Predicted Reversal in N-Methylazepine/N-Methyl-7-azanorcaradiene Equilibrium Upon Formation of Their N-Oxides.

Density functional calculations and up to five different basis sets have been applied to the exploration of the structural, enthalpy and free energy changes upon conversion of the azepine to the corresponding N-oxide. Although it is well known that azepines are typically much more stable than their 7-azanorcaradiene valence isomers, the stabilities are reversed for the corresponding N-oxides. Structural, thermochemical as well as nucleus-independent chemical shift (NICS) criteria are employed to probe the potential aromaticity, antiaromaticity and nonaromaticity of N-methylazepine, its 7-azanorcaradiene valence isomer. For the sake of comparison, analogous studies are performed on N-methylpyrrole and its N-oxide.

Computational Study of Selected Amine and Lactam N-Oxides Including Comparisons of N-O Bond Dissociation Enthalpies with Those of Pyridine N-Oxides.

A computational study of the structures and energetics of amine N-oxides, including pyridine N-oxides, trimethylamine N-oxide, bridgehead bicyclic amine N-oxides, and lactam N-oxides, allowed comparisons with published experimental data. Most of the computations employed the B3LYP/6-31G* and M06/6-311G+(d,p) models and comparisons were also made between the results of the HF 6-31G*, B3LYP/6-31G**, B3PW91/6-31G*, B3PW91/6-31G**, and the B3PW91/6-311G+(d,p) models. The range of calculated N-O bond dissociation energies (BDE) (actually enthalpies) was about 40 kcal/mol. Of particular interest was the BDE difference between pyridine N-oxide (PNO) and trimethylamine N-oxide (TMAO). Published thermochemical and computational (HF 6-31G*) data suggest that the BDE of PNO was only about 2 kcal/mol greater than that of TMAO. The higher IR frequency for N-O stretch in PNO and its shorter N-O bond length suggest a greater difference in BDE values, predicted at 10-14 kcal/mol in the present work. Determination of the enthalpy of sublimation of TMAO, or at least the enthalpy of fusion and estimation of the enthalpy of vaporization might solve this dichotomy. The "extra" resonance stabilization in pyridine N-oxide relative to pyridine was consistent with the 10-14 kcal/mol increase in BDE, relative to TMAO, and was about half the "extra" stabilization in phenoxide, relative to phenol or benzene. Comparison of pyridine N-oxide with its acyclic model nitrone ("Dewar-Breslow model") indicated aromaticity slightly less than that of pyridine.

Phenotypic landscape of granulocytes and monocytes by multiparametric flow cytometry: A prospective study of a 1-tube panel strategy for diagnosis and prognosis of patients with MDS.

BACKGROUND: Multiparametric flow cytometry (MFC) was recently reported to be a helpful additional tool in the diagnosis of myelodysplastic syndromes (MDS). However, numerous aberrancies have been reported that makes their evaluation difficult as part of a routine diagnosis. METHODS: Here, we validated a 1-tube panel for the evaluation of granulocytic and monocytic maturation by MFC and correlated our findings with diagnosis and prognosis of MDS. A total of 251 samples with MDS suspicion were prospectively analyzed and compared to an internal reference database leading to the calculation of the Diff score. RESULTS: The associated specificity and sensitivity values of this scoring system were 92.1% and 60.4% in a first learning cohort and 96.7% and 65.2% in a second independent validation cohort. The combination of the Diff score with the concomitantly calculated Ogata score increased the sensitivity to 74.2% and 78.3% in the learning and validation cohorts, respectively. Finally, a normal Diff score in MDS patients was associated with a significant prolonged progression-free survival. CONCLUSIONS: Taken together, the present data indicate that our strategy is a sensitive and specific MFC tool for the diagnosis of MDS-related cytopenia(s) which could be also useful for predicting evolution of these diseases.

FLT3 inhibitors: clinical potential in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy that is cured in as few as 15%-40% of cases. Tremendous improvements in AML prognostication arose from a comprehensive analysis of leukemia cell genomes. Among normal karyotype AML cases, mutations in the FLT3 gene are the ones most commonly detected as having a deleterious prognostic impact. FLT3 is a transmembrane tyrosine kinase receptor, and alterations of the FLT3 gene such as internal tandem duplications (FLT3-ITD) deregulate FLT3 downstream signaling pathways in favor of increased cell proliferation and survival. FLT3 tyrosine kinase inhibitors (TKI) emerged as a new therapeutic option in FLT3-ITD AML, and clinical trials are ongoing with a variety of TKI either alone, combined with chemotherapy, or even as maintenance after allogenic stem cell transplantation. However, a wide range of molecular resistance mechanisms are activated upon TKI therapy, thus limiting their clinical impact. Massive research efforts are now ongoing to develop more efficient FLT3 TKI and/or new therapies targeting these resistance mechanisms to improve the prognosis of FLT3-ITD AML patients in the future.

Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia.

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.

Targeting CHK1 inhibits cell proliferation in FLT3-ITD positive acute myeloid leukemia.

CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in normal cell cycle progression. In this study, we investigate how CHK1 participates to proliferation of acute myeloid leukemia cells expressing the mutated FLT3-ITD tyrosine kinase receptor. Pharmacological inhibition of CHK1 as well as its shRNA mediated down regulation reduced the proliferation rate of FLT-ITD expressing leukemic cell lines in a cytostatic manner. Flow cytometry analysis revealed no accumulation in a specific phase of the cell cycle upon CHK1 inhibition. Accordingly, lentiviral-mediated CHK1 overexpression increased the proliferation rate of FLT3-ITD expressing cells, as judged by cell viability and [3H] thymidine incorporation experiments. By contrast, expression of a ser280 mutant did not, suggesting that phosphorylation of this residue is an important determinant of CHK1 proliferative function. Clonogenic growth of primary leukemic cells from patients in semi-solid medium was reduced upon CHK1 inhibition, confirming the data obtained with leukemic established cell lines. Surprisingly, 3 out of 4 CHK1 inhibitory compounds tested in this study were also potent inhibitors of the FLT3-ITD tyrosine kinase receptor. Altogether, these data identify CHK1 as a regulator of FLT3-ITD-positive leukemic cells proliferation, and they open interesting perspectives in terms of new therapeutic strategies for these pathologies.

Inhibiting glutamine uptake represents an attractive new strategy for treating acute myeloid leukemia.

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.

High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study.

BACKGROUND: Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34(+)CD38(low/-) compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34(+)CD38(low/-)CD123(+) phenotype. DESIGN AND METHODS: The percentage of CD34(+)CD38(low/-)CD123(+) cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. RESULTS: A proportion of CD34(+)CD38(low/-)CD123(+) cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34(+)CD38(low/-)CD123(+) higher than 15% retained significance with an odds ratio of 0.33 (0.1-0.97; P=0.044). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells retained prognostic significance for both parameters after multivariate analysis. CONCLUSIONS: The percentage of CD34(+)CD38(low/-)CD123(+) leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.

LKB1/AMPK/mTOR signaling pathway in hematological malignancies: from metabolism to cancer cell biology.

The link between cancer and metabolism has been suggested for a long time but further evidence of this hypothesis came from the recent molecular characterization of the LKB1/AMPK signaling pathway as a tumor suppressor axis. Besides the discovery of somatic mutations in the LKB1 gene in certain type of cancers, a critical emerging point was that the LKB1/AMPK axis remains generally functional and could be stimulated by pharmacological molecules such as metformin in cancer cells. Notably, most of experimental evidence of the anti-tumor activity of AMPK agonists comes from the study of solid tumors such as breast or prostate cancers and only few data are available in hematological malignancies, although recent works emphasized the potential therapeutic value of AMPK agonists in this setting. Further basic research work should be conducted to elucidate the molecular targets of LKB1/AMPK responsible for its anti-tumor activity in parallel of conducting clinical trials using metformin, AICAR or new AMPK activating agents to explore the potential of the LKB1/AMPK signaling pathway as a new target for anticancer drug development.

Dual inhibition of PI3K and mTORC1/2 signaling by NVP-BEZ235 as a new therapeutic strategy for acute myeloid leukemia.

PURPOSE: The growth and survival of acute myeloid leukemia (AML) cells are enhanced by the deregulation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Major efforts have thus been made to develop molecules targeting these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes: mTORC1 and mTORC2. The mTORC1 pathway is rapamycin sensitive and controls protein translation through the phosphorylation of 4E-BP1 in most models. In AML, however, the translation process is deregulated and rapamycin resistant. Furthermore, the activity of PI3K/Akt and mTOR is closely related, as mTORC2 activates the oncogenic kinase Akt. We therefore tested, in this study, the antileukemic activity of the dual PI3K/mTOR ATP-competitive inhibitor NVP-BEZ235 compound (Novartis). EXPERIMENTAL DESIGN: The activity of NVP-BEZ235 was tested in primary AML samples (n = 21) and human leukemic cell lines. The different signaling pathways were analyzed by Western blotting. The cap-dependent mRNA translation was studied by 7-methyl-GTP pull-down experiments, polysomal analysis, and [(3)H]leucine incorporation assays. The antileukemic activity of NVP-BEZ235 was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation, and survival. RESULTS: The NVP-BEZ235 compound was found to inhibit PI3K and mTORC1 signaling and also mTORC2 activity. Furthermore, NVP-BEZ235 fully inhibits the rapamycin-resistant phosphorylation of 4E-BP1, resulting in a marked inhibition of protein translation in AML cells. Hence, NVP-BEZ235 reduces the proliferation rate and induces an important apoptotic response in AML cells without affecting normal CD34(+) survival. CONCLUSIONS: Our results clearly show the antileukemic efficiency of the NVP-BEZ235 compound, which therefore represents a promising option for future AML therapies.

