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In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, that recruit much of the cell’s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase–binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, which suggests a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.

The Notch signaling pathway is a regulator of self-renewal and differentiation in several tissues and cell types. Notch is a binary cell-fate determinant, and its hyperactivation has been implicated as oncogenic in several cancers including breast cancer and T-cell acute lymphoblastic leukemia (T-ALL). Recently, several studies also unraveled tumor-suppressor roles for Notch signaling in different tissues, including tissues where it was before recognized as an oncogene in specific lineages. Whereas involvement of Notch as an oncogene in several lymphoid malignancies (T-ALL, B-chronic lymphocytic leukemia, splenic marginal zone lymphoma) is well characterized, there is growing evidence involving Notch signaling as a tumor suppressor in myeloid malignancies. It therefore appears that Notch signaling pathway’s oncogenic or tumor-suppressor abilities are highly context dependent. In this review, we summarize and discuss latest advances in the understanding of this dual role in hematopoiesis and the possible consequences for the treatment of hematologic malignancies.

The TP53 tumour suppressor is activated in response to distinct stimuli, including an ARF-dependent response to oncogene stress and an ATM/ATR-dependent response to DNA damage. In human T-cell acute lymphoblastic leukaemia (T-ALL), TP53-dependent tumour suppression is typically disabled via biallelic ARF deletions. In murine models, loss of Arf (Cdkn2a) or Tp53 markedly accelerates the onset of Myc-induced lymphoblastic malignancies. In zebrafish, no ARF ortholog has been identified, but the sequence of ARF is very poorly conserved evolutionarily, making it difficult to exclude the presence of a zebrafish ARF ortholog without functional studies. Here we show that tp53 mutations have no significant influence on the onset of myc-induced T-ALL in zebrafish, consistent with the lack of additional effects of Tp53 loss on lymphomagenesis in Arf-deficient mice. By contrast, irradiation leads to complete T-ALL regression in tp53 wild-type but not homozygous mutant zebrafish, indicating that the tp53-dependent DNA damage response is intact. We conclude that tp53 inactivation has no impact on the onset of myc-induced T-ALL in the zebrafish, consistent with the lack of a functional ARF ortholog linking myc-induced oncogene stress to tp53-dependent tumour suppression. Thus, the zebrafish model is well suited to the study of ARF-independent pathways in T-ALL pathobiology.

Background Protein phosphatase 2A (PP2A) represents a family of potent tumor suppressors that are often suppressed in human cancers by upregulation of proteins that inhibit subunit assembly into active enzyme complexes. Thus, restoration of PP2A has assumed increasing importance for cancer treatment. In earlier work, we found that perphenazine (PPZ) kills leukemic T cells by activating PP2A (Gutierrez et al. 2014. J Clin Invest.). PPZ also acts to inhibit dopamine D2 receptor (DRD2) in the basal ganglia, which causes movement disorders at dosages less than those needed to kill T-ALL cells, effectively precluding "repurposing" of PPZ for the therapy of T-ALL. Hypothesis We sought to identify PPZ analogues that activate PP2A and induce apoptosis in T-ALL cells but lack the ability to bind and inhibit DRD2. Such analogues would not induce the movement disorders that have limited the usefulness of PPZ as an anti-leukemic drug. Methods PP2A is a heterotrimeric phosphatase that is assembled from three classes of subunits, encoded by two possible "A" genes, 15 "B" genes and 2 "C" genes, providing 60 distinct PP2A holoenzymes that can potentially form in the cell. To identify the key subunits of PP2A required for the antitumor activity of PPZ in T-ALL cells, we knocked out each subunit in human T-ALL cell lines and treated these cells with PPZ. Biochemical reporter assays were established to identify a phenothiazine derivative, iHAP1, that efficiently reactivates PP2A but does not inhibit dopamine signaling. iHAP1 preclinical studies included in vivo efficacy and toxicity in T-ALL xenograft models and in vitro efficacy in 248 cancer cell lines from 35 distinct tumor types. Results Using this approach, we found that knockout of each of three specific subunit genes of PP2A - PPP2R1A, PPP2CA and PPP2R5E - uniquely conferred resistance to PPZ treatment in T-ALL cell lines,. An independent Immunoprecipitation followed by western blotting indicated that all three subunits form a functional PP2A heterotrimeric holoenzyme complex in response to PPZ treatment. Narla and coworkers have published extensively about a series of compounds called "small molecule PP2 activators" or SMAPs (Sangodkar et al. 2017 J Clin Invest.). Intriguingly, we showed that activities of SMAP compounds depend on a different "B subunit" - PPP2R2A - and target different signal transduction pathways. Based on this finding, we sought to identify analogues of PPZ that more potently activate PP2A through this mechanism and kill T-ALL cells but lack inhibitory activity against DRD2. Testing more than 80 analogues of PPZ revealed a highly potent PP2A activator, iHAP1 (improvedHeterocyclic Activators of PP2A 1). iHAP1 is ten times more potent than PPZ in its ability to activate PP2A and kill tumor cells, but does not measurably inhibit dopamine signalling. iHAP1 is highly active as an antitumor drug human T-ALL xenograft models, without causing untoward movement disorders or other toxicity in vivo. Phosphoproteomics analysis followed by detailed biochemical assays revealed that the potent antitumor activity of PPZ and iHAP1 is mediated by dephosphorylation of MYBL2, a transcription factor that is essential for expression of genes whose products mediate prometaphase, and thus for cancer cell growth and survival. SMAP compounds do not dephosphorylate the same transcription factor and rather target other phosphoproteins. Thus, the potent PP2A activator iHAP1 drives three specific PP2A subunits into an active trimeric phosphatase and this drug is highly active against T-ALL and other hematologic malignancies. iHAP1 did not exhibit measurable toxicity up to 80 mg/kg/day PO or IM for 30 days in preclinical studies of human leukemia xenografts growing in immunosuppressed mice, providing a therapeutic index for this lead compound in preclinical studies. Conclusions Our findings show that small molecules promote the assembly of unique PP2A complexes with different regulatory subunits and substrates, allowing detailed structure and function studies of PP2A family members. A goal is to identify small molecules that assemble PP2A enzymes containing each of the remaining 13 regulatory PP2A subunits, thereby targeting a diverse array of substrates crucial to the pathogenesis of cancer and other diseases. Disclosures Gray: Gatekeeper: Equity Ownership; Syros: Equity Ownership; Petra: Equity Ownership; C4: Equity Ownership; B2S: Equity Ownership; Soltego: Equity Ownership; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Janssen: Research Funding; Kinogen: Research Funding; Voronoi: Research Funding; Her2llc: Research Funding; Deerfield: Research Funding; Sanofi: Research Funding. Fischer:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Deerfield: Consultancy, Research Funding; Astellas: Research Funding.

