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The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors,identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count,based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions,and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity,according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10,it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50,thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors. View Publication

Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL),calling for novel strategies that counter apoptosis resistance. Here,we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations,but not a structurally related control compound,synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells,whereas they do not affect viability of normal peripheral blood lymphocytes,suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases,loss of mitochondrial membrane potential,and cytochrome c release in a caspase-dependent manner,indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note,XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly,XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo,they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus,XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL. View Publication

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently,hES cell lines are derived from surplus embryos from assisted reproduction cycles,independent of their quality or morphology. Here,we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore,we show that the self-renewal,pluripotency,and differentiation ability of hES cell lines derived from either source are comparable. Finally,we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine. View Publication

Cyclophosphamide (CYC) can control diffuse proliferative lupus nephritis (DPLN) by potent immunosuppression but remains associated with serious and life-threatening complications. Drugs that specifically target mediators of DPLN may help to reduce CYC dose and side effects. Monocyte chemoattractant protein (MCP-1)/CCL2 mediates monocyte and T cell recruitment in DPLN and Ccl2-specific l-enantiomeric RNA Spiegelmer mNOX-E36 neutralizes the biological effects of murine Ccl2 in vitro and in vivo. We injected MRL(lpr/lpr) mice with DPLN from 14 weeks of age with vehicle,weekly 30 mg/kg CYC (full dose),monthly 30 mg/kg CYC (one-fourth full dose),pegylated control Spiegelmer,pegylated anti-Ccl2 Spiegelmer (3/week),pegylated anti-Ccl2 Spiegelmer plus CYC one-fourth full dose and mycophenolate mofetil. At week 24,DPLN and autoimmune lung injury were virtually abolished with CYC full dose but not with CYC one-fourth full dose. The CYC one-fourth full dose/Spiegelmer combination was equipotent to CYC full dose on kidney and lung injury. CD3(+)CD4(-)CD8(-) and CD3(+)CD4(+)CD25(+) T cells and serum interleukin-12p40 and tumor necrosis factor-alpha levels were all markedly affected by CYC full dose but not by CYC one-fourth full dose. No additive effects of anti-Ccl2 Spiegelmer were noted on bone marrow colony-forming unit-granulocyte macrophage counts and 7/4(high) monocyte counts,lymphoproliferation,and spleen T cell depletion. In summary,anti-Ccl2 Spiegelmer permits 75% dose reduction of CYC for controlling DPLN and pneumonitis in MRL-Fas(lpr) mice,sparing suppressive effects of full-dose CYC on myelosuppression and T cell depletion. We propose anti-Ccl2 Spiegelmer therapy as a novel strategy to reduce CYC toxicity in the treatment of severe lupus. View Publication

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3,which also contains the structurally related proteases testisin,tryptase epsilon,tryptase gamma,and EOS. To gain insight into the biological functions of marapsin,we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues,including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus,tonsil,cervix,larynx,and cornea. In the keratinizing stratified squamous epidermis of skin,however,its expression was induced only during epidermal hyperproliferation,such as in psoriasis and in murine wound healing. In fact,marapsin was the second most strongly up-regulated protease in psoriatic lesions,where expression was localized to the upper region of the hyperplastic epidermis. Similarly,in the hyperproliferative epithelium of regenerating murine skin wounds,marapsin localized to the suprabasal layers,where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin,which closely correlated with re-epithelialization,was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore,in reconstituted human epidermis,a model system of epidermal differentiation,members of the IL-20 subfamily of cytokines,such as IL-22,induced marapsin expression. Consistent with a physiologic role in marapsin regulation,IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression,localization,and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia. View Publication

The human transferrin receptor (hTfR) is a target for cancer immunotherapy due to its overexpression on the surface of cancer cells. We previously developed an antibody-avidin fusion protein that targets hTfR (anti-hTfR IgG3-Av) and exhibits intrinsic cytotoxicity against certain malignant cells. Gambogic acid (GA),a drug that also binds hTfR,induces cytotoxicity in several malignant cell lines. We now report that anti-hTfR IgG3-Av and GA induce cytotoxicity in a new broader panel of hematopoietic malignant cell lines. Our results show that the effect of anti-hTfR IgG3-Av is iron-dependent whereas that of GA is iron-independent in all cells tested. In addition,we observed that GA exerts a TfR-independent cytotoxicity. We also found that GA increases the generation of reactive oxygen species that may play a role in the cytotoxicity induced by this drug. Additive cytotoxicity was observed by simultaneous combination treatment with these drugs and synergy by using anti-hTfR IgG3-Av as a chemosensitizing agent. In addition,we found a concentration of GA that is toxic to malignant hematopoietic cells but not to human hematopoietic progenitor cells. Our results suggest that these two compounds may be effective,alone or in combination,for the treatment of human hematopoietic malignancies. View Publication

BACKGROUND AND AIMS: Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however,recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-kappaB (NF-kappaB) signalling. Several chemopreventive agents are able to inhibit NF-kappaB,and favourable results have been obtained--for example,for the broccoli compound sulforaphane-in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents. METHODS: TICs were defined by expression patterns of a CD44(+)/CD24(-),CD44(+)/CD24(+) or CD44(+)/CD133(+) phenotype and correlation to growth in immunodeficient mice,differentiation grade,clonogenic growth,sphere formation,aldehyde dehydrogenase (ALDH) activity and therapy resistance. RESULTS: Mechanistically,specific binding of transcriptionally active cRel-containing NF-kappaB complexes in TICs was observed. Sulforaphane prevented NF-kappaB binding,downregulated apoptosis inhibitors and induced apoptosis,together with prevention of clonogenicity. Gemcitabine,the chemopreventive agents resveratrol and wogonin,and the death ligand TRAIL were less effective. In a xenograft model,sulforaphane strongly blocked tumour growth and angiogenesis,while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxicity. CONCLUSION: The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs. View Publication

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants,the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system,precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However,HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells,but not more mature lineage-restricted progenitors. Overall,these data are consistent with the hypothesis that TCDD,through AhR activation,alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2,a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation,is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells,providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor,SD-208,inhibits smad2 activation in hematopoietic progenitors,suppresses TGF-beta-mediated gene activation in BM stromal cells,and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore,SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover,in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS. View Publication