Neuroblastoma and Its Zebrafish Model.

Neuroblastoma, an important developmental tumor arising in the peripheral sympathetic nervous system (PSNS), accounts for approximately 10 % of all cancer-related deaths in children. Recent genomic analyses have identified a spectrum of genetic alterations in this tumor. Amplification of the MYCN oncogene is found in 20 % of cases and is often accompanied by mutational activation of the ALK (anaplastic lymphoma kinase) gene, suggesting their cooperation in tumor initiation and spread. Understanding how complex genetic changes function together in oncogenesis has been a continuing and daunting task in cancer research. This challenge was addressed in neuroblastoma by generating a transgenic zebrafish model that overexpresses human MYCN and activated ALK in the PSNS, leading to tumors that closely resemble human neuroblastoma and new opportunities to probe the mechanisms that underlie the pathogenesis of this tumor. For example, coexpression of activated ALK with MYCN in this model triples the penetrance of neuroblastoma and markedly accelerates tumor onset, demonstrating the interaction of these modified genes in tumor development. Further, MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. In the context of MYCN overexpression, activated ALK provides prosurvival signals that block this apoptotic response, allowing continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. This application of the zebrafish model illustrates its value in rational assessment of the multigenic changes that define neuroblastoma pathogenesis and points the way to future studies to identify novel targets for therapeutic intervention.

KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia.

This study explored the anti-leukaemic efficacy of novel irreversible inhibitors of the major nuclear export receptor, chromosome region maintenance 1 (CRM1, also termed XPO1). We found that these novel CRM1 antagonists, termed SINE (Selective Inhibitors of Nuclear Export), induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukaemia (T-ALL) cell lines representing different molecular subtypes of the disease. To assess in vivo anti-leukaemia cell activity, we engrafted immunodeficient mice intravenously with the human T-ALL MOLT-4 cells, which harbour activating mutations of NOTCH1 and NRAS as well as loss of function of the CDKN2A, PTEN and TP53 tumour suppressors and express a high level of oncogenic transcription factor TAL1. Importantly, we examined the in vivo anti-leukaemic efficacy of the clinical SINE compound KPT-330 against T-ALL and acute myeloid leukaemia (AML) cells. These studies demonstrated striking in vivo activity of KPT-330 against T-ALL and AML cells, with little toxicity to normal murine haematopoietic cells. Taken together, our results show that SINE CRM1 antagonists represent promising 'first-in-class' drugs with a novel mechanism of action and wide therapeutic index, and imply that drugs of this class show promise for the targeted therapy of T-ALL and AML.

Oncogenic fusion E2A-HLF sensitizes t(17;19)-positive acute lymphoblastic leukemia to TRAIL-mediated apoptosis by upregulating the expression of death receptors.

t(17;19)-acute lymphoblastic leukemia (ALL) shows extremely poor prognosis. E2A-HLF derived from t(17;19) blocks apoptosis induced by the intrinsic mitochondrial pathway and has a central role in leukemogenesis and chemoresistance. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed on cytotoxic T cells and natural killer cells and binds with death receptors (DR4/DR5), inducing apoptosis by dual activation of intrinsic and extrinsic pathways, and TRAIL mediates the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT). We found that cell lines and patients' samples of t(17;19)-ALL expressed death receptors for TRAIL, and recombinant soluble TRAIL immediately induced apoptosis into t(17;19)-ALL cell lines. E2A-HLF induced gene expression of DR4/DR5, which was dependent on the DNA-binding and transactivation activities of E2A-HLF through the 5' upstream region of the start site at least in the DR4 gene. Introduction of E2A-HLF into non-t(17;19)-ALL cell line upregulated DR4 and DR5 expression, and sensitized to proapoptotic activity of recombinant soluble TRAIL. Finally, a newly diagnosed t(17;19)-ALL patient underwent allo-SCT immediately after induction of first complete remission, and the patient has survived without relapse for over 3-1/2 years after allo-SCT. These findings suggest that E2A-HLF sensitizes t(17;19)-ALL to the GVL effect by upregulating death receptors for TRAIL.

The effect of a depth gradient on the mating behavior, oviposition site preference, and embryo production in the zebrafish, Danio rerio.

Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436 +/- 35 tilted vs. 362 +/- 34 untilted; p < 0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity.

Gene expression profiling: will it complement or replace immunophenotyping?

Gene expression profiling using DNA microarrays represents a major advance in the diagnosis of haematological malignancies; it enables the analysis of RNA expression by clonal populations of leukaemia and lymphoma cells on a genome-wide scale. Currently, flow cytometric analysis of differentiation-linked surface antigens expressed by malignant cells is essential for the accurate diagnosis and optimal therapeutic planning for patients with leukaemias and lymphomas. Given the astonishing pace of research in microarray technology, a key question for the haematopathologist is: will gene expression profiling using DNA microarrays replace immunophenotyping, or will these two technologies evolve together as complementary approaches to improve diagnosis and therapy? In this chapter, we review the technical advantages and pitfalls of each of these two approaches and provide an assessment of their differences and potentially synergistic applications.