TET2 is among the most frequently mutated genes in hematopoietic malignancies. Inactivating mutations in TET2 are found in ∼30% of patients with myelodysplastic syndrome (MDS), ∼19% with de novo acute myeloid leukemia (AML), and ~15% with myeloproliferative neoplasm (MPN). TET2 mutations are also identified in a subset of individuals over 50 years of age with clonal hematopoiesis of indeterminate potential (CHIP), a condition that predisposes affected individuals to progression to myeloid malignancy and atherosclerotic heart disease with heart attack or stroke. TET2 mutations represent an early genetic lesion in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state of clonal dominance that predisposes to the acquisition of additional mutations. Thus, effective therapy for patients with TET2 mutations in HSPCs will require identifying drugs that are selectively lethal to TET2 mutant HSPCs but spare normal HSPCs, a property analogous to the widely studied genetic relationship called “synthetic lethality”. Using a tet2-mutant zebrafish model created in our laboratory, we screened for drugs from libraries of FDA-approved compounds and drugs in Phase I/II testing. We found that nuclear exporter XPO1 inhibitors, selinexor (KPT-330) and eltanexor (KPT-8602), were among the most promising, in that they are selectively lethal to HSPCs in tet2-mutant compared to wild-type fish. Moreover, we treated HSPCs of wild-type and Tet2-deficient mice with both selinexor and eltanexor in a methylcellulose colony formation assay. WT HSPCs lose their replating capacity at passage 3 (P3), while Tet2-mutant clones demonstrate aberrant sustained self-renewal capacity over multiple replatings. We found that both drugs selectively kill primary Tet2-mutant murine HSPCs and also block the aberrant self-renewal of these cells. By transplanting WT and Tet2-mutant CD45.2 donor cells into CD45.1 recipient mice, we monitored peripheral blood cells before and after treatment with selinexor or eltanexor to explore synthetic lethality of these drugs in vivo. Moreover, the human AML cell line K562 with TET2 homozygous mutations generated with CRISPR-Cas9, was more sensitive to selinexor and eltanexor than the parental cell line. To elucidate the mechanism behind the selective targeting of Tet2-mutant blood stem cells, we will use PRO-Seq and START-Seq methods to identify transcriptional elongation and initiation sites at single-nucleotide resolution. These preclinical studies must be done to show a therapeutic efficacy and mechanism of action of these new drugs before they can be translated to improve therapy of patients who have TET2 inactivating mutations. Citation Format: Nicole Prutsch, Chang-Bin Jing, Julia Etchin, Alla Berezovskaya, Hong Tiv, Michael J. Poitras, Prafulla Gokhale, Yosef Landesman, A. Thomas Look. Selective activity of XPO1 inhibitors in TET2-mutant myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2953.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug's antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

