Abstract 3463
Epigenetic and apoptosis-regulating mechanisms have been implicated as critical factors contributing to the progression from myelodysplastic syndromes (MDS) to secondary acute myeloid ...leukemia (AML). However, the exact molecular mechanisms and genes involved in disease evolution have not been identified yet. We screened for epigenetically regulated pro-apoptotic effector molecules in neoplastic cells in patients with MDS (n=50) and AML (n=30). Among a series of potential regulators, we identified FAS (CD95) as an epigenetically regulated critical death regulator in neoplastic cells. As assessed by qPCR, bone marrow cells obtained from patients with low risk MDS were found to display high levels of FAS, whereas FAS mRNA levels were lower or undetectable in patients with advanced MDS (with excess of blasts) or secondary AML. Moreover, we were able to show by multicolor flow cytometry that CD34+/CD38+ progenitor cells and CD34+/CD38- stem cells in MDS and AML display measurable FAS (CD95) on their surface, with slightly higher levels detectable in progenitor cells in low risk MDS compared to high risk MDS and secondary AML. Methylation-specific PCR and qPCR revealed that the FAS-promoter is hypermethylated in primary AML cells as well as in various AML cell lines including KG1 and HL60 thus repressing mRNA-synthesis. In addition, we found that exposure to 5-Azacytidine or Decitabine leads to demethylation of CpG-rich regions closest to the transcription starting sites, and thus to re-expression of FAS in AML cells. In vitro-targeting of AML cells by demethylating drugs was also found to revert epigenetic inactivation of other tumor suppressor genes such as CDKN2B (P15), CDKN2A (P16), ESR1 (estrogen-receptor alpha), with subsequent normalization of mRNA expression levels. Next, we asked whether CD95 acts as a critical death regulator involved in drug-induced apoptosis in neoplastic cells. In these experiments, both demethylating agents, 5-Azacytidine or Decitabine, were found to induce dose-dependent apoptosis and growth inhibition in primary AML cells, primary MDS cells, and in all AML cell lines examined. Drug-induced apoptosis in AML cells was accompanied by activation of caspase 8 and caspase 3 as well as increased expression of proapoptotic FAS/CD95 as determined by qPCR, Western blotting, and flow cytometry. Moreover, both drugs were found to induce expression of the FAS-ligand and DAPK1 in neoplastic cells. We then applied a siRNA against FAS. The siRNA-induced knock-down of FAS was found to block drug-induced FAS expression and FAS-induced apoptosis in KG1 cells and HL60 cells. In conclusion, our data show that FAS is hypermethylated in neoplastic cells in patients with advanced MDS and AML, that demethylating agents lead to re-expression of FAS, and that drug-induced FAS expression mediates apoptosis in leukemic cells. As FAS is also expressed on neoplastic stem cells, these observations may have clinical implications and may explain beneficial effects seen with 5-Azacytidine or Decitabine in patients with advanced MDS.
Valent:Novartis: Consultancy, Honoraria, Research Funding.
Abstract 961
In Philadelphia-positive (Ph+) chronic myeloid leukemia (CML), leukemic stem cells (LSC) supposedly reside in a CD34+/CD38−/Lin− fraction of the leukemic clone. However, little is known ...about phenotypic properties of LSC in CML. We screened for novel LSC markers and targets in CML by gene chip studies and extensive flow cytometry analyses using monoclonal antibodies against various surface antigens (n=50). A total number of 240 bone marrow or peripheral blood samples (CML, n=95; AML, n=103; CMML, n=10, control marrow, n=32) were examined. In common with normal SC, CD34+/CD38− CML LSC were found to co-express the homing-receptor CD44, G-CSF-R (CD114), KIT (CD117), FLT3 (CD135), and CXCR4 (CD184). Similar to LSC in AML and CMML, CML LSC were found to display higher levels of Siglec-3 (CD33) and IL-3RA (CD123). Most significantly, however, we found that in contrast to normal CD34+/CD38− stem cells, CD34+/CD38− CML LSC aberrantly express IL-2RA (CD25), dipeptidylpeptidase IV (DPPIV=CD26), and IL-1RAP. In other myeloid leukemias (AML, CMML), CD34+/CD38− LSC also co-expressed CD25, but usually did not express CD26 or IL-1RAP. Whereas CD26 was expressed almost invariably on CD34+/CD38− cells in all CML patients tested, the surface enzyme was neither detectable in more mature CD34+/CD38+ progenitor cells nor on