Working ethics: William Beaumont, Alexis St. Martin, and medical research in antebellum America.

Analyzing William Beaumont's relationship with his experimental subject, Alexis St. Martin, this article demonstrates how the "research ethics" of antebellum America were predicated on models of employment, servitude, and labor. The association between Beaumont and St. Martin drew from and was understood in terms of the ideas and practices of contract labor, informal domestic servitude, indentures, and military service. Beaumont and St. Martin lived through an important period of transition in which personal master-servant relations existed alongside the "free" contract labor of market capitalism. Their relationship reflected and helped constitute important developments in nineteenth-century American labor history.

IκB kinase overcomes PI3K/Akt and ERK/MAPK to control FOXO3a activity in acute myeloid leukemia.

The FOXO transcription factors are involved in multiple signaling pathways and have tumor-suppressor functions. In acute myeloid leukemia (AML), deregulation of oncogenic kinases, including Akt, extra-signal-regulated kinase, or IκB kinase, is frequently observed, which may potentially inactivate FOXO activity. We therefore investigated the mechanism underlying the regulation of FOXO3a, the only FOXO protein constantly expressed in AML blast cells. We show that in both primary AML samples and in a MV4-11/FOXO3a-GFP cell line, FOXO3a is in a constant inactive state due to its cytoplasmic localization, and that neither PI3K/Akt nor extra-signal-regulated kinase-specific inhibition resulted in its nuclear translocation. In contrast, the anti-Nemo peptide that specifically inhibits IKK activity was found to induce FOXO3a nuclear localization in leukemic cells. Furthermore, an IKK-insensitive FOXO3a protein mutated at S644 translocated into the nucleus and activated the transcription of the Fas-L and p21(Cip1) genes. This, in turn, inhibited leukemic cell proliferation and induced apoptosis. These results thus indicate that IKK activity maintains FOXO3a in the cytoplasm and establishes an important role of FOXO3a inactivation in the proliferation and survival of AML cells. The restoration of FOXO3a activity by interacting with its subcellular distribution may thus represent a new attractive therapeutic strategy for AML.

The LKB1/AMPK signaling pathway has tumor suppressor activity in acute myeloid leukemia through the repression of mTOR-dependent oncogenic mRNA translation.

Finding an effective treatment for acute myeloid leukemia (AML) remains a challenge, and all cellular processes that are deregulated in AML cells should be considered in the design of targeted therapies. We show in our current study that the LKB1/AMPK/TSC tumor suppressor axis is functional in AML and can be activated by the biguanide molecule metformin, resulting in a specific inhibition of mammalian target of rapamycin (mTOR) catalytic activity. This induces a multisite dephosphorylation of the key translation regulator, 4E-BP1, which markedly inhibits the initiation step of mRNA translation. Consequently, metformin reduces the recruitment of mRNA molecules encoding oncogenic proteins to the polysomes, resulting in a strong antileukemic activity against primary AML cells while sparing normal hematopoiesis ex vivo and significantly reducing the growth of AML cells in nude mice. The induction of the LKB1/AMPK tumor-suppressor pathway thus represents a promising new strategy for AML therapy.

Role of the PI3K/AKT and mTOR signaling pathways in acute myeloid leukemia.

The PI3K/AKT and mTOR signaling pathways are activated in acute myeloid leukemia, including in the more immature leukemic populations. Constitutive PI3K activation is detectable in 50% of acute myeloid leukemia samples whereas mTORC1 is activated in all cases of this disease. In leukemic cells, the PI3K activity relates to the expression of the p110delta isoform of class IA PI3K. Constitutive PI3K activation is the result of autocrine IGF-1/IGF-1R signaling in 70% of acute myeloid leukemia samples but specific inhibition of this pathway does not induce apoptosis. Specific inhibition of PI3K/AKT or mTORC1 alone in vitro has anti-leukemic effects which are essentially exerted via the suppression of proliferation. However, as mTORC1 activation is independent of PI3K/AKT in acute myeloid leukemia, dual PI3K and mTOR inhibitors may induce apoptosis in blast cells. Moreover, mTORC1 inhibition using sirolimus overactivates PI3K/AKT via the upregulation of IRS2 expression and by favoring IGF-1/IGF-1R autocrine signaling. Recent data also indicate that mTORC1 does not control protein translation in acute myeloid leukemia. These results open the way for the design of direct inhibitors of protein synthesis as novel acute myeloid leukemia therapies and also for the development of second generation mTOR inhibitors (the TORKinhibs).