ALBERT

Description: In addition to their role in desensitization and internalization of G protein-coupled receptors (GPCRs), β-arrestins are essential scaffolds linking GPCRs to Erk1/2 signaling. However, their role in GPCR-operated Erk1/2 activation differs between GPCRs and the underlying mechanism remains poorly characterized. Here, we show that activation of serotonin 5-HT2C receptors, which engage Erk1/2 pathway via a β-arrestin-dependent mechanism, promotes MEK-dependent β-arrestin2 phosphorylation at Thr383, a necessary step for Erk recruitment to the receptor/β-arrestin complex and Erk activation. Likewise, Thr383 phosphorylation is involved in β-arrestin-dependent Erk1/2 stimulation elicited by other GPCRs such as β2-adrenergic, FSH and CXCR4 receptors, but does not affect the β-arrestin-independent Erk1/2 activation by 5-HT4 receptor. Collectively, these data show that β-arrestin2 phosphorylation at Thr383 underlies β-arrestin-dependent Erk1/2 activation by GPCRs.

Description: We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units–fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

Description: Introduction Bone marrow niche of acute myeloid leukemia (AML) is a highly specialized microenvironment that regulates leukemic progression by favoring leukemic cell quiescence and chemoresistance. Niche components, especially mesenchymal stromal/stem cells (MSCs), have pro-survival effects on leukemia cells by protecting them from chemotherapy-induced apoptosis. The protection mechanisms of AML cells by the microenvironment are extensively studied to identify pharmacological targets to prevent AML relapse after chemotherapy. Direct intercellular communications between hematopoietic stem cells and MSCs involve connexins, such as connexin 43 (Taniguchi Ishikawa E et al. Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells. Proc Natl Acad Sci U S A. 2012,109:9071-6). The connexins are key components of gap junctions and we postulated in this study that blocking their assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. To this end we evaluated the effects of carbenoxolone (CBX), a glycyrrhetinic acid derivative known to block gap junctions (Rozental R et al. How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 2001;154:447-76), already evaluated in the treatment of human diseases. Methods Primary AML blasts isolated from bone marrow patients and 8 AML cell lines (KG1-a, KG1, HL-60, THP-1, MOLM-13, MV4-11, ML2, NB4) were exposed to CBX to assess cell viability and proliferation (Trypan blue exclusion and MTT assays). To study the combined drug effects of CBX and cytarabine (Ara-C), isobolograms were determined by co-treatment of AML cell lines with various doses of these two molecules. The metabolic effects of CBX on AML live cells were investigated with Seahorse® analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and high-throughput metabolic profiling was performed by OmniLog® technology. Direct cell-cell interactions were studied in a co-culture system of AML cells and bone marrow primary MSCs combining or not CBX (150 µM) and Ara-C (1 µM). After 48h (37°C, 5% CO2), AML cells adherent to MSCs were collected and the two populations were discriminated by flow cytometry studies (AML cells: CD45+CD90- / MSCs: CD45-CD90+) to quantify cell number apoptosis/necrosis (AnnexinV/7AAD assay). Gap junction inhibition by CBX was checked by calcein transfer (dye transferred mainly by gap junction channels) from labeled MSCs to AML cells in the co-culture system. Statistical analyses were performed with Mann-Whitney-Wilcoxon test. Results Treatment of AML cell lines with CBX reduced cell growth and viability in a time- and dose-dependent