CD34+/CD38− stem cells in reactive bone marrow or healthy controls. During successful treatment with imatinib or nilotinib (patients examined at CCyR and/or MMR), CD34+/CD38− stem cells invariably showed a ‘normal’ phenotype (CD25−, CD26−, IL-1RAP−), whereas in relapsing CML, CD34+/CD38− cells were again found to co-express CD25 and CD26. Sorted Lin−/CD26− stem cells obtained from CML patients (at diagnosis) engrafted irradiated NOD-SCID IL-2Rγ−/− (NSG) mice with normal multilineage BCR/ABL1− hematopoiesis, whereas Lin−/CD26+ stem cells were found to engraft NSG mice with BCR/ABL+ cells. We next examined the regulation of expression of CD25 and CD26 on CML LSC. Whereas expression of CD25 was found to depend on BCR/ABL1 and STAT5-activity, CD26 expression was found to be expressed independent of BCR/ABL1 and independent of STAT5-signaling. In a next step, we examined the potential function of CD26 on CML LSC. In these studies, CD26 was identified as a target-enzyme disrupting the niche-related SDF-1α/CXCR4 axis by degrading SDF-1α. Correspondingly, CD26-targeting gliptins (sitagliptin, 1 μM; vildagliptin, 1 μM) were found to revert recombinant DPPIV/CD26-induced or cellular CD26-induced inhibition of SDF-1α-mediated in vitro migration of CD26+ leukemic cells. Finally, we found that in a CML patient treated with nilotinib, in whom uncontrolled diabetes mellitus required therapy with saxagliptin, BCR/ABL1 levels (in percent of ABL according to IS) that were found to increase before the start of saxagliptin (IS before saxagliptin: 1.6 -4 months, 2.3 -3 months, and 2.4 at therapy-start), decreased over time during saxagliptin-therapy (IS: 1.0 +1 month, 1.0 +3 months, 0.8 +5 months). Together, the CML-initiating LSC is a CD34+/CD38− cell that exhibits aberrant expression of IL-1RAP, CD25, and DPPIV/CD26. All three markers may be useful for purification of CML LSC. DPPIV/CD26 appears to be a functionally and pathogenetically relevant antigen that may facilitate niche-independent uncontrolled redistribution and thus extramedullary spread of LSC and LSC-derived progenitor cells in CML. Whether CD26 can be developed as a novel therapeutic target in CML is currently under investigation.
Valent:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.
In order to satisfy the demanding SWaP requirements of modern microwave/MMW instruments, designers are looking for novel integrated solutions. In particular, phased array packaging, especially at Ka ...and higher frequencies, is extremely challenging as half-wavelength spacing is prohibitively small. The cost of implementing high density phased array packaging is another impediment to wider use. This communication presents a new electronically beam-steering technology that is compatible with highly integrated antenna design and dramatically simplifies packaging. It is based on the coherent scattering of the evanescent field associated with waves propagating through a dielectric waveguide. The antenna scattering elements are controlled electronically and constitute a dynamically reconfigurable hologram. The switching time from one hologram pattern (one beam position) to another is on the order of tens of nanoseconds. Because of the hologram nature of this approach, the beam-forming capabilities of the electronically reconfigurable aperture (ERA) approach are comparable to those of phased arrays: 1D and 2D beam-forming and beam-steering, multiple simultaneous individually controlled beams, steerable nulls and variable beam width(s).
PAMIR-3U magnetohydrodynamic generator results Price, David W.; Swallom, Daniel W.; Goldfarb, Victor M. ...
Digest of Technical Papers. Tenth IEEE International Pulsed Power Conference,
1995, Letnik:
2
Conference Proceeding
The Air Force's Phillips Laboratory has acquired a high power magnetohydrodynamic (MHD) generator for possible use with advanced weapons applications. This MHD generator is a PAMIR-3U, a modified ...Russian-built MHD generator that uses a modified rocket fuel to produce a DC electrical pulse of 100 MJ. The PAMIR-3U generator produces tens of kA at 800 V for an optimized load of (20/spl plusmn/5) m/spl Omega/. A review of the MHD generator design and results of the generator acceptance testing is presented. The PAMIR-3U generator was constructed by the Institute of High Temperatures of the Russian Academy of Sciences (IVTAN) and delivered to the Air Force's Phillips Laboratory under contract with Textron Defense Systems (TDS) of Everett MA.
