,
Rose Hurren,
Jeremy Parfitt,
Lesley Larsen,
Robin A J Smith,
Michael P Murphy,
Aaron D Schimmer,
Shana O Kelley
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ABSTRACT: We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.
PLoS ONE 01/2013; 8(4):e60253. · 4.09 Impact Factor
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Mahadeo A Sukhai,
Swayam Prabha,
Rose Hurren,
Angela C Rutledge,
Anna Y Lee,
Shrivani Sriskanthadevan,
Hong Sun, Xiaoming Wang,
Marko Skrtic,
Ayesh Seneviratne, [......],
Pascal Sonnet,
William Y Ellis,
Seth J Corey,
Connie Eaves,
Mark D Minden,
Jean C Y Wang,
John E Dick,
Corey Nislow,
Guri Giaever,
Aaron D Schimmer
[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
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Reza Beheshti Zavareh,
Mahadeo A Sukhai,
Rose Hurren,
Marcela Gronda, Xiaoming Wang,
Craig D Simpson,
Neil Maclean,
Francis Zih,
Troy Ketela,
Carol J Swallow,
Jason Moffat,
David R Rose,
Harry Schachter,
Aaron D Schimmer,
James W Dennis
[show abstract]
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ABSTRACT: Oncogenic signaling promotes tumor invasion and metastasis, in part, by increasing the expression of tri- and tetra- branched N-glycans. The branched N-glycans bind to galectins forming a multivalent lattice that enhances cell surface residency of growth factor receptors, and focal adhesion turnover. N-acetylglucosaminyltransferase I (MGAT1), the first branching enzyme in the pathway, is required for the addition of all subsequent branches. Here we have introduced MGAT1 shRNA into human HeLa cervical and PC-3-Yellow prostate tumor cells lines, generating cell lines with reduced transcript, enzyme activity and branched N-glycans at the cell surface. MGAT1 knockdown inhibited HeLa cell migration and invasion, but did not alter cell proliferation rates. Swainsonine, an inhibitor of α-mannosidase II immediately downstream of MGAT1, also inhibited cell invasion and was not additive with MGAT1 shRNA, consistent with a common mechanism of action. Focal adhesion and microfilament organization in MGAT1 knockdown cells also indicate a less motile phenotype. In vivo, MGAT1 knockdown in the PC-3-Yellow orthotopic prostate cancer xenograft model significantly decreased primary tumor growth and the incidence of lung metastases. Our results demonstrate that blocking MGAT1 is a potential target for anti-cancer therapy.
PLoS ONE 01/2012; 7(9):e43721. · 4.09 Impact Factor
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Marko Skrtić,
Shrivani Sriskanthadevan,
Bozhena Jhas,
Marinella Gebbia, Xiaoming Wang,
Zezhou Wang,
Rose Hurren,
Yulia Jitkova,
Marcela Gronda,
Neil Maclean, [......],
Jessie H Cameron,
Jeffery Wrana,
Connie J Eaves,
Mark D Minden,
Jean C Y Wang,
John E Dick,
Keith Humphries,
Corey Nislow,
Guri Giaever,
Aaron D Schimmer
[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
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Xinliang Mao,
Biyin Cao,
Tabitha E Wood,
Rose Hurren,
Jiefei Tong, Xiaoming Wang,
Wenjie Wang,
Jie Li,
Yueping Jin,
Wenxian Sun,
Paul A Spagnuolo,
Neil MacLean,
Michael F Moran,
Alessandro Datti,
Jeffery Wrana,
Robert A Batey,
Aaron D Schimmer
[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.
Blood 02/2011; 117(6):1986-97. · 9.90 Impact Factor
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Sumaiya Sharmeen,
Marko Skrtic,
Mahadeo A Sukhai,
Rose Hurren,
Marcela Gronda, Xiaoming Wang,
Sonali B Fonseca,
Hong Sun,
Tabitha E Wood,
Richard Ward,
Mark D Minden,
Robert A Batey,
Alessandro Datti,
Jeff Wrana,
Shana O Kelley,
Aaron D Schimmer
[show abstract]
[hide abstract]
ABSTRACT: To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.