manner and the CBX IC50 was around 150 µM. Moreover, the exposure for 48h of primary AML blast to this dose of CBX induced a two time decrease in the number of viable cells. Interestingly, the isobolograms of the 8 AML cells lines identified three different profiles of resistance to chemotherapy and a synergistic effect between CBX and Ara-C. Regarding the energy metabolism, the analysis of all AML cell lines showed a major reduction of OCR and ECAR after treatment with CBX (150 µM, 48h) regardless their chemoresistance to Ara-C. Moreover, treated AML cells lacked their metabolic capacities for utilization of numerous substrates. In the co-culture experiments, contact with MSCs induced resistance to Ara-C; the apoptosis/necrosis rate observed after Ara-C exposure was 4.3- and 6.4-times decreased by MSC-contact for KG1a cells (n=5, p=0.0115) and primary blasts (n=5, p=0.0001), respectively. Co-treatment of leukemic cells with CBX and Ara-C reversed these deleterious effects of MSC-contact; the apoptosis/necrosis rate observed in MSC-contact leukemic cells was 5- and 2-times increased by CBX co-treatment for KG1a cells (n=5, p=0.007) and primary blasts (n=5, p=0.042), respectively. Finally, CBX induced a 48%-decrease in calcein transfer between leukemic cells and MSCs, highlighting its role as a gap junction inhibitor. Conclusion Niche-induced chemoresistance is associated with the mechanism of AML relapse after initial well-conducted chemotherapy. Combined to chemotherapy, CBX could be of interest to reduce the deleterious effects of leukemic niche by targeting gap junctions. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 951 AML cells with CD34+CD38-CD123+ phenotype represent a subset enriched in leukemic stem cells. Moreover, this subpopulation has been described to be resistant to genotoxic agents as compared with leukemic bulk. However, the clinical impact of the amount of CD34+CD38-CD123+ remains poorly described. In this study we evaluated the prognostic impact of the amount of CD34+CD38-CD123+ cells detected at diagnosis in a series of AML patients treated by intensive chemotherapy according to trials from the French GOELAMS group. Quantification of blast cells with the CD34+CD38-CD123+ phenotype was achieved by flow cytometry in 111 patients less than 66 years old with de novo AML treated by 3+7-like chemotherapy. The characteristics of the patients are shown in table 1. Age, WBC count, NPM1 mutation and FLT3-ITD had no impact on achievement of complete response (CR) whereas, CD34+CD38-CD123+ (〉15%) and unfavourable karyotype were significantly correlated with lack of CR. By logistic regression, CD34+CD38-CD123+ (〉15%) retains significance for CR achievement with an OR of 0.3 (0.11-0.84) (p=0.02). For the 91 complete responders, age, WBC count, karyotype, NPM1 mutation had no impact on disease-free survival (DFS).Interestingly, patients with 15% CD34+CD38-CD123+ had a median DFS of 57.6 (SE 6.6), 11.2 (SE 7.5) and 9.2 (SE 13.4) months, respectively (p

Description: The PI3K/Akt pathway is an important signaling pathway governing cell survival, proliferation and drug resistance in AML. However, the prognosis impact of Akt phosphorylation in AML has yielded some controversies as far as the phosphorylation on Ser473 is concerned. Because full activation of Akt requires phosphorylation on both Thr308 and Ser473, the assessment of these two sites would allow a more complete analysis of Akt activation in AML cells. We have studied the level of phosphorylation on both sites in primary AML samples by flow cytometry. We retrospectively assessed samples from 58 AML patients treated between 12/2000 and 02/2005. The clinical characteristics were: median