Genomic tools for dissecting oncogenic transcriptional networks in human leukemia.

Chromatin immunoprecipitation (ChIP)-chip and ChIP-seq technologies are rapidly expanding our capacity to interrogate the location of transcription factor-binding sites in the human genome and to map the pattern of chromatin modifications associated with the regulation of gene expression. The application of these techniques to the study of hematologic malignancies will complement gene expression profiling studies to elucidate the structure and function of oncogenic transcriptional networks involved in the pathogenesis of leukemias and lymphomas.

Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.

CUTLL1, a novel human T-cell lymphoma cell line with t(7;9) rearrangement, aberrant NOTCH1 activation and high sensitivity to gamma-secretase inhibitors.

Activating mutations in NOTCH1 are present in over 50% of human T-cell lymphoblastic leukemia (T-ALL) samples and inhibition of NOTCH1 signaling with gamma-secretase inhibitors (GSI) has emerged as a potential therapeutic strategy for the treatment of this disease. Here, we report a new human T-cell lymphoma line CUTLL1, which expresses high levels of activated NOTCH1 and is extremely sensitive to gamma-secretase inhibitors treatment. CUTLL1 cells harbor a t(7;9)(q34;q34) translocation which induces the expression of a TCRB-NOTCH1 fusion transcript encoding a membrane-bound truncated form of the NOTCH1 receptor. GSI treatment of CUTLL1 cells blocked NOTCH1 processing and caused rapid clearance of activated intracellular NOTCH1. Loss of NOTCH1 activity induced a gene expression signature characterized by the downregulation of NOTCH1 target genes such as HES1 and NOTCH3. In contrast with most human T-ALL cell lines with activating mutations in NOTCH1, CUTLL1 cells showed a robust cellular phenotype upon GSI treatment characterized by G1 cell cycle arrest and increased apoptosis. These results show that the CUTLL1 cell line has a strong dependence on NOTCH1 signaling for proliferation and survival and supports that T-ALL patients whose tumors harbor t(7;9) should be included in clinical trials testing the therapeutic efficacy NOTCH1 inhibition with GSIs.

Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio).

Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.

Modulation of human erg K+ channel gating by activation of a G protein-coupled receptor and protein kinase C.

1. Modulation of the human ether-à-go-go-related gene (HERG) K+ channel was studied in two-electrode voltage-clamped Xenopus oocytes co-expressing the channel protein and the thyrotropin-releasing hormone (TRH) receptor. 2. Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization-induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH. 3. The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+. 4. The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with beta-phorbol 12-myristate, 13-acetate (PMA). The TRH-induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA-dependent regulation of the channels. 5. It is concluded that a PKC-dependent pathway links G protein-coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C-activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.

A G protein beta gamma dimer-mediated pathway contributes to mitogen-activated protein kinase activation by thyrotropin-releasing hormone receptors in transfected COS-7 cells.

Activation of mitogen-activated protein kinase (MAPK) is induced by adding thyrotropin-releasing hormone (TRH) to COS-7 cells cotransfected with TRH receptors and an epitope-tagged MAPK. Long term treatment of the cells with pertussis toxin has no effect on TRH-induced MAPK activation. Incubation of the cells with the protein kinase C (PKC) inhibitor GF109203X causes an almost complete inhibition of MAPK activation by the PKC activator phorbol-12-myristate-13-acetate. In contrast, only approximately 50% of the TRH-induced MAPK activity is inhibited by GF109203X, indicating that activation of MAPK by TRH is only partially dependent on PKC. The inhibitory effect of GF109203X is additive with that of p21(N17ras), a dominant negative mutant of p21(ras) that exerts little effect on PKC-dependent MAPK activation by phorbol-12-myristate-13-acetate. The TRH-induced activation of MAPK also is inhibited partially by overexpression of transducin alpha subunits (alpha t), an agent known to sequester free G protein beta gamma dimers. However, the inhibitory potency of alpha t on TRH-induced activation is about half of that obtained in cells transfected with m2 muscarinic receptors, which activate MAPK exclusively through beta gamma dimers. The effect of alpha t is also additive with that of GF109203X but not with that of p21(N17ras). MAPK activation is not induced by the constitutively active form of G alpha q due to an inhibitory effect of its expression at a step downstream of that at which PKC-dependent and -independent routes to MAPK converge. Our results demonstrate that TRH receptors activate MAPK by a pathway only partially dependent on PKC activity. Furthermore, they indicate that beta gamma dimers of a pertussis and cholera toxin-insensitive G protein are involved in the PKC-independent fraction of the dual signaling route to MAPK initiated in the TRH receptor.

Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells.

Reduction of an inwardly rectifying K+ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH3 rat anterior pituitary cells. However, the existence of an inwardly rectifying K+ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH3 cells. In this report, an inwardly rectifying component of K+ current is indeed demonstrated in perforated-patch voltage-clamped GH3 cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K+ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K+ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K+ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH3 cells.