Deciphering the Pathogenesis of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant disorder of the blood system. Hematopoietic stem cells (HSCs) supply and maintain this system by differentiating via intermediates into lineage-restricted progenitors that strongly proliferate to keep up with the high turn-over of mature blood cells. In AML, the mechanisms controlling differentiation and proliferation of myeloid cells are disturbed leading to the accumulation of undifferentiated cells that interfere with the production of normal blood cells. Mutations of the transcription factor C/EBPα have been observed in 10 percent in AML with normal cytogenetics. In addition, internal tandem duplications (ITD) of FLT3 are frequently observed alterations in AML and coincide with mutations of C/EBPα. The effects of FLT3-ITD cooperation with C/EBPα mutations in AML are not fully understood. To address this, knockin mouse strains harboring different Cebpa mutations and Flt3-ITD were used to generate an AML mouse model. This model demonstrated a block at the transition from pGMP to GMP due to disrupted C/EBPα function. The cooperative effect of FLT3-ITD is composed of enhancing the generation of leukemia-initiating GMPs and activation of STAT5 targets. In in vitro studies it was demonstrated that FLT3-ITD reduces the cytokine-requirements for cell growth and that leukemic cells harboring FLT3-ITD are more sensitive to inhibition of the FLT3 pathway in vitro. To address the impact of FLT3-ITD gene dosage and loss of Flt3 wild type allele in vivo the Flt3-ITD knockin mouse was crossed to the Flt3 receptor knockout mouse. These studies demonstrated that the myeloproliferative phenotype was FLT3-ITD dosage-dependent and independent of FL. In summary, the data presented provide deeper insights into oncogene cooperation and FLT3-ITD dosage in AML

Die Wirkung des Sekretolytikums Ambroxol an neuronalen Natriumkanälen durch die Interaktion mit der Lokalanästhetikumbindungsstelle

