ALBERT

Beschreibung: Abstract 2995 Poster Board II-973 Introduction: The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX. Methods and Results: A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding and diminished ability to mediate cell adhesion to VWF under flow conditions. Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3188 Poster Board III-125 The pervasive gram-positive bacterial pathogen Staphylococcus aureus has evolved and maintained a number of proteins that directly engage the host hemostatic system, including factors that directly interact with the host coagulation factor fibrinogen. Here, we investigate the hypothesis that binding of host fibrin(ogen) through the bacterial fibrinogen receptor, clumping factor A (ClfA), significantly contributes to virulence in the context of acute bacteremia. To directly test this view, we compared survival following intravenous injection of ClfA-postive S. aureus into fibrinogen-deficient and control mice. The genetic elimination of fibrinogen resulted in a dramatic prolongation in survival relative to fibrinogen sufficient control animals. To determine if this prolongation in survival was related to the ability of the pathogen to directly bind fibrinogen, we performed identical survival studies in mice expressing a mutant form of fibrinogen that retains normal clotting function but lacks the binding motif for ClfA located on the fibrinogen γ chain (termed Fib γΔ5). A profound survival advantage was observed for Fib γΔ5 mice relative to wild-type animals over a wide bacterial dose range corresponding to an LD50 to an LD100 for control animals. This difference in survival appeared to be linked to ClfA, as control and Fib γΔ5 mice revealed an identical survival profile when infected with ClfA-deficient S. aureus. Analysis of bacterial burden after infection in various organ systems revealed that ClfA-positive S. aureus rapidly marginalized from the circulation and colonized target organs in mice of both genotypes. The highest numbers of colony forming units (cfu) were found in the heart and kidney of challenged mice, but the cfu values observed in both of these tissues were significantly reduced in Fib γΔ5 mice, relative to control animals at 24 and 48 hours after initial infection. The cfu values also tended to be reduced in Fib γΔ5 mice over wild-type controls in the liver, lung, spleen and blood, but this difference did not achieve statistical significance. Complementary studies revealed a comparable systemic inflammatory response in control and Fib γΔ5 animals following initial infection; plasma levels of Il-6 and fibrinogen (an established acute phase reactant) were similarly elevated in both genotypes following infection. Notably, increased histological evidence of cardiac damage secondary to S. aureus bacteremia in wild-type mice relative to Fib γΔ5 animals correlated with significantly higher plasma levels of markers of cardiac damage (i.e., lactose dehydrogenase and muscle creatine kinase enzyme activity) 48 hours after infection. These data suggest that bacterial engagement of host fibrinogen via the bacterial fibrinogen receptor ClfA significantly contributes to S. aureus virulence following intravenous infection. Strategies designed to disrupt this interaction may be of significant clinical benefit to limit disease progression in S. aureus sepsis and could be achieved without necessarily compromising host hemostatic function. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 3081 Poster Board III-18 Novel treatment strategies are needed for patients with ALL diagnosed with resistant phenotypes or after relapse. The glucose analogue 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that induces growth arrest and cell death by inhibiting the key glycolytic enzymes phosphoglucose isomerase (PGI) and hexokinase (HK). Cancer cells and hypoxic cells are more sensitive to 2-DG due to their reliance on glycolysis for ATP generation. In this study, we evaluated the antileukemic activity of 2-DG in T- and Bp-ALL subtypes characterized by non-random translocations. The cytotoxicity of 2-DG was determined on CCRF-CEM (PTEN mutant T-ALL), NALM6 (Bp-ALL), REH (TEL/AML1+ Bp-ALL), and the BCR/ABL+ ALL cell lines SupB15 and TOM1. Cells were treated with 4mM 2-DG for 72 h under normoxic vs. hypoxic (0.5% O2) conditions using a hypoxia box chamber (BioSpherix), and growth arrest and cell death were assessed. 