Targeting BET proteins improves the therapeutic efficacy of BCL-2 inhibition in T-cell acute lymphoblastic leukemia.

Inhibition of anti-apoptotic BCL-2 (B-cell lymphoma 2) has recently emerged as a promising new therapeutic strategy for the treatment of a variety of human cancers, including leukemia. Here, we used T-cell acute lymphoblastic leukemia (T-ALL) as a model system to identify novel synergistic drug combinations with the BH3 mimetic venetoclax (ABT-199). In vitro drug screening in primary leukemia specimens that were derived from patients with high risk of relapse or relapse and cell lines revealed synergistic activity between venetoclax and the BET (bromodomain and extraterminal) bromodomain inhibitor JQ1. Notably, this drug synergism was confirmed in vivo using T-ALL cell line and patient-derived xenograft models. Moreover, the therapeutic benefit of this drug combination might, at least in part, be mediated by an acute induction of the pro-apoptotic factor BCL2L11 and concomitant reduction of BCL-2 upon BET bromodomain inhibition, ultimately resulting in an enhanced binding of BIM (encoded by BCL2L11) to BCL-2. Altogether, our work provides a rationale to develop a new type of targeted combination therapy for selected subgroups of high-risk leukemia patients.

Complementary activities of DOT1L and Menin inhibitors in MLL-rearranged leukemia.

Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.

CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage.

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, 'monocytoids' share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14(neg)CD19(pos)CD34(pos) B lymphoblasts and CD14(pos)CD19(neg)CD34(neg) 'monocytoids' were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.

The E2A-HLF oncogenic fusion protein acts through Lmo2 and Bcl-2 to immortalize hematopoietic progenitors.

The oncogenic fusion protein E2A-HLF is a chimeric transcription factor that arises from the t(17;19) translocation in childhood B-cell acute lymphoblastic leukemias (B-precursor ALL) and is associated with very poor outcome. We show that retroviral-mediated expression of E2A-HLF alone is sufficient to immortalize primary lymphoid progenitors. We identify Lmo2 and Bcl-2 as direct target genes downstream of E2A-HLF. We use real-time PCR analysis to show that LMO2 and BCL-2 expression is preferentially upregulated both in biopsy material from t(17;19) B-precursor ALL patients and lymphoid cell lines derived from t(17;19) leukemias. Co-expression of Lmo2 and Bcl-2 was sufficient to immortalize lymphoid progenitor cells resulting in a similar phenotype to that induced by E2A-HLF alone. Both shRNA-mediated knockdown of Lmo2 expression and pharmacological inhibition of BCL-2 function in E2A-HLF immortalized cells severely compromised their viability. These data suggest that both Lmo2 and Bcl-2 are required for the action of E2A-HLF in leukemogenesis.

MSAP enhances migration of C6 glioma cells through phosphorylation of the myosin regulatory light chain.

A key regulatory mechanism in cell motility is the control of myosin activity, which in non-muscle cells is determined by phosphorylation of the myosin regulatory light chain (MRLC). Here we show that MRLC-interacting protein (MIR)-interacting saposin-like protein (MSAP) enhances cell spreading in fibroblasts and migration of rat C6 glioma cells through increases in MRLC phosphorylation. Overexpression of MSAP enhanced the motility of glioma cells measured in matrigel invasion chambers and using a scratch assay. Downregulation of MSAP by RNA interference significantly decreased glioma cell migration and phosphorylation of MRLC. Inhibition of the corresponding MRLC kinase by ML-7 did not affect migration of MSAP-overexpressing cells. The present results show that MSAP controls glioma cell migration via enhancement of MRLC phosphorylation. This effect is independent of the activity of MRLC kinase. Thus, MSAP is a novel modulator of cell motility that influences migration of glioma cells and possibly other tumors.

Differential expression of utrophin and dystrophin in CNS neurons: an in situ hybridization and immunohistochemical study.

The cellular distribution of utrophin, the autosomal homologue of dystrophin, was investigated in developing and adult rat and mouse brain by in situ hybridization and immunohistochemistry. Digoxigenin-labeled cRNA probes complementary to N-terminal, rod-domain, and C-terminal encoding sequences of utrophin were used to differentiate between full-length and short C-terminal isoforms. Largely overlapping distribution patterns were seen for the three probes in neurons of cerebral cortex, accessory olfactory bulb, and several sensory and motor brainstem nuclei as well as in blood vessels, pia mater, and choroid plexus. The C-terminal probe was detected in addition in the main olfactory bulb, striatum, thalamic reticular nucleus, and hypothalamus, suggesting a selective expression of G-utrophin in these neurons. Western blot analysis with isoform-specific antisera confirmed the expression of both full-length and G-utrophin in brain. Immunohistochemically, only full-length utrophin was detected in neurons, in close association with the plasma membrane. In addition, intense staining was seen in blood vessels, meninges, and choroid plexus, selectively localized in the basolateral membrane of immunopositive epithelial cells. The expression pattern of utrophin was already established at early postnatal stages and did not change thereafter. Double-labeling analysis revealed that utrophin and dystrophin are differentially expressed on the cellular and subcellular levels in juvenile and adult brain. Likewise, in mice lacking full-length dystrophin isoforms (mdx mice), no change in utrophin expression and distribution could be detected in brain, although utrophin was markedly up-regulated in muscle cells. These results suggest that utrophin and dystrophin are independently regulated and have distinct functional roles in CNS neurons.