BACKGROUND This study sought to investigate biological/clinicopathological characteristics of neuroblastoma, undifferentiated subtype (NBUD). METHODS This study examined 157 NBUD cases filed at the Children's Oncology Group Neuroblastoma Pathology Reference Laboratory, and survival rates of the patients were analyzed with known prognostic factors. Immunostainings for MYCN and MYC protein were performed on 68 tumors. RESULTS NBUD cases had a poor prognosis (48.4% ± 5.0% 3-year event-free survival [EFS]; 56.5% ± 5.0% overall survival [OS]), and were often associated with high mitosis-karyorrhexis index (MKI, 65%), prominent nucleoli (PN, 83%), ≥ 18 months of age (75%), MYCN amplification (MYCN-A, 83%), diploid pattern (63%), and 1pLOH (loss of heterozygosity (72%). However, these prognostic indicators, except for MYCN status, had no significant impact on survival. Surprisingly, EFS for patients with MYCN-A tumors (53.4% ± 5.6%) was significantly better (P = .0248) than for patients with MYCN-nonamplified (MYCN-NA) tumors (31.7% ± 11.7%), with MYCN-NA and PN (+) tumors having the worst prognosis (9.3% ± 8.8%, P = .0045). Immunohistochemically, MYCN expression was found in 42 of 48 MYCN-A tumors. In contrast, MYC expression was almost exclusively found in the MYCN-NA tumors (9 of 20) especially when they had PN (8 of 11). Those patients with only MYC-positive tumors had the worst EFS (N = 8, 12.5% ± 11.7%) compared with only MYCN-positive (N = 39, 49.9% ± 17.7%) and both negative tumors (N = 15, 70.0% ± 17.1%) (P = .0029). High MKI was often found in only MYCN-positive (30 of 38) but rarely in only MYC-positive (2 of 8) tumors. CONCLUSIONS NBUD represents a unique subtype of neuroblastoma associated with a poor prognosis. In this subtype, MYC protein expression may be a new prognostic factor indicating more aggressive clinical behavior than MYCN amplification and subsequent MYCN protein expression. Cancer 2013;119:3718–3726. © 2013 American Cancer Society.

Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P = 7.47 × 10(-29), odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P = 0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P

Contains fulltext : 177057.pdf (Publisher’s version ) (Closed access) A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dbetah promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

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.

Malignant peripheral nerve sheath tumors (MPNST) are tumors derived from Schwann cells or Schwann cell precursors. Although rare overall, the incidence of MPNST has increased with improved clinical management of patients with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome. Unfortunately, current treatment modalities for MPNST are limited, with no targeted therapies available and poor efficacy of conventional radiation and chemotherapeutic regimens. Many murine and zebrafish models of MPNST have been developed, which have helped to elucidate the genes and pathways that are dysregulated in MPNST tumorigenesis, including the p53, and the RB1, PI3K-Akt-mTOR, RAS-ERK and Wnt signaling pathways. Preclinical results have suggested that new therapies, including mTOR and ERK inhibitors, may synergize with conventional chemotherapy in human tumors. The discovery of new genome editing technologies, like CRISPR-cas9, and their successful application to the zebrafish model will enable rapid progress in the faithful modeling of MPNST molecular pathogenesis. The zebrafish model is especially suited for high throughput screening of new targeted therapeutics as well as drugs approved for other purposes, which may help to bring enhanced treatment modalities into human clinical trials for this devastating disease.

Over 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publically available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment called the Public Repository of Xenografts (PRoXe; www.proxe.org). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional and proteomic biomarkers in both treatment-naïve and relapsed/refractory disease.

SummaryD-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains that allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals’ health. Ablation of cyclin D3 in mice bearing Notch1-driven T cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anticancer strategy that specifically targets cancer cells without significantly affecting normal tissues.

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.

SummaryThe oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Background Of 4,706 peripheral neuroblastic tumors (pNTs) registered on the Children’s Cancer Group and Children’s Oncology Group Neuroblastoma Study between 1989 and 2010, 51 cases (1.1%) had genotype-phenotype discordance characterized by MYCN amplification (indicating poor prognosis) and Favorable Histology (indicating better prognosis).

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy(1,2). Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells(3-8). Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1)(9), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18hi1727/+ embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

Summary Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5′ to 3′ UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68–100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0–2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.