2-DG induced 2-13 fold higher cell death in all ALL cells tested under normal O2 conditions compared to hypoxia. Similarly, growth inhibition was greater under normoxia. Both BCR/ABL+ ALL cell lines TOM1 and SupB15 were among the most sensitive, while the PTEN mutant CCRF-CEM (T-ALL) cell model was the least. To analyze the mechanisms of higher sensitivity of ALL cells under normoxia, we first used mannose to assess the role of glycosylation in 2-DG induced ALL cell death. Mannose reversed 2-DG induced cell death in CCRF-CEM cells but only partially in NALM6 cells, indicating inhibition of glycosylation mainly mediated death in CCRF-CEM cells, while inhibition of both glycolysis and glycosylation mediated cell death in NALM6 cells. To further assess other mechanisms leading to cell death in ALL, we evaluated mitochondrial integrity in representative Bp- and T-ALL models by determining cellular respiration with a Clark electrode (Hansatech Instruments). Our data show that cellular respiration in NALM6 was 50% lower compared to CCRF-CEM cells, suggesting that an intrinsic mitochondrial dysfunction was also responsible for 2-DG induced cell death in NALM6 cells. We then evaluated the observed higher sensitivity of BCR/ABL+ ALL cells to 2-DG. The AMPK and PI3K/Akt/mTOR pathways regulate protein, fatty acid and glucose metabolism, and key glycolytic enzymes are known to be upregulated in BCR/ABL+ cells. Therefore, we determined changes on AMPK and PI3K/Akt/mTOR signaling following exposure to 2-DG in SupB15 (BCR/ABL+) and in CCRF-CEM cells, the least sensitive ALL model examined. Western immunoblotting showed that 2-DG led to upregulation of p-AMPK (Thr172) and downregulation of p-mTOR (S2448) and p-p70S6K (Thr389). In both cell lines, 2-DG also led to cell death by triggering an unfolded protein response (UPR) evidenced by CHOP expression and PARP cleavage. We previously demonstrated that activation of AMPK leads to upregulation of Akt as a compensatory survival mechanism in ALL cells (Mol Cancer 6: 46, 2007). On this basis, we then tested induction of cell death by the combination of 2-DG (4 mM) plus Akt inhibitor X (AIX) (12 μM) in CCRF-CEM vs. SupB15 cells under normoxic conditions. Again, SupB15 cells were more sensitive to 2-DG, while AIX alone at low dose (12 μM) had minimal effect on either cell line. When both drugs were used in combination at these same concentrations, significant synergism was seen in CCRF-CEM cells but not in SupB15 cells. Analysis of AMPK, mTOR and Akt in CCRF-CEM cells treated with the combination of 2-DG plus AIX showed higher p-AMPK activation and mTOR downregulation compared to 2-DG alone, while these signaling changes were lower in SupB15 cells. Consistent with these findings, this combination led to higher induction of UPR and PARP cleavage in CCRF-CEM vs. SupB15 cells. We conclude that the greater sensitivity of ALL cells to 2-DG under normoxia is due to concomitant inhibition of glycolysis and/or glycosylation, an inherent mitochondrial dysfunction in some phenotypes, and 2-DG induced changes in AMPK and PI3K/Akt/mTOR signaling leading to the induction of UPR. Concomitant Akt inhibition sensitizes the relatively 2-DG resistant CCRF-CEM cells to death with 2-DG. Chemotherapy resistant BCR/ABL+ ALL cells exhibit significant sensitivity to 2-DG, likely due to their increased reliance on glycolysis for ATP generation. On this basis, we propose that glycolytic inhibitors represent a promising novel therapeutic strategy for ALL, particularly for BCR/ABL+ ALL. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 824 Background: Although acute myeloid leukemia (AML) with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) [hereafter referred to as inv(16)] is considered as a favorable AML subset, 30-40% of the patients (pts) are not cured by the current treatment strategies. Various genetic markers have been assessed to identify pts who are at high risk to fail therapy. However, the results among the different studies were not fully consistent. Methods: We studied diagnostic bone marrow and/or blood specimens from 179 adult AML pts with inv(16) for secondary chromosome abnormalities and gene mutations in FLT3 [internal tandem duplications (ITD) and tyrosine kinase domain mutations (TKD)], KIT, JAK2 (V617F) and in N-and K-RAS. All pts were treated on one of 7 prospective protocols of the German-Austrian AML Study Group (AMLSG) including anthracycline- and cytarabine-based induction therapy, and consolidation therapy incorporating higher doses of cytarabine in various settings, but also autologous and allogeneic stem cell transplantation. Multivariable analyses were performed to assess the prognostic value of gene mutations on relapse-free (RFS) and overall survival (OS) and were stratified for treatment protocols. Results: At least one gene mutation was found in 84% of the pts with 21% of the pts having multiple mutations. Pts with gene mutations ≥2 showed no significant differences in WBC, incidence of trisomy 22 and age. Mutations were most frequent in RAS (53%), followed by mutations in KIT (37%) and FLT3 (17%) [FLT3-TKD (15%) and FLT3-ITD (4%)] genes. No mutations were detected in JAK2. Concurrent mutations of KIT and RAS in the same leukemia sample were less likely to occur (P=0.003) than expected based on their frequencies as single markers; no other significant interactions between the gene mutations were observed. Median age of the cohort was 41 years (yrs; range, 18-74 yrs), and median white blood count (WBC) was 38.3 × 109/l (range, 1.1 to 294.9 × 109/l). Median follow-up for survival according to Korn was 4.9 yrs [95%-confidence interval (CI), 4.3-6.0 yrs]. While one multivariable model was constructed using the mutations in KIT, FLT3 and RAS as covariates (model I), the other included the number of mutated genes (≥2 vs