Blood 11/2010; 116(18):3593-603. · 9.90 Impact Factor
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Xiaoming Li,
Tabitha E Wood,
Remco Sprangers,
Gerrit Jansen,
Niels E Franke,
Xinliang Mao, Xiaoming Wang,
Yi Zhang,
Sue Ellen Verbrugge,
Hans Adomat, [......],
Tomasz L Religa,
Nazir Jamal,
Hans Messner,
Jacqueline Cloos,
David R Rose,
Ami Navon,
Emma Guns,
Robert A Batey,
Lewis E Kay,
Aaron D Schimmer
[show abstract]
[hide abstract]
ABSTRACT: Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance.
5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided.
5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002).
5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.
CancerSpectrum Knowledge Environment 07/2010; 102(14):1069-82. · 14.07 Impact Factor
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G Wei Xu,
Mohsin Ali,
Tabitha E Wood,
Derek Wong,
Neil Maclean, Xiaoming Wang,
Marcela Gronda,
Marko Skrtic,
Xiaoming Li,
Rose Hurren,
Xinliang Mao,
Meenakshi Venkatesan,
Reza Beheshti Zavareh,
Troy Ketela,
John C Reed,
David Rose,
Jason Moffat,
Robert A Batey,
Sirano Dhe-Paganon,
Aaron D Schimmer
[show abstract]
[hide abstract]
ABSTRACT: The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.
Blood 03/2010; 115(11):2251-9. · 9.90 Impact Factor
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Paul A Spagnuolo,
Jiayi Hu,
Rose Hurren, Xiaoming Wang,
Marcela Gronda,
Mahadeo A Sukhai,
Ashley Di Meo,
Jonathan Boss,
Iman Ashali,
Reza Beheshti Zavareh,
Noah Fine,
Craig D Simpson,
Sumaiya Sharmeen,
Rob Rottapel,
Aaron D Schimmer
[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.
Blood 03/2010; 115(23):4824-33. · 9.90 Impact Factor
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Yanina Eberhard,
Sean P McDermott, Xiaoming Wang,
Marcela Gronda,
Amudha Venugopal,
Tabitha E Wood,
Rose Hurren,
Alessandro Datti,
Robert A Batey,
Jeffrey Wrana,
William E Antholine,
John E Dick,
John Dick,
Aaron D Schimmer
[show abstract]
[hide abstract]
ABSTRACT: Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds, we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia, myeloma, and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore, oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition, oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models, thereby establishing its ability to target leukemia stem cells. Mechanistically, CPX bound intracellular iron, and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance, CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus, in summary, CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore, CPX could be rapidly repurposed for the treatment of malignancies, including leukemia and myeloma.
Blood 08/2009; 114(14):3064-73. · 9.90 Impact Factor
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Craig D Simpson,
Imtiaz A Mawji,
Kika Anyiwe,
Moyo A Williams, Xiaoming Wang,
Amudha L Venugopal,
Marcela Gronda,
Rose Hurren,
Sonia Cheng,
Stefano Serra,
Reza Beheshti Zavareh,
Alessandro Datti,
Jeffrey L Wrana,
Shereen Ezzat,
Aaron D Schimmer
[show abstract]
[hide abstract]
ABSTRACT: Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed "anoikis." However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na(+)/K(+) ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na(+)/K(+) ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides.
Cancer Research 05/2009; 69(7):2739-47. · 7.86 Impact Factor
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Xinliang Mao,
Sheng-ben Liang,
Rose Hurren,
Marcela Gronda,
Sue Chow,
G Wei Xu, Xiaoming Wang,
Reza Beheshti Zavareh,
Nazir Jamal,
Hans Messner,
David W Hedley,
Alessandro Datti,
Jeff L Wrana,
Yuanxiao Zhu,
Chang-xin Shi,
Kyle Lee,
Rodger Tiedemann,
Suzanne Trudel,
A Keith Stewart,
Aaron D Schimmer
[show abstract]
[hide abstract]
ABSTRACT: 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 G(0)/G(1) 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.
Blood 06/2008; 112(3):760-9. · 9.90 Impact Factor