Introduction: Pediatric and young adult patients (pts) with relapsed/refractory (R/R) acute leukemia have a poor prognosis. Selinexor is a first-in-class antagonist of exportin (XPO1) with single agent activity in adults with acute myelogenous leukemia (AML). Dose-defining non-hematologic toxicities in adult studies included nausea, weight loss and anorexia. Methods: DFCI Protocol 13-563 was a Phase I dose-escalation trial conducted at nine institutions to test the safety, tolerability and pharmacokinetic (PK) characteristics of selinexor administered to pediatric and young adult pts with R/R acute leukemias. Selinexor was orally administered twice weekly during 4-week cycles. Three dose levels (DLs) were tested (30 mg/m2, 40 mg/m2 and 56 mg/m2) with dose-escalation decisions based on standard 3+3 Phase I design rules. Required supportive care medications included antiemetics and bacterial prophylaxis. All grade ≥2 non-hematologic adverse events (AEs) were recorded with the exception of non-clinically significant grade 2 laboratory abnormalities. Grades 1-4 hematologic AEs were expected in a relapsed leukemia patient population and were not considered AEs. Results: Sixteen pts enrolled on study, including 5 with primary refractory disease and 7 with second or greater relapse; 6 pts had received prior hematopoietic stem cell transplant. Median age was 9.5 years (range 2.3-21.8). Twelve pts had AML and 4 had acute lymphoblastic leukemia (ALL). Median number of completed cycles was 1 (range 0-3). Thirteen pts (81%) were evaluable for dose limiting toxicity (DLT) assessment, having received at least 7 doses of selinexor during the first cycle. Of 9 pts enrolled at DL1 (30 mg/m2), 6 were DLT-evaluable; 1 pt experienced a DLT of grade 5 pancreatitis (possibly related to study treatment). Three pts enrolled at DL1 were not DLT-evaluable; 2 pts were removed from study due to disease progression and 1 pt died from complications of a pulmonary infection. Six pts enrolled at DL2 (40 mg/m2) and none experienced a DLT. One pt was treated at DL3 (56 mg/m2) and developed a DLT of cognitive impairment (grade 3). Further enrollment onto DL3 was suspended secondary to an amendment capping dose escalation at 40 mg/m2 (equivalent to recommended single-agent dose in adult AML). During Cycle 1, the most common selinexor treatment-related AE's were hyponatremia (31%), elevated alanine aminotransferase (25%), nausea (25%), hyperkalemia (25%), elevated aspartate aminotransferase (19%) and diarrhea (19%). The most common grade ≥ 3 selinexor treatment-related AE's were hyponatremia (31%), elevated alanine aminotransferase (19%), hyperglycemia (13%) and hyperkalemia (13%). In contrast to adults treated with selinexor, there were no cases of grade ≥ 3 weight loss or nausea and only one case of grade 3 anorexia. One AML pt, after 2 cycles, achieved a complete remission with incomplete recovery of platelet count and one ALL pt, after 1 cycle, achieved a partial response, resulting in a protocol-defined overall response rate of 12.5%. Two of 4 pts with ALL (one each in DLs 1 and 2) developed clinically significant (grade 4) TLS. These included the pt who achieved a PR and another pt with early T-cell precursor ALL. Overall, 9 pts (56%), including 6 AML and 3 ALL, experienced an objective response and/or an investigator-defined clinical benefit (e.g., reduction in transfusions, clearance of peripheral blasts, decreased pain) from therapy. Conclusions: Selinexor, at a doses of 30 or 40 mg/m2 given twice weekly, was well tolerated in pediatric and young adult pts with R/R acute leukemia, demonstrating single agent clinical activity and less GI toxicity than had been seen in adults. The high frequency of TLS in ALL pts is particularly notable as treatment of ALL with selinexor has not been previously reported. Analysis of pharmacokinetic and correlative biology studies are underway. Based on the results of this Phase I study, the recommended Phase II dose of single-agent selinexor in children with R/R acute leukemia is 40 mg/m2 administered twice weekly. These results also support the continued investigation of selinexor in combination with multiagent chemotherapy. Disclosures Burke: AMGEN: Speakers Bureau; JAZZ: Speakers Bureau; Shire: Speakers Bureau.

The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Beisi Xu, Shizhen Zhu, Xiaoling Zhang, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. MYC activation through enhancer hijacking or focal enhancer amplification drives a subset of high-risk pediatric neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA19.

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid–rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.

Many children with metastatic or recurrent pediatric solid tumors continue to have poor survival, and there is an immense need to identify novel therapeutic approaches. Moreover, these cancers typically have simple genomes with limited known druggable molecular events. In order to discover new vulnerabilities in pediatric solid tumors, we have performed genome-scale CRISPR-Cas9 loss-of-function screening and deep “omic” characterization in over 60 pediatric cancer cell lines to date, including neuroblastoma, medulloblastoma, Ewing sarcoma, malignant rhabdoid tumor and rhabdomyosarcoma lines, to begin defining a pediatric cancer dependency map. Global analyses of the pediatric dependency landscape have identified emerging classes of pediatric cancers, including epigenetic-driven, aberrant transcription factor-driven and receptor tyrosine kinase-driven malignancies. For example, the preferential dependencies identified in a subset of neuroblastoma, which has aberrantly high expression of the transcription factor MYCN, are highly enriched for an interconnected network of genes annotated to have transcription factor activity. In addition to the global evaluation, we have developed methods and tools for prioritizing targets for further validation within a cancer type. These tools computationally integrate the pediatric dependency data across multiple datasets to identify categories of genetic dependencies that are especially strong hits or enriched hits in a specific pediatric malignancy. As an example, the intersection of MYCN-amplified neuroblastoma specific dependencies and H3-lysine 27 acetylation (H3K27ac) profiling across MYCN-amplified neuroblastoma allowed us to identify a transcriptional core regulatory circuit (CRC) that may drive the malignant state. Furthermore, targeting transcription with the BRD4 inhibitor JQ1 and CDK7 inhibitor THZ1 caused synergistic killing of neuroblastoma cells suggesting a novel therapeutic approach to treating this disease. Thus, defining a comprehensive pediatric cancer dependency map and developing the methods and tools to prioritize vulnerabilities in different cancer types will allow us to discover both novel biology and new therapeutic opportunities in childhood malignancies. Citation Format: Neekesh V. Dharia, Clare Malone, Amanda Balboni Iniguez, Lillian Guenther, Liying Chen, Gabriela Alexe, Adam D. Durbin, Mark W. Zimmerman, Andrew Hong, Pratiti Bandopadhayay, Mariella G. Filbin, Thomas Howard, Brenton Paolella, Iris Fung, Josephine Lee, Phil Montgomery, John M. Krill-Burger, Brian J. Abraham, Jennifer Roth, David E. Root, Richard A. Young, A. Thomas Look, Rameen Beroukhim, Jesse S. Boehm, William C. Hahn, Todd R. Golub, Aviad Tsherniak, Francisca Vazquez, Kimberly Stegmaier. Defining a pediatric cancer dependency map [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2352.