1. Background and Purpose Voltage-gated sodium channels (Nav) are critical for the generation and propagation of action potentials and thus for neuronal excitability. In sensory neurons, several Nav-subtypes are involved in transmitting nociceptive information from the periphery into the central nervous system (Chevrier et al., 2004). The tetrodotoxin-resistant (TTXr) subtype Nav1.8 is selectively expressed in nociceptive sensory neurons and is regarded a promising target for the development of novel analgesics. Several Na+ channel blockers are established as analgesics or co-analgesics in clinical practise. However, all these substances are non-selective Nav-blockers und thus their use is hampered by dose-limiting side effects (Gaida et al., 2005). With properties resembling those of classical local anesthetics, the secreolytic ambroxol has been reported to inhibit Na+ currents in sensory neurons with a preference for the TTXr current generated by Nav1.8 (Gaida et al., 2005; Weiser, 2006; Weiser et al., 2002). Accordingly, ambroxol reduces pain-related behaviour in rodents and alleviates pain in humans (Fischer et al., 2002; Schutz et al., 2002, Gaida et al., 2005). The present study was performed to explore molecular determinants for ambroxol-induced block of Na+ channels and to study the proposed preferential block of Nav1.8 compared to tetrodotoxin-sensitive (TTXs) Nav-subtypes in more detail. 2. Methods In this study, the effects of ambroxol and local anesthetics on the recombinant TTXr subtype Nav1.8, on neuronal TTXs subtypes and on local anesthetic insensitive mutants of Nav1.4 were investigated using the whole-cell patch clamp technique. 3. Key results Tonic and use-dependent block by ambroxol were strongly alleviated in local anesthetic-insensitive mutant-constructs of the TTXs Na+ channel Nav1.4. Use-dependent block, but not tonic block was significantly stronger on Nav1.8 than on TTXs channels. The TTXs subtype Nav1.3 displayed the least degree of use-dependent block by ambroxol. The classical local anesthetics mepivacaine and S(-)-bupivacaine also blocked Nav1.8 and TTXs channels differentially. While mepivacaine displayed a preferential use-dependent block of Nav1.8, S(-)-bupivacaine displayed a preference for TTXs Na+ channels. 4. Conclusions Ambroxol indeed acts similarly to local anesthetic on Na+ channels by interacting with specific residues in the S6 segments. This property most likely meditates the analgesic effect of ambroxol. Ambroxol preferentially blocks Nav1.8, however shares this property with commonly used local anesthetics like mepivacaine.1. Hintergrund und Ziele Spannungsabhängige Natriumkanäle (Nav) in Nervenzellmembranen sind wesentlich an der Erregungsbildung und der Ausbreitung von Aktionspotentialen beteiligt und spielen somit auch eine wichtige Rolle in der Weiterleitung von Schmerzreizen im peripheren bis in das zentrale Nervensystem (Chevrier et al., 2004). Unter den neuronalen Subtypen der Natriumkanäle wird der tetrodotoxin-resistente (TTXr) Nav1.8 Kanal selektiv in nozizeptiven, sensorischen Neuronen exprimiert und stellt mit dieser Eigenschaft ein viel versprechendes Ziel für die Entwicklung neuer Analgetika dar. Viele im klinischen Gebrauch etablierte Analgetika und Co-Analgetika sind zwar potente Natriumkanal-Blocker, durch ihre fehlende Selektivität für bestimmte Kanalsubtypen verursachen sie jedoch häufig dosis-limitierende Nebenwirkungen (Gaida et al., 2005). Das Sekretolytikum Ambroxol inhibiert hingegen bevorzugt jene TTXr Natriumströme, welche durch Nav1.8 Kanäle in sensorischen Neuronen generiert werden (Gaida et al., 2005; Weiser, 2006; Weiser et al., 2002). Des Weiteren reduziert Ambroxol im Tierversuch das Schmerzverhalten von Säugetieren und weist auch beim Menschen eine schmerzlindernde Komponente auf, beispielsweise in der Behandlung einer Pharyngitis (Fischer et al., 2002; Schutz et al., 2002, Gaida et al., 2005). Die vorliegende Studie wurde durchgeführt, um die molekularen Wirkmechanismen von Ambroxol an Natriumkanälen näher zu untersuchen sowie die vorbeschriebene Präferenz von Ambroxol für Nav1.8 Kanäle im Unterschied zu tetrodotoxin-sensiblen (TTXs) Natriumkanälen zu prüfen. 2. Methoden Um den Einfluss von Ambroxol auch im Vergleich zu anderen Lokalanästhetika studieren zu können, wurden der Block an dem TTXr Natriumkanalsubtyp Nav1.8, der Block an TTXs Natriumkanälen sowie die Inhibition des Wildtyps und der lokalanästhetika-unempfindlichen Mutationen des Nav1.4 Kanals mit der Whole-cell Patch-Clamp-Methode untersucht. 3. Ergebnisse und Beobachtungen An den für Lokalanästhetika unempfindlichen Mutationen des Nav1.4 Kanals stellte sich der tonische sowie der phasische „use-dependent“ Block stark abgeschwächt dar. Der „use-dependent“ Block, nicht aber der tonische Block, war an Nav1.8 Kanälen signifikant stärker ausgeprägt als an TTXs Kanälen in ND7/23 Zellen. Der TTXs Kanalsubtyp Nav1.3 zeigte den geringsten Grad an „use-dependent“ Block durch Ambroxol. Auch die im Vergleich verwendeten Lokalanästhetika Mepivacain und S(-)-Bupivacain hatten eine unterschiedliche Wirkung an den verschiedenen Kanälen. Während Mepivacain einen bevorzugten „use-dependent“ Block am Nav1.8 Kanal aufwies, war die S(-)-Bupivacainblockade an TTXs Kanälen ausgeprägter. 4. Schlussfolgerungen Unsere Daten zeigen, dass Ambroxol in der Tat ähnlich wie ein typisches Lokalanästhetikum fungiert, indem es mit spezifischen Aminosäuren im Segment S6 des Natriumkanals interagiert. Diese Eigenschaft vermittelt wahrscheinlich den analgetischen Effekt des Sekretolytikums. Ambroxol inhibiert dabei bevorzugt den Natriumkanalsubtyp Nav1.8, teilt diesen Effekt allerdings mit etablierten Lokalanästhetika, wie Mepivacain

Rapid testing leads to the underestimation of the scrapie prevalence in an affected sheep and goat flock

To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrPSc in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrPSc depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrPSc was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrPSc exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy

Hypoxic induction of vascular endothelial growth factor regulates murine hematopoietic stem cell function in the low-oxygenic niche.