age, 46.5 y (15–61); sex ratio (M/F) 1.24; FAB, 1 M0, 29 M1/M2, 25 M4/M5, 1 M6, 2 unclassified; median WBC, 33.4 G/L (1.2–225); Hb, 9.2 g/dL (5–13.5); platelets, 48 G/L (6–328); % marrow blasts, 78 (20–97); cytogenetic risk group (favorable, 28%; intermediate, 31%; unfavorable, 19%); FLT3-ITD, 10/54 (19%). The complete response rate after induction chemotherapy (anthracyclines + ara-C) was 83%. Patients in CR1 were referred to consolidation chemotherapy, autologous or allogeneic stem-cell transplantion according to the cytogenetic risk. The global overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) were of 22.1, 14.1, and 15.6 months, respectively. The levels of Akt phosphorylation (Thr308 and Ser473) were evaluated on pretreatment marrow samples and expressed by the ratio between mean fluorescence intensity of the stained AML sample and non-relevant IgG control (rMFI). The values of both Thr308 and Ser473 represented a continuum ranging from 0.3 to 5.6 and 0.4 to 2.87, respectively. The median level of phosphorylation was 2.3 (±0.89) and 1.3 (±0.27) for Thr308 and Ser473, respectively. There were no significant correlations between age, gender, FAB, WBC, FLT3-ITD and Akt phosphorylation. However, the level of phosphorylation on Thr308 but not on Ser473 was significantly correlated with the cytogenetic risk group. Indeed, the median rMFIs on Thr308 were 1.95 (± 0.37); 2.2 (±1) and 3.1 (±0.49) for favorable, intermediate and unfavorable karyotype, respectively (p=0.0069). In univariate analysis, unfavorable karyotype, FLT3-ITD and Thr308high correlated with poor OS, EFS, and RFS. Thr308high patients (〉 median value) had significantly shorter OS (11 vs 47 months; p=0.01), EFS (9 vs 26 months; p=0.005) and RFS (10 months vs not reached; p=0.02) than Thr308low patients. In the Cox model for multivariate analysis, only the cytogenetic risk independently predicted worse RFS, EFS and OS. Neither mutation screening for AKT1 E17K in an independant series of 148 AML cases nor changes in the level of PTEN expression and phosphorylation could explain the increased phosphorylation of Thr308 in high-risk cytogenetics AML cells. However, we found a statistically significant correlation between protein phosphatase 2A (PP2A) activity and complex karyotypes. Indeed, PP2A activity was markedly reduced by 40% in complex karyotypes compared with normal karyotypes. To specifically assess the role of Akt in the survival and proliferation of complex karyotype AML cells, we used the allosteric Akt kinase inhibitor targeting Akt1 and Akt2 (Akt-i). Akt-i inhibited the phosphorylation of Akt on both sites in these cells. Akt-i inhibited the clonogenic activity of AML cells from normal and complex karyotype samples in a dose-dependent manner, but this effect was significantly more potent in complex than in normal karyotype samples. After 24 h incubation with 10 μM Akt-i, features of apoptosis were detected by Annexin V staining (25% ±3,38 in treated vs 14% in untreated cells). Moreover, Akt-i enhanced the toxicity of daunorubicin (37% ±2,9, Akt-i+dnr vs 25% ±3,5, dnr). We also detected Akt phosphorylation in the immature leukemic compartment (CD34+ CD38− CD123+). Accordingly, Akt-i also induced apoptosis and enhanced dnr activity in CD34+ CD38− CD123+ cells from complex karyotype samples. This study shows that AML cells with complex karyotype display high level of phosphorylation of AktThr308 and suggests that this anti-apoptotic pathway may represent a valuable therapeutic target. Thus, it will be important to determine in clinical trials if this aberrant activation of Akt sensitizes AML cells to PI3K/Akt inhibitors specifically in this subgroup of very high-risk patients.

Description: The number of peripheral white blood cells (WBC) shows a wide range in acute myeloblastic leukemia (AML) patients. A high WBC count constitutes an adverse prognosis factor when at presentation and (myelo)monocytic subtypes were more frequently associated with extramedullar tumor sites. Marrow endothelial cells express CD31 and CD31/CD31 interaction is known to promote the migration in physiological mechanisms, as for example transendothelial migration of neutrophiles and monocytes in diapedesis. CD31 is a specific receptor of CD38 which is associated with numerous molecules on the surface of blood cells. Moreover, CD38 could interact with hyaluronate, a component of the extracellular matrix, 2 hyaluronate-binding sites having been reported in its extracellular domain. These elements lead us to postulate that CD31 and CD38 are colocalized on AML cells and that an excess of CD31 promotes the egress of the cells from the marrow compartment, while an excess of CD38 favors their anchorage to marrow microenvironment. The CD38/CD31 colocalization was demonstrated using FRET and cocapping strategies. FRET experiments were performed with a 488 nm laser flow cytometer, Cy3-conjugated anti-CD38 mAb and FITC-conjugated mAb against CD31. For cocapping experiments, we induced capping of CD31 with anti-CD31 mAb at 37°C; the cells were then fixed and labeled with anti-CD38 mAb. Transendothelial migration has been studied using anti-CD31 mAb and anti-CD38 blocking mAbs in Transwell® experiments performed with TrHBMEC cell line. To study the CD38/hyaluronate interaction, cells were treated or not with all-trans retinoic acid to induce CD38 overexpression just before plating the cells onto hyaluronate-coated dish. The in vivo influence of CD31 and CD38 coexpression level was evaluated by studying the phenotype of marrow leukemic cells (S/N ratio of the MFI) and the WBC count of 78 consecutive patients with newly diagnosed de novo AML. In all experiments, P value

Description: Abstract 659 The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a critical role in a variety of tumor cells including hematological malignancies. Class IA PI3Ks are heterodimers that consist of a p85 regulatory and a p110 catalytic subunit. There are several isoforms of both the catalytic (p110α, p110β and p110δ) and regulatory subunits. The p110α isoform of the class IA PI3-Ks was recently genetically validated as a promising target for anticancer therapy. To date, only one compound (imidazo[1,2-a]pyridine, PIK-75) has been described as a very potent and selective inhibitor of this isoform (〉100-fold selectivity over p110α and p110δ). In acute myeloid leukemia (AML), aberrant PI3-K activation is detectable in most of cases both in leukemic bulk and in the immature compartment of the leukemic clone. This activation contributes to cell growth, proliferation, survival, and drug-resistance. Furthermore, we have previously shown that the level of Akt phosphorylation on threonine 308, the major target of PI3-K, is correlated with poor outcome in AML patients (Gallay et al, Leukemia 2009, 23(6):1029-38). Therefore, effective targeting of this pathway with pharmacological inhibitors could improve therapeutic outcome in AML. Here, we studied the anti-neoplastic activity of several inhibitors of the PI3-K p110 subunits in AML cell lines and primary patient specimens. Treatment with PIK-75 led to a decrease of the proliferation in all cell lines at low dose (MTT assay, IC50: 62 nM, 144 nM, 173 nM in KG1, HL60 and KG1a cell lines, respectively). This inhibition of proliferation was due to massive apoptosis of KG1 and KG1a cells in both liquid culture but also after adhesion of leukemic cells on a fibronectin matrix. By contrast, p110ß (TGX221, 10 μM) and p110γ (AS252424, 10 μM) inhibitors only slightly decreased cell proliferation in KG1 and HL60 cells while p110δ inhibitor (IC87114) has no effect up to 10 μM. PIK-75 inhibited the phosphorylation of Akt on Thr308, and downstream effectors (4-EBP1 and RPS6) in these cells. These results strongly suggest a major role of p110α subunit which is highly express in AML cell lines and 19/19 patients samples. Next, we assessed the PIK-75 efficacy in 1 AML cell lines, 9 AML samples and 1 normal bone marrow CD34+ cells using clonogenic assays. PIK-75 inhibited