Somatic structural variations (SV) play an important role in tumorigenesis as they may cause oncogenic gene fusions or transcriptional activation of oncogenes by introducing aberrant promoter-enhancer interactions. Evaluating the oncogenic implications of SVs is challenging, especially for SVs in noncoding regions, which requires integrating data from 1) whole-genome sequencing (WGS), 2) RNA-seq from the tumor sample and series patient samples with same tumor type to compare with, 3) epigenetic profiling (ChIP-seq), and 4) genome-wide chromosome conformation capture studies such as Hi-C. To facilitate the discovery of oncogenic SVs, we developed GenomePaint, an interactive browser to integrate and visualize somatic SVs and copy number alterations (CNAs) analyzed by WGS, and gene expression data analyzed by RNA-seq in >2,000 pediatric cancers, as well as ChIP-seq and Hi-C data from pediatric tumors and cell lines. The genomic profiling of patient samples was generated by the St Jude/Washington University Pediatric Cancer Genome Project and the NCI-TARGET project, and includes all major subtypes of pediatric leukemia, solid tumors and brain tumors. A global view of the entire patient cohort using GenomePaint revealed SV hotspots (e.g. within first intron of TP53 in osterosarcoma) and recurrent CNAs (e.g. TAL1 deletion in T-ALL) in each cancer type. Each SV was integrated with results on gene expression, ChIP-seq and Hi-C, on the rearranged chromosome. Our discovery of the mechanism underlying aberrant MYC overexpression, in a subset of neuroblastomas (NBL) lacking MYCN amplification, proves the power of this tool in facilitating the discovery of oncogenic SV drivers. The sample with the highest MYC expression in this cohort harbors an SV between chr 8 and 4, with breakpoint on chr 8 located 50 Kb downstream of MYC. Similar SVs were detected in multiple NBL cell lines by WGS, which also express high levels of MYC. By analyzing Hi-C data from these cell lines, a new topologically associating domain (TAD) extending beyond the SV breakpoint was observed along the chimeric chromosome. This new TAD shows an aberrant interaction of a super-enhancer with a broad H3K27ac peak associated with HAND2/FBXO8 on chr 4 and the MYC promoter, demonstrating the rewired regulatory architecture introduced by SV as the driver for MYC dysregulation. In conclusion, our new GenomePaint interactive browser facilitates the analysis of SVs, CNAs and gene expression in concert studies by WGS and Hi-C in pediatric tumors. Coding mutations are rarely identified in many types of childhood tumors, even in the presence of pronounced chromosomal SVs, indicating new approaches are needed to unveil oncogenic mechanisms. GenomePaint provides an integrative analysis tool to evaluate the pathogenic basis underlying the often complex SVs in noncoding genome and function as oncogenic drivers in a large fraction of pediatric malignancies. Citation Format: Yu Liu, Xin Zhou, Jian Wang, Ying Shao, John Easton, Mark W. Zimmerman, Brian J. Abraham, A. Thomas Look, Jinghui Zhang. Exploring somatic DNA structural alteration and aberrant genomic interactions in cancer through GenomePaint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1287.

Childhood neuroblastomas with MYCN gene amplification form a particularly high-risk subset of this disease and are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that reflect important pathways in tumorigenesis, and thus could provide valuable targets for the development of novel therapeutics. Using genome-scale CRISPR-Cas9 approaches that allow unbiased detection of genes critically involved in tumor cell growth and survival, we identified 147 candidate genes associated with selective vulnerabilities in nine MYCN-amplified neuroblastoma cell lines, compared to findings in over 300 other human cancer cell lines representing multiple tumor cell types. We then used genome-wide ChIP-seq analysis to test the hypothesis that a small number of transcription factors - MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, all represented in the selective dependency group - are members of the transcriptional core regulatory circuitry (CRC) that underlies cell state in MYCN-amplified neuroblastoma. We show that these transcription factors bind as dense clusters at defined epicentres within the enhancers of their own genes, as well as those of the other CRC transcription factor genes, creating a positive feed-forward autoregulatory loop that establishes and maintains high levels of gene expression. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which we postulated would act synergistically by targeting both transcriptional initiation and elongation required to synthesize regulatory transcription factors. MYCN-amplified neuroblastoma cells treated with both drugs were killed synergistically, in vitro and in vivo, and accompanied by rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma, a subset of which comprises the oncogenic transcriptional regulatory circuitry that underlies cell state and survival in this tumor. Citation Format: Adam D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia, Brian J. Abraham, Brian J. Abraham, Amanda Balboni-Iniguez, Nina Weichert-Leahey, Shuning He, John M. Krill-Burger, David E. Root, Francisca Vazquez, Aviad Tsherniak, William C. Hahn, Todd R. Golub, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2062.

The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that c-MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Xu Beisi, Shizhen Zhu, Xiaoling Zhang, Zhaodong Li, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. c-MYC drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4135.

Comprehensive analysis of the cancer genome has become a standard approach to identifying new disease loci, and ultimately will guide therapeutic decisions. A key technology in this effort, single nucleotide polymorphism arrays, has been applied in hematologic malignancies to detect deletions, amplifications, and loss of heterozygosity (LOH) at high resolution. An inherent challenge of such studies lies in correctly distinguishing somatically acquired, cancer-specific lesions from patient-specific inherited copy number variations or segments of homozygosity. Failure to include appropriate normal DNA reference samples for each patient in retrospective or prospective studies makes it difficult to identify small somatic deletions not evident by standard cytogenetic analysis. In addition, the lack of proper controls can also lead to vastly overestimated frequencies of LOH without accompanying loss of DNA copies, so-called copy-neutral LOH. Here we use examples from patients with myeloid malignancies to demonstrate the superiority of matched tumor and normal DNA samples (paired studies) over multiple unpaired samples with respect to reducing false discovery rates in high-resolution single nucleotide polymorphism array analysis. Comparisons between matched tumor and normal samples will continue to be critical as the field moves from high resolution array analysis to deep sequencing to detect abnormalities in the cancer genome.

Summary The tyrosine phosphatase SHP2 ( PTPN11 ) regulates cellular proliferation, survival, migration, and differentiation during development. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, which have overlapping clinical features. Paradoxically, Noonan syndrome mutations increase SHP2 phosphatase activity, while LEOPARD syndrome mutants are catalytically impaired, raising the possibility that SHP2 has phosphatase-independent roles. By comparing shp2 -deficient zebrafish embryos with those injected with mRNA encoding LEOPARD syndrome point mutations, we identify a phosphatase- and Erk-dependent role for Shp2 in neural crest specification and migration. We also identify an unexpected phosphatase- and Erk-independent function, mediated through its SH2 domains, which is evolutionarily conserved and prevents p53-mediated apoptosis in the brain and neural crest. Our results indicate that previously enigmatic aspects of LEOPARD syndrome pathogenesis can be explained by the combined effects of loss of Shp2 catalytic function and retention of an SH2 domain-mediated role that is essential for neural crest cell survival.

To identify dysregulated pathways in distinct phases of NOTCH1-mediated T-cell leukemogenesis, as well as small-molecule inhibitors that could synergize with or substitute for γ-secretase inhibitors (GSIs) in T-cell acute lymphoblastic leukemia (T-ALL) therapy, we compared gene expression profiles in a Notch1-induced mouse model of T-ALL with those in human T-ALL. The overall patterns of NOTCH1-mediated gene expression in human and mouse T-ALLs were remarkably similar, as defined early in transformation in the mouse by the regulation of MYC and its target genes and activation of nuclear factor-κB and PI3K/AKT pathways. Later events in murine Notch1-mediated leukemogenesis included down-regulation of genes encoding tumor suppressors and negative cell cycle regulators. Gene set enrichment analysis and connectivity map algorithm predicted that small-molecule inhibitors, including heat-shock protein 90, histone deacetylase, PI3K/AKT, and proteasome inhibitors, could reverse the gene expression changes induced by NOTCH1. When tested in vitro, histone deacetylase, PI3K and proteasome inhibitors synergized with GSI in suppressing T-ALL cell growth in GSI-sensitive cells. Interestingly, alvespimycin, a potent inhibitor of the heat-shock protein 90 molecular chaperone, markedly inhibited the growth of both GSI-sensitive and -resistant T-ALL cells, suggesting that its loss disrupts signal transduction pathways crucial for the growth and survival of T-ALL cells.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

Von Recklinghausen neurofibromatosis is a common autosomal dominant genetic disorder characterized by benign and malignant tumors of neural crest origin. Significant progress in understanding the pathophysiology of this disease has occurred in recent years, largely aided by the development of relevant animal models. Von Recklinghausen neurofibromatosis is caused by mutations in the NF1 gene, which encodes neurofibromin, a large protein that modulates the activity of Ras. Here, we describe the identification and characterization of zebrafish nf1a and nf1b , orthologues of NF1 , and show neural crest and cardiovascular defects resulting from morpholino knockdown, including vascular and cardiac valvular abnormalities. Development of a zebrafish model of von Recklinghausen neurofibromatosis will allow for structure-function analysis and genetic screens in this tractable vertebrate system.

Nuclear factor interleukin-3 (Nfil3; also known as E4-binding protein 4) is a basic region leucine zipper transcription factor that has antiapoptotic activity in vitro under conditions of growth factor withdrawal. To study the role of Nfil3 in vivo, we generated gene-targeted Nfil3-deficient (Nfil3−/−) mice. Nfil3−/− mice were born at normal Mendelian frequency and were grossly normal and fertile. Although numbers of T cells, B cells, and natural killer (NK) T cells were normal in Nfil3−/− mice, a specific disruption in NK cell development resulted in severely reduced numbers of mature NK cells in the periphery. This defect was NK cell intrinsic in nature, leading to a failure to reject MHC class I–deficient cells in vivo and reductions in both interferon γ production and cytolytic activity in vitro. Our results confirm the specific and essential requirement of Nfil3 for the development of cells of the NK lineage.

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase II alpha-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

Background The International Neuroblastoma Pathology Classification (INPC) was the first to clearly define prognostic subgroups in ganglioneuroma (GN) and ganglioneuroblastoma (GNB).

Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.

Stromal contamination is one of the major confounding factors in the analysis of solid tumor samples by single nucleotide polymorphism (SNP) arrays. As we propose to use genome-wide SNP microarray analysis as a diagnostic platform for neuroblastoma, the sensitivity, specificity, and accuracy of these studies must be optimized. To investigate the effects of stromal contamination, we derived early-passage cell lines from nine primary tumors and compared their genomic signature with that of the primary tumors using 100K SNP arrays. The average concordance between tumor and cell line for raw loss of heterozygosity (LOH) calls was 96% (range, 91–99%) and for raw copy number alterations, 71% (range, 43–87%). In general, there were a larger number of LOH events identified in the cell lines compared with the matched tumor samples (mean increase, 3.2% ± 1.9%). We have developed an algorithm that shows that the presence of stroma contributes to under-reporting of LOH and copy number loss. Notable findings in this sample set were uniparental disomy of chromosome arms 11p, 1q, 14q, and 15q and a novel area of amplification on chromosome band 11p15. Our analysis shows that LOH was identified significantly more often in derived cell lines compared with the original tumor samples. Although these may in part be due to clonal selection during adaptation to tissue culture, our study indicates that stromal contamination may be a major contributing factor in underestimation of LOH and copy number loss events. [Cancer Res 2009;69(10):4143–9]

The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome-wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5-Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end-sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development.

Factor for adipocyte differentiation 24 (fad24) is a novel gene that has been implicated in adipocyte differentiation and DNA replication. In a screen for zebrafish mutants that have an abnormal tissue distribution of neutrophils, we identified an insertional allele of fad24, fad24hi1019. Homozygous fad24hi1019 larvae exhibit muscle degeneration accompanied by leukocyte infiltration. Muscle degeneration was extensive and included tissue apoptosis and disorganized, poorly striated muscle fibers. Blocking apoptosis using pan-caspase inhibitors resulted in decreased neutrophil recruitment into the body of the larva, suggesting a causative link between apoptosis and leukocyte infiltration. These findings suggest that zebrafish is a powerful genetic model system to address the interplay between muscle degeneration and leukocyte infiltration, and indicate that tissue apoptosis may contribute to neutrophil recruitment in some inflammatory states.

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.

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

VHL, NF-1, c-Ret, and Succinate Dehydrogenase Subunits B and D act on a developmental apoptotic pathway that is activated when nerve growth factor (NGF) becomes limiting for neuronal progenitor cells and requires the EglN3 prolyl hydroxylase as a downstream effector. Germline mutations of these genes cause familial pheochromocytoma and other neural crest-derived tumors. Using an unbiased shRNA screen we found that the kinesin KIF1Bβ acts downstream from EglN3 and is both necessary and sufficient for neuronal apoptosis when NGF becomes limiting. KIF1Bβ maps to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors including neuroblastomas. We identified inherited loss-of-function KIF1Bβ missense mutations in neuroblastomas and pheochromocytomas and an acquired loss-of-function mutation in a medulloblastoma, arguing that KIF1Bβ is a pathogenic target of these deletions.