Beschreibung: Abstract 4856 Objective The multiparametric flow cytometry is becoming a very useful tool of the diagnosis and prognostication for patients with Myelodysplastic Syndrome(MDS). This study was aimed at using multiparametric flow cytometry to explore the immunophenotypic abnormalities of bone marrow cells from patients with RAEB. Methods We collected BM samples from 12 MDS-RAEB patients (6 male, 6 female, Median Age 67.5) and 20 non-MDS patients (11 male, 9 female, median age 32.5, 7 AA, 5 PNH, 3 IDA, 1 ALL, 2 CML, 2 MM). The multiparametric flow cytometric analysis was performed using an extensive panel of monoclonal antibodies. We used the conventional and secondary gating strategies to analysis the BM cells compartments such as the percents of blast cells and the expression of lineage and maturation-associated antigens of BM hemopoietic cells quantified. Results Compared with the non-MDS group, the proportion of blast cells increased significantly in the MDS-RAEB group (P=0.001), but the percentages of nucleated erythrocyte, lymphocyte, monocyte and granulocyte were no significant difference (P=0.954, P=0.893, P=0.730 and P=0.182). As the percentage of blasts cells increasing, the survival time became shorter (13 ± 6 vs 35 ± 15 months; P=0.02). The expressions of haemopoietic stem/progenitor cell surface marker CD34+ and T lymphocyte surface marker CD7+ on blast cells were much higher by secondary gating method than non-MDS group (P=0.009, P=0.002, respectively), while no significant difference of the expression of CD56 (P=0.375). The expressions of CD7+ and CD56+ on lymphocyte were no significant difference between the two groups (P=0.195, P=0.369, respectively), however the expression of CD19+ may be different (P=0.039). The expressions of CD33+ and CD13+ on granulocyte were no significant difference between the two groups (P=0.289, P=0.744, respectively). However, the expression levels of CD15+CD11b+, CD15+CD11b-, CD10+, HLA-DR, CD56+ in the MDS-RAEB group were significantly higher than those in the non-MDS group, specially, the levels of CD10+, HLA-DR and CD56+ were much higher (P=0.016, P=0.011, P=0.005, P=0.005 and P=0.005, respectively) and these patients showed a shorter median overall survival (15 ± 5 vs 36 ± 10 months; p = 0.03). Conclusions The percentage of blast cells increased in MDS-RAEB patients and the expressions of CD34+, CD7+ on blast cells and the expressions of CD10+, HLA-DR and CD56+ on granulocyte is higher than non-MDS group. It will be necessary to increase the number of cases (MDS-RAEB) to confirm our findings. Using multiparametric flow cytometry can find the immunophenotypic abnormalities more sensitively, accurately and objectively, and this new approach could provide much more useful information in the diagnosis and prognosis of MDS-RAEB patients. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 4161 Acute myeloid leukemia (AML) is a serious and often lethal hematopoietic malignant disease arising from stem cells. Leukemic stem cells (LSCs) play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population. We previous found that LSC were resistant to chemotherapy and NK-mediated cytotoxicity, which were resulted from the apoptosis defect and NKG2D ligands change. Our previous data also showed that Manumycin induced apoptosis in leukemia cells through mitochondria pathway. Here, we demonstrated that manumycin increased the sensitivity of LSC to chemotherapy and NK cell-mediated cytotoxicity. Manumycin enhanced sensitivity to chemotherapy in LSCs via induced apoptosis. Analysis of signaling pathways revealed that manumycin enhanced mitoxantrone activation of caspase 3 and significantly decreasing Bcl2 protein. These finding indicated that manumycin sensitized mitoxantrone to chemotherapy via down-regulating Bcl2. Importantly, manumycin enhanced the sensitivities of LSCs to NK cells cytotoxicity through up-regulating the expressions of NKG2D ligands MICA/B and ULBP3. Thus, Manumycin present a promising novel therapeutic approach for AML therapy especially when resistant to chemotherapy and NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: To evaluate internal tandem duplication (ITD) insertion sites and length as well as their clinical impact in younger adult patients with FLT3-ITD–positive acute myeloid leukemia (AML), sequencing after DNA-based amplification was performed in diagnostic samples from 241 FLT3-ITD–mutated patients. All patients were treated on 3 German-Austrian AML Study Group protocols. Thirty-four of the 241 patients had more than 1 ITD, leading to a total of 282 ITDs; the median ITD length was 48 nucleotides (range, 15-180 nucleotides). ITD integration sites were categorized according to functional regions of the FLT3 receptor: juxtamembrane domain (JMD), n = 148; JMD hinge region, n = 48; beta1-sheet of the tyrosine kinase domain-1 (TKD1), n = 73; remaining TKD1 region, n = 13. ITD length was strongly correlated with functional regions (P 〈 .001). In multivariable analyses, ITD integration site in the beta1-sheet was identified as an unfavorable prognostic factor for achievement of a complete remission (odds ratio, 0.22; P = .01), relapse-free survival (hazard ratio, 1.86; P 〈 .001), and overall survival (hazard ratio, 1.59; P = .008). ITD insertion site in the beta1-sheet appears to be an important unfavorable prognostic factor in young adult patients with FLT3-ITD–positive AML. The clinical trials described herein have been registered as follows: AML HD93 (already published in 2003), AML HD98A (NCT00146120; http://www.ClinicalTrials.gov), and AMLSG 07-04 (NCT00151242; http://www.ClinicalTrials.gov).

Beschreibung: FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a “clean” FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM−/− cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM−/− cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM−/− cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex–dependent and –independent manner.

Beschreibung: Dyskeratosis congenita (DC) is a rare inherited form of bone marrow failure (BMF) caused by mutations in telomere maintaining genes including TERC and TERT. Here we studied the prevalence of TERC and TERT gene mutations and of telomere shortening in an unselected population of patients with BMF at our medical center and in a selected group of patients referred from outside institutions. Less than 5% of patients with BMF had pathogenic mutations in TERC or TERT. In patients with BMF, pathogenic TERC or TERT gene mutations were invariably associated with marked telomere shortening (≪ 1st percentile) in peripheral blood mononuclear cells (PBMCs). In asymptomatic family members, however, telomere length was not a reliable predictor for the presence or absence of a TERC or TERT gene mutation. Telomere shortening was not pathognomonic of DC, as approximately 30% of patients with BMF due to other causes had PBMC telomere lengths at the 1st percentile or lower. We conclude that in the setting of BMF, measurement of telomere length is a sensitive but nonspecific screening method for DC. In the absence of BMF, telomere length measurements should be interpreted with caution.

Beschreibung: Abstract 3098 Poster Board III-35 In a subset of AML with a normal karyotype, a frame-shift mutation in the extreme C-terminal of the nucleophosmin (NPM) gene results in the creation of a nuclear export signal, generating a mutant NPM protein (NPMc) that is permanently dislocated in the cytoplasm. In the present study, we have analyzed the interaction between NPMc and a cytoplasmic subcomplex of Fanconi anemia (FA) proteins. Sequence analysis of bone marrow samples from 46 FA patients shows that NPMc mutations were excluded from FA genome. NPMc was degraded more rapidly in AML bone marrow cells from FA patients (t1/2 〈 30 min) than in AML cell line HL60 (t1/2 〉 90 min). Further analysis revealed that inducible knockdown of FANCA or FANCC in leukemic OCI/AML3 cells carrying the NPMc mutation induced degradation of the cytoplasmic NPMc protein. Forced localization of FANCC to the nucleus also caused rapid NPMc degradation. We also show that NPMc degradation was mediated by the proteasome and that correction of mutant lymphoblasts from FA-A or FA-C patients with a functional FANCA or FANCC protein prevented NPMc ubiquitination and consequently degradation. Moreover, we demonstrate that the cytoplasmic FANCA and FANCC interacted with NPMc in the cytosolic fractions of normal human lymphoblaststic cells and that the acidic domains of NPM were required for the cytoplasmic FA-NPMc complex formation. Finally, using patient-derived FANCC mutant, a nuclearized FANCC and a NOG/SGM3 xenotansplant model, we present evidence that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability and biological function. Thus, these findings reveal the potential molecular mechanism involved in the cytoplasmic retention of the leukemic NPMc. Disclosures No relevant conflicts of interest to declare.