AML-CFU in both KG1 cell line and all patient samples tested with an IC50 of 214 nM and 72 nM, respectively. Interestingly, PIK-75 has no effect on normal CFU-GM colonies, even at high dose (IC50 not reached at 1 μM), a result consistent with the normal haematopoiesis observed in p110αfnKO mice (Gritsman et al, Blood (ASH Annual Meeting Abstracts) 2009 114: Abstract 3620). Since leukemic subpopulation bearing the CD34+CD38-CD123+ phenotype is thought to be more resistant to chemotherapy than the leukemic bulk, we have assessed the apoptosis of 17 AML primary cells treated with increasing doses of cytarabine (Ara-C). We found significant differences of IC50 with 9.5 μM and 43 μM in bulk and CD34+38-123+ subpopulation, respectively. By contrast, PIK-75 demonstrated potent activity in both leukemic compartments of 42 AML samples with IC50 of 589 nM and 638 nM, respectively. Interestingly, the effect of PIK75 was not altered at relapse neither in bulk (IC50: 513 nM vs 492 nM at diagnosis and at relapse) nor in CD34+38-123+ subpopulation (IC50: 567 nM vs 254 nM at diagnosis and at relapse) in 8 AML samples. In NOD/SCID mice engrafted with HL60 cells, PIK-75 delivered at 1 and 10 mg/kg/d for 4 days induced a significant decrease in tumor burden after apoptosis induction detected ex vivo by annexin V staining. Further in vivo studies using NSG mice engrafted with primary AML specimens are ongoing. These results demonstrate that PIK-75 is the most potent inhibitor of PI3-K in leukemic cells suggesting that the selective inhibition of the p110α subunit could be a critical target in AML. Moreover, PIK-75 targets both leukemic bulk and chemoresistant leukemic subpopulations paving the way for clinical studies assessing the combination of selective p110α inhibitor with conventional chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Cytokins, N6-subsitued adenine derivatives play an important role in many different processes in plant development (Mok et al, Annu Rev Plant. Physiol Plant Mol Biol2001, 52:89–118), including the cell growth and division control, the vegetable cell differentiation with auxin and the storage of various metabolites as alkaloids (Yahia et al, Plant Science1998, 133:9–15). Furthermore, several studies suggest that cytokinins and their purin derivated are able to control mammalian cell apoptosis and differentiation of human leukemic cells (HL-60 myeloblastic cell line) into mature granulocytes (Ishii et al, Cell Growth Differ2002, 13: 19–26). All these compounds exert their biological activity via Cyclin-Dependant Kinase (CDK) inhibition and particulary CDK1 and CDK2. The aim of our study was the synthesis of new 7-azaindole derivates as cytokinin analogues and the evaluation of their biological effects on HL-60 cells. Eight analogues of 7-azaindole were prepared by the condension of (N-methyl-)4-chloropyrrolo[2,3-b]pyridine with corresponding amines using palladium-catalyzed reaction (Hartwig et al, Angew Chem IN Ed1998, 37: 2046–2067). The four derivates from 1H-pyrrolo[2,3-b]pyridine were 4-phenylamino-7-azaindole, 4-benzylamino-7-azaindole, 4-phenethylamino-7-azaindole and 4-phenylpiperazylamino-7-azaindole. The four derivates from 1-methyl-pyrrolo[2,3-b]pyridine were 4-phenylamino-N-methyl-7-azaindole, 4-benzylamino-N-methyl-7-azaindole, 4-phenetylamino-N-methyl-7-azaindole and 4-phenylpiperazylamino-N-methyl-7-azaindole. HL-60 cells were exposed to three concentrations of these compounds (10–100–500 μM) during 72h at 37°C. The number of viable cells was determined by Trypan blue exclusion, and the cell cycle was assessed by propidium iodide staining followed by flow cytometric analysis. All these compounds decreased the number of viable cells. The compounds of the NH serie were more active than their methyl-analogues, especially 4-phenylamino-N-methyl-7-azaindole and 4-phenethylamino-7-azaindole which presented an estimated IC50 of 2 μM. Moreover, when used at 10 μM, 4-phenylamino-N-methyl-7-azaindole induced apoptosis whereas 4-phenethylamino-7-azaindole promoted inhibition of the cell cycle without pro-apoptotic effect. These results suggest that cytokinin analogues derived from 1H-pyrrolo[2,3-b]pyridine may present interesting therapeutic potential as cytostatic agents. Further studies will clarify their biological effects on leukemic cells.