Beat C. Bornhauser

Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance

In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL.

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 α1 and α2 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.

CD133 Positive Embryonal Rhabdomyosarcoma Stem-Like Cell Population Is Enriched in Rhabdospheres

Cancer stem cells (CSCs) have been identified in a number of solid tumors, but not yet in rhabdomyosarcoma (RMS), the most frequently occurring soft tissue tumor in childhood. Hence, the aim of this study was to identify and characterize a CSC population in RMS using a functional approach. We found that embryonal rhabdomyosarcoma (eRMS) cell lines can form rhabdomyosarcoma spheres (short rhabdospheres) in stem cell medium containing defined growth factors over several passages. Using an orthotopic xenograft model, we demonstrate that a 100 fold less sphere cells result in faster tumor growth compared to the adherent population suggesting that CSCs were enriched in the sphere population. Furthermore, stem cell genes such as oct4, nanog, c-myc, pax3 and sox2 are significantly upregulated in rhabdospheres which can be differentiated into multiple lineages such as adipocytes, myocytes and neuronal cells. Surprisingly, gene expression profiles indicate that rhabdospheres show more similarities with neuronal than with hematopoietic or mesenchymal stem cells. Analysis of these profiles identified the known CSC marker CD133 as one of the genes upregulated in rhabdospheres, both on RNA and protein levels. CD133+ sorted cells were subsequently shown to be more tumorigenic and more resistant to commonly used chemotherapeutics. Using a tissue microarray (TMA) of eRMS patients, we found that high expression of CD133 correlates with poor overall survival. Hence, CD133 could be a prognostic marker for eRMS. These experiments indicate that a CD133+ CSC population can be enriched from eRMS which might help to develop novel targeted therapies against this pediatric tumor.

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. J. Comp. Neurol. 422:594–611, 2000.

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

Abstract.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.

The activating STAT5B N642H mutation is a common abnormality in pediatric T-cell acute lymphoblastic leukemia and confers a higher risk of relapse

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that accounts for approximately 15% of pediatric acute lymphoblastic leukemias. A variety of genetic events affecting cellular processes such as the cell cycle, differentiation and survival have been identified in

Fibroblast growth factor-21 (FGF21) regulates low-density lipoprotein receptor (LDLR) levels in cells via the E3-ubiquitin ligase Mylip/Idol and the Canopy2 (Cnpy2)/Mylip-interacting Saposin-like Protein (Msap)

Background: LDLR is regulated by the E3 ubiquitin ligase Mylip/Idol. Results: FGF21 down-regulates Mylip/Idol expression and up-regulates its inhibitor Cnpy2/Msap. Conclusion: Increased LDLR by FGF21 results in increased lipoprotein uptake in human hepatocyte cells. Significance: The FGF21-mediated effect on cholesterol uptake is additive to that of statin, which may be of clinical significance. The LDLR is a critical factor in the regulation of blood cholesterol levels that are altered in different human diseases. The level of LDLR in the cell is regulated by both transcriptional and post-transcriptional events. The E3 ubiquitin ligase, myosin regulatory light chain-interacting protein (Mylip)/inducible degrader of the LDL-R (Idol) was shown to induce degradation of LDLR via protein ubiquitination. We have here studied novel factors and mechanisms that may regulate Mylip/Idol in human hepatocyte cells and in mouse macrophages. We observed that FGF21 that is present in serum in different conditions reduced Mylip/Idol at the RNA and protein level, and increased LDLR levels and stability in the cells. FGF21 also enhanced expression of Canopy2 (Cnpy2)/MIR-interacting Saposin-like protein (Msap) that is known to interact with Mylip/Idol. Overexpression of Cnpy2/Msap increased LDLRs, and knockdown experiments showed that Cnpy2/Msap is crucial for the FGF21 effect on LDLRs. Experiments using DiI-labeled LDL particles showed that FGF21 increased lipoprotein uptake and the effect of FGF21 was additive to that of statins. Our results are consistent with an important role of FGF21 and Cnpy2/Msap in the regulation of LDLRs in cultured cells, which warrants further studies using human samples.

Xenografts of highly resistant leukemia recapitulate the clonal composition of the leukemogenic compartment

Clonal evolution of the leukemogenic compartment may contribute to alter the therapeutic response in acute lymphoblastic leukemia (ALL). Using xenotransplantation of primary leukemia cells, we evaluated the phenotypic and genetic composition of de novo resistant very high risk precursor B-cell ALL, a subgroup defined by the persistence of minimal residual disease despite intensive chemotherapy. Analysis of copy number alterations (CNAs) showed that the xenografted leukemia, even when reconstituted from 100 cells, remained highly related to the diagnostic sample, with minor changes in CNAs, mostly deletions, emerging in most cases in the first passage into mice. At the single-cell level, the pattern of monoallelic and biallelic deletions of the CDKN2A locus revealed distinct leukemia subpopulations, which were reproducibly tracked in xenografts. In most very high risk ALL cases, the predominant diagnostic clones were reconstituted in xenografts, as shown by multiplex polymerase chain reaction analysis of immunoglobulin and T-cell receptor loci. In other cases, the pattern in CNAs and immunoglobulin and T-cell receptor rearrangement was less concordant in xenografts, suggesting the outgrowth of subclones. These results unequivocally demonstrate the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for drug development and preclinical disease modeling.

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models

A CBFA2T3-GLIS2 fusion gene was identified in 31% of non–Down syndrome AMKL.