Identification and characterisation of transcript and protein of a new short N-terminal utrophin isoform.

Dystrophin and utrophin are known to link the intracellular cytoskeleton to the extracellular matrix via a transmembraneous glycoprotein complex. Four short C-terminal isoforms (Dp71, Dp116, Dp140, and Dp260) are described for dystrophin and three for utrophin (Up71, Up113, and Up140). We describe here for the first time the existence of a 3.7-kb transcript and a 62-kDa protein in C6 glioma cells representing a short N-terminal isoform unique for utrophin (N-utrophin). More than 20 clones covering the entire coding region of utrophin were isolated from a rat C6 glioma cell cDNA library. Two clones were found to code for a protein with 539 amino acids. Its sequence is identical to that of the full-length utrophin, except for the last residue where Cys is replaced by Val. This isoform contains the actin binding domain (consisting of two calponin homology subdomains), followed by two spectrin-like repeats. A recombinant fragment corresponding to N-utrophin binds to F-actin in vitro with an equilibrium constant (affinity) K of 4.5 x 10(5) M(-1) and a stoichiometry of one fragment per around five actin monomers. Immunocytochemical staining of C6 glioma cells with antisera specific for different utrophin regions localised full-length utrophin in the submembraneous cortical actin layer as revealed by confocal microscopy. A distinct staining pattern for the N-utrophin was not detectable, although it was expected to localise at the actin stress fibers. It is assumed that it co-localises via the two spectrin-like repeats with the full-length utrophin at the cell membrane.

Neuronal expression of the ERM-like protein MIR in rat brain and its localization to human chromosome 6.

The ERM proteins, ezrin, radixin, and moesin, regulate cell motility by linking cortical F-actin to the plasma membrane in different cell types. Myosin regulatory light chain interacting protein (MIR) is a recently cloned ERM-like protein which was shown to be involved in neurite outgrowth. Here we have studied the occurrence and expression of MIR in rats during brain development. As shown using Western blotting, MIR is present in different regions both in developing and adult brain. Immunohistochemistry and double labelling studies showed that MIR is localized especially to neurons in hippocampus and cerebellum. A search using the gene bank showed that the MIR gene localised to human chromosome 6 in the interval 6p22.3-23, the loss of which is characterized by mental retardation and different malformations in man. The presence of MIR in brain neurons during development together with its known effects on neurite outgrowth suggest an important function of the protein in the regulation of nerve cell motility and cytoskeletal interactions.

Short communication: altered synaptic clustering of GABAA receptors in mice lacking dystrophin (mdx mice).

Dystrophin is selectively localized in the postsynaptic density of neurons in cerebral cortex, hippocampus and cerebellum. Here, we show by double-immunofluorescence staining that dystrophin is extensively colocalized with GABAA receptor subunit clusters in these brain regions. To determine the relevance of this observation, we investigated in mdx mice, which provide a model of Duchenne muscular dystrophy, whether the absence of dystrophin affects the synaptic clustering of GABAA receptors. A marked reduction in the number of clusters immunoreactive for the alpha1 and alpha2 subunits was observed in, respectively, cerebellum and hippocampus of mdx mice, but not in striatum, which is normally devoid of dystrophin. Furthermore, these alterations were not accompanied by a change in gephyrin staining, although gephyrin is colocalized with the majority of GABAA receptor clusters in these regions. These results indicate that dystrophin may play an important role in the clustering or stabilization of GABAA receptors in a subset of central inhibitory synapses. These deficits may underlie the cognitive impairment seen in Duchenne patients.

Histamine-like immunoreactivity in the visual system and brain of an orthopteran and a hymenopteran insect.

The distribution of histamine-like immunoreactivity has been analyzed in the visual system and brain of the cricket Gryllus campestris and of the bee Apis mellifera by using an antiserum against histamine. Specific immunolabeling of the photoreceptors has been found in the compound eyes and ocelli of both examined species. Intense immunostaining can be also detected in the midbrain of these species. The axons of immunoreactive cells innervate almost every area in the protocerebrum. Most of the reactive neurons are typically wide-field neurons with bilateral ramifications that form dense arborizations. Numerous small buttons on the arborizations probably represent pre- and postsynaptic sites. The histamine-like immunoreactive neurons are apparently connected to many postsynaptic neurons. In both bees and crickets, some regions of the nervous system such as the first two optic neuropils and the central body show the same labeling pattern, whereas the mushroom bodies exhibit no immunoreactivity. Nevertheless, several differences in the staining pattern can be seen: the glomeruli of the antennal lobe are invaded by histamine-like immunoreactive fibers in the bee but not in the cricket. Furthermore, an interneuron connects the second and third optic neuropil in the cricket, whereas no histamine-like immunoreactive interneuron is found in the second optic neuropil in the bee. In accord with the work of other authors on the distribution histamine in the insect nervous system, we suggest that histamine is not only a transmitter within the visual system, but also a transmitter or co-transmitter in the insect midbrain.