Hypoxia is emerging as an important characteristic of the hematopoietic stem cell (HSC) niche, but the molecular mechanisms contributing to quiescence, self-renewal, and survival remain elusive. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis and hematopoiesis. Its expression is commonly regulated by hypoxia-inducible factors (HIF) that are functionally induced in low-oxygen conditions and that activate transcription by binding to hypoxia-response elements (HRE). Vegfa is indispensable for HSC survival, mediated by a cell-intrinsic, autocrine mechanism. We hypothesized that a hypoxic HSC microenvironment is required for maintenance or upregulation of Vegfa expression in HSCs and therefore crucial for HSC survival. We have tested this hypothesis in the mouse model Vegfa(δ/δ), where the HRE in the Vegfa promoter is mutated, preventing HIF binding. Vegfa expression was reduced in highly purified HSCs from Vegfa(δ/δ) mice, showing that HSCs reside in hypoxic areas. Loss of hypoxia-regulated Vegfa expression increases the numbers of phenotypically defined hematopoietic stem and progenitor cells. However, HSC function was clearly impaired when assessed in competitive transplantation assays. Our data provide further evidence that HSCs reside in a hypoxic microenvironment and demonstrate a novel way in which the hypoxic niche affects HSC fate, via the hypoxia-Vegfa axis

Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML

The key myeloid transcription factor (TF), CEBPA, is frequently mutated in acute myeloid leukemia (AML), but the direct molecular effects of this leukemic driver mutation remain elusive. To investigate mutant AML, we performed microscale, in vivo chromatin immunoprecipitation sequencing and identified a set of aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. Comparing gene expression changes in human mutant AML and the corresponding mouse model, we identified , encoding CD73, as a cross-species AML gene with an upstream leukemic enhancer physically and functionally linked to the gene. Increased expression of CD73, mediated by the CEBPA-p30 isoform, sustained leukemic growth via the CD73/A2AR axis. Notably, targeting of this pathway enhanced survival of AML-transplanted mice. Our data thus indicate a first-in-class link between a cancer driver mutation in a TF and a druggable, direct transcriptional target

Induction chemotherapy followed by concurrent standard radiotherapy and daily low-dose cisplatin in locally advanced non-small-cell lung cancer

Both induction chemotherapy and concurrent low-dose cisplatin have been shown to improve results of thoracic irradiation in the treatment of locally advanced non-small-cell lung cancer (NSCLC). This phase II study was designed to investigate activity and feasibility of a novel chemoradiation regimen consisting of induction chemotherapy followed by standard radiotherapy and concurrent daily low-dose cisplatin. Previously untreated patients with histologically/cytologically proven unresectable stage IIIA/B NSCLC were eligible. Induction chemotherapy consisted of vinblastine 5 mg m−2 intravenously (i.v.) on days 1, 8, 15, 22 and 29, and cisplatin 100 mg m−2 i.v. on days 1 and 22 followed by continuous radiotherapy (60 Gy in 30 fractions) given concurrently with daily cisplatin at a dose of 5 mg m−2 i.v. Thirty-two patients were enrolled. Major toxicity during induction chemotherapy was haematological: grade III–IV leukopenia was observed in 31% and grade II anaemia in 16% of the patients. The most common severe toxicity during concurrent chemoradiation consisted of grade III leukopenia (21% of the patients); grade III oesophagitis occurred in only two patients and pulmonary toxicity in one patient who died of this complication. Eighteen of 32 patients (56%, 95% CI 38–73%) had a major response (11 partial response, seven complete response). With a median follow-up of 38.4 months, the median survival was 12.5 months and the actuarial survival rates at 1, 2 and 3 years were 52%, 26% and 19% respectively. The median event-free survival was 8.3 months with a probability of 40%, 23% and 20% at 1, 2 and 3 years respectively. Induction chemotherapy followed by concurrent daily low-dose cisplatin and thoracic irradiation, in patients with locally advanced NSCLC, is active and feasible with minimal non-haematological toxicity. Long-term survival results are promising and appear to be similar to those of more toxic chemoradiation regimens, warranting further testing of this novel chemoradiation strategy. © 1999 Cancer Research Campaig

Synthesis of Indomorphan Pseudo Natural Product Inhibitors of Glucose Transporters GLUT‐1 and ‐3

Bioactive compound design based on natural product (NP) structure may be limited due to partial coverage of NP‐like chemical space and biological target space. These limitations can be overcome by combining NP‐centered strategies with fragment‐based compound design through combination of NP‐derived fragments to structurally unprecedented “pseudo natural products” (pseudo‐NPs). We describe the design, synthesis and biological evaluation of a collection of indomorphan pseudo‐NPs that combine biosynthetically unrelated indole‐ and morphan‐alkaloid fragments. Biological investigation in a cell‐based screen for modulators of glucose uptake identified the indomorphane derivative Glupin as potent inhibitor of glucose uptake. Glupin selectively targets and upregulates both, glucose transporters GLUT‐1 and GLUT‐3. Glupin suppresses glycolysis, reduces the levels of glucose‐derived metabolites and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT‐1 and GLUT‐3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity