No abstract

Clinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cellmediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8 ؉ T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overex-

Background We previously demonstrated upregulation of c-myc, survivin, and cyclin D1 in CD34+ bone marrow mononuclear cells (BMMNCs) of patients with trisomy 8 and monosomy 7 myelodysplastic syndromes (MDS). “Knockdown” of cyclin D1 by RNA interference decreased trisomy 8 cell growth, suggesting that this might be a therapeutic target in MDS. Experimental Design We performed preclinical studies using BMMNCs from patients with MDS and AML to examine the effects of the styryl sulfone ON 01910.Na on cyclin D1 accumulation, aneuploidy, and CD34+ blast percentage. We next treated twelve patients with higher risk MDS and two trisomy 8 AML patients with ON01910.Na on a phase I clinical protocol (NCT00533416). Results ON 01910.Na inhibited cyclin D1 expression, and was selectively toxic to trisomy 8 cells in vitro. Flow cytometry studies demonstrated increased mature CD15+ myeloid cells and decreased CD34+ blasts. Three patients treated with ON01910.Na on a clinical had decreased bone marrow blasts by ≥50%, and three patients had hematologic improvements, one of which was sustained for 33 months. Patients with hematologic responses to ON 01910.Na had decreased cyclin D1 expression in their CD34+ cells. Conclusions The preclinical results and responses of patients on a clinical trial warrant further investigation of ON 01910.Na as a potential novel targeted therapy for higher risk MDS patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients’ plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 mg/g and maximal expression at approximately 400 mg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non–ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2V617F-positive leukemic cells and blocks the IL-3–mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABLT315I and JAK2V617F. Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

Graft-versus-host disease (GVHD) is a major risk factor for secondary malignancy after hematopoietic stem cell transplantation. Squamous cell carcinoma (SCC) of the skin and mucous membranes are especially frequent in this setting where aneuploidy and tetraploidy are associated with aggressive disease. The current study is directed to the mechanism of neoplasia in this setting. Un-manipulated keratinocytes from areas of oral GVHD in 9 patients showed tetraploidy in 10–46% of cells when examined by florescence in situ hybridization (FISH). Keratinocytes isolated from biopsy sites of GVHD but not from normal tissue showed even greater numbers of tetraploid cells (mean 78%, range 15–85%; N=9) after culture. To mimic the inflammatory process in GVHD, allogeneic HLA- mismatched lymphocytes were mixed with normal keratinocytes. After two weeks, substantial numbers of aneuploid and tetraploid cells were evident in cultures with lymphocytes and with purified CD8 but not CD4 cells. Telomere length was substantially decreased in the lymphocyte-treated sample. No mutations were present in p53 gene, although haploinsufficiency for p53 due to loss of chromosome 17 was common in cells exposed to lymphocytes. These findings suggest that in GVHD, inflammation and repeated cell division correlates with the development of karyotypic abnormalities.

The initiation of viral RNA replication by the transfection of viral RNA is an integral tool in dissecting the life cycles, susceptibility, and pathogenesis of numerous RNA viruses. Many different transfection methods deliver viral RNA into mammalian cells, including DEAE-dextran and lipidbased reagents, but electroporation is one of the most popular methods. Unfortunately, electroporation suffers from many limitations, including high cell death, serum-free transfection conditions, and requires many cells and relatively large amounts of RNA. To optimize and facilitate the introduction of viral RNAs into mammalian cells, different commercially available RNA transfection reagents were compared for their ability to deliver yellow fever virus (YFV) and hepatitis C virus (HCV) RNA replicons into Huh7 cells. The performance of the commercial transfection reagents was also compared directly to electroporation. When properly optimized, certain reagents were superior to electroporation, with much less cell death, less RNA required and increased transfection efficiency. The factors associated with high efficiency transfection, and the advantage of being able to deliver RNA in the presence of serum are discussed.