Purpose MYCN amplification is rarely detected in patients with favorable-stage neuroblastoma (NB). To determine the clinical significance of MYCN amplification in children with favorable-stage NB, we performed a retrospective review of data from the Pediatric Oncology Group (POG) biology study 9047. Patients and Methods MYCN status, tumor cell ploidy, treatment, and outcome of patients with stage A, B, or Ds NB, enrolled on POG 9047 between 1990 and 1999 were analyzed. Event-free survival (EFS) and overall (OS) survival rates were analyzed using the Kaplan-Meier method. Results Of the 1,667 patients enrolled on POG 9047, 643 had favorable-stage disease. Of these, follow-up data were available on 568 (34%) with stage A, B, or Ds disease and normal MYCN copy number, and 32 (1.9%) patients with MYCN-amplified, stage A, B, or Ds tumors. Within the cohort lacking MYCN amplification, the 7-year EFS and OS rates (± SE) were 91% ± 1% and 96% ± 1%, respectively. Patients with MYCN amplification had significantly worse EFS and OS (50% ± 9% and 59% ± 9%, respectively, P < .0001). Within the cohort of children with MYCN amplification, the 7-year EFS and OS rates were 80% ± 10% and 87% ± 9%, respectively for patients with hyperdiploid tumors and 25% ± 11% and 38% ± 12% for patients with diploid/hypodiploid NBs (P = .0063 and P = .0074, respectively). Conclusion Tumor cell ploidy may be a clinically useful factor for prognostication and treatment stratification in children with MYCN-amplified, favorable-stage NB tumors.

Germ line gain-of-function mutations in several members of the RAS/ERK pathway, including PTPN11, KRAS, and RAF1, cause the autosomal dominant genetic disorder Noonan Syndrome (NS). NS patients are at increased risk of leukemia/myeloproliferative disease and possibly some solid tumors, such as neuroblastoma. Recently, SOS1 gain of function mutations have also been shown to cause NS. Somatic PTPN11, KRAS, and RAF1 mutations occur (although at different frequencies) in a variety of sporadic neoplasms, but whether SOS1 mutations are associated with human cancer has not been evaluated. We sequenced DNA from a total of 810 primary malignancies, including pancreatic, lung, breast, and colon carcinomas, and acute myelogenous leukemia, as well as several neuroblastoma cell lines. From this large, diverse series, missense SOS1 mutations were identified in a single pancreatic tumor, one lung adenocarcinoma, and a T-cell acute lymphoblastic leukemia cell line. Our findings suggest that SOS1 is not a significant human oncogene in most cancers. Furthermore, NS patients with SOS1 mutations may not be at increased risk of developing cancer.

Earlier reports showed that hyperplasia of sympathoadrenal cell precursors during embryogenesis in Nf1-deficient mice is independent of Nf1’s role in down-modulating RAS-MAPK signaling. We demonstrate in zebrafish that nf1 loss leads to aberrant activation of RAS signaling in MYCN-induced neuroblastomas that arise in these precursors, and that the GTPase-activating protein (GAP)-related domain (GRD) is sufficient to suppress the acceleration of neuroblastoma in nf1-deficient fish, but not the hypertrophy of sympathoadrenal cells in nf1 mutant embryos. Thus, even though neuroblastoma is a classical “developmental tumor”, NF1 relies on a very different mechanism to suppress malignant transformation than it does to modulate normal neural crest cell growth. We also show marked synergy in tumor cell killing between MEK inhibitors (trametinib) and retinoids (isotretinoin) in primary nf1a-/- zebrafish neuroblastomas. Thus, our model system has considerable translational potential for investigating new strategies to improve the treatment of very high-risk neuroblastomas with aberrant RAS-MAPK activation. DOI: http://dx.doi.org/10.7554/eLife.14713.001, eLife digest Neuroblastoma is one of the most common childhood cancers and is responsible for about 15% of childhood deaths due to cancer. The neuroblastoma tumors arise in cells that develop into and form part of the body’s nervous system. Many researchers have studied the genetics of this disease and have recognised common patterns. In particular, neuroblastomas can occur when a protein called MYCN is over-produced and a tumor suppressor protein called NF1 is lost. NF1 is a large protein with several distinct parts or domains. The most studied domain of NF1 is called the GRD, and it is mainly responsible for dampening down signals that cause cells to grow, specialize and survive. However, experiments in mice have revealed that this protein uses its other domains to control the normal development of part of the nervous system. He et al. wanted to know which domains of NF1 are important for suppressing the growth of neuroblastomas. The experiments were conducted in zebrafish that had been engineered to produce an excess of the human version of MYCN. When He et al. also deleted the gene for the zebrafish’s version of NF1, the fish quickly developed neuroblastomas. Supplying the zebrafish with just the GRD of NF1 was enough to supress the growth of the tumors. These experiments show that NF1 uses different domains and signalling pathways to regulate the normal development of part of the nervous system and to prevent formation of neuroblastoma. These engineered zebrafish represent an animal model of neuroblastoma that mimics the human disease in many ways. This model will make it possible to test new drug combinations and to find more effective treatments for neuroblastoma patients. DOI: http://dx.doi.org/10.7554/eLife.14713.002

The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers., United States. National Institutes of Health (HG002668), United States. National Institutes of Health (CA109901)