We found that c-myc, encoded by a gene present on chromosome 8, was upregulated in trisomy 8 cells, likely a gene-dosage effect and a direct result of the trisomy. Upregulation of cyclin D1 mRNA and protein in these cells results from upregulated c-myc. Cyclin D1 increases cell proliferation and inhibits apoptosis via upregulated anti-apoptotic proteins. WT1 m-RNA is also high in trisomy 8 and may elicit an immune response directed against trisomy 8 cells. Knock-down studies of these proteins in trisomy 8 showed preferential killing of the trisomy 8 clone in vitro, suggesting that a potential avenue for therapeutic intervention might be inhibition of these transcription factors. A drug developed by Onconova Pharmaceuticals, ON1910, suppresses c-myc as well as cyclin D1. In vitro, this compound inhibited trisomy 8 cell proliferation and promoted apoptosis while increasing growth of normal diploid cells and promoting their maturation. Monosomy 7 cells express increased amounts of the granulocyte colony-stimulating factor receptor (GCSFR) isoform IV, which accounts for the cells inferior responses to physiologic levels of granulocyte colony stimulating factor. However, monosomy 7 cells proliferate at higher concentrations of this cytokine, as occur in bone marrow failure or with pharmacologic administration of GCSF. Recently, we have attempted to develop targeted therapy for each of these cytogenetic abnormalities using laboratory data linking regulation of important transcription factors favoring survival of trisomy 8 cells or monosomy 7 cells. Potential therapies for monosomy 7 include drugs that inhibit tyrosine kinase and Jak2, while inhibitors of cyclin D or c-myc might be effective in trisomy 8. We conducted a phase I study of an anti-cyclin D1 drug, ON1910, in MDS. ON 01910.Na inhibited cyclin D1 accumulation in MDS bone marrow-derived mononuclear cells, and was selectively toxic to trisomy 8 cells while promoting maturation of diploid cells. Three of four patients with refractory anemia with excess blasts and trisomy 8 and 2 of 2 patients with monosomy 7 treated on a pilot clinical study with ON 01910.Na had transient and two had sustained responses to the drug. Responders showed decreased bone marrow blasts and improved blood counts. Patients who exhibited hematologic responses to ON 01910.Na had decreased cyclin D1 levels in their CD34+ cells. Modulation of cell cycle control enzymes such as cyclin D1 therefore may represent a novel targeted approach for trisomy 8 MDS. We previously demonstrated abnormalities in the granulocyte colony stimulating factor receptor in monosomy 7 cells, which result in abnormal transduction of growth factor signals. This altered signaling results in resistance to apoptosis and increased proliferation in the presence of high concentrations of GCSF, ultimately resulting in a survival advantage compared to normal diploid cells. We assesed the effect of inhibition of Jak2 on the survival of these cells, using two types of drugs: Jak-2 inhibitors currently available for treatment of myeloproliferative disorder and tyrosine kinase inhibitors which might have non-specific activity against phosphylated Jak2, which has tyrosine kinase activity. Dasatinib, licensed for treatment of chronic myelogenous leukemia, has non-specific activity against tyrosine kinases other than bcr-abl. These drugs have much less toxicity compared to chemotherapeutic drugs, particularly important in elderly and debilitated patients. We examined the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in marrow cells, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of marrow with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus controls, while diploid cell numbers were stable in flow cytometry experiments, incubation with TG101348 decreased the number of CD34+CD13- stem cells and increased more differentiated CD34-CD13+ myeloid cells. We have applied for a patent for the use of this drug in patients with monosomy 7 and excess blasts. In vitro studies of dasatinib demonstrated decreased numbers of monosomy 7 cells while dramatically increasing proliferation of diploid hematopoietic colony formation. We secured from Bristol Myers a donation of dasatinib and are currently preparing a phase II study of this drug in aplastic anemia patients who have developed monosomy 7. Similar to aplastic anemia, some individuals with MDS (usually younger patients) show features of immune activation and respond to immunosuppressive therapy. Wilms tumor antigen1 (WT1) is up-regulated in MDS but not in healthy CD34+ stem cells and expression levels increase as the disease progress8. We have recently identified cytotoxic T lymphocytes (CTL) directed against WT1 protein. These CTL characterize patients who recover hematopoietic function after immunosuppressive therapy, and they specifically recognize MDS cells with the trisomy 8 abnormality that express WT1. We plan to clone and sequence the WT1-specific TCR (T cell receptor) for use in gene transfer experiments. Much of the Branchs work has been directed toward evaluating the factors that favor survival of aneuploid cells in an inflammatory environment such as AA. Inflammatory conditions such as ulcerative colitis or Barretts esophagus predispose to chromosomal instability. We documented the presence of aneuploidy and tetraploidy in skin GVHD and were able to reproduce these genomic changes by adding mis-matched allogeneic lymphocytes or interferon gamma. Aplastic anemia also shows some features of chronic inflammation. We postulated that chromosomal abnormalities might be a consequence of genomic damage caused by effector T-cells as well as of telomere shortening from accelerated cell turnover. We were able to demonstrate aneuploidy, which appeared to be induced by the inflammatory environment, and which was ameliorated by addition of free radical scavengers, alpha-tocopherol, and desferrioxamine, and by an inhibitor of T cell activation, cyclosporine. In paroxysmal nocturnal hemoglobinuria, there is expansion of a clone of hematopoietic stem cells mutant in X chromosome gene, Pig-A, resulting in failure to present glycosylphilphasphinositol-anchored proteins on the cell surface. The most prominent feature of PNH is intravascular hemolysis due to absence of complement inactivating proteins on the red cell surface, but PNH clonal expansion occurs in the setting of bone marrow failure and many patients concurrently suffer aplastic anemia. The mechanisms by which PNH and aplastic anemia associate and the pathophysiology of GPI-anchored protein negative cell clonal expansion in aplastic anemia are not known. Recent experiments have tested a hypothesis that failure to produce the GPI moiety would affect protein degradation, as large quantities of proteins would be anchorless and require degradation via the proteasome. Experiments using paired hematopoietic cell lines and isolated human bone marrow cells have disclosed that proteasome inhibitors selectively affect the PNH clone, inducing an unfolded protein response and apoptosis. Treatment with proteasome inhibitors might eliminate PNH clones in patients.
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