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The quest for effective cancer therapeutics has traditionally centered on targeting mutated or overexpressed oncogenic proteins. However, challenges arise in cancers with low mutational burden or when the mutated oncogene is not conventionally targetable, which are common situations in childhood cancers. This obstacle has sparked large-scale unbiased screens to identify collateral genetic dependencies crucial for cancer cell growth. These screens have revealed promising targets for therapeutic intervention in the form of lineage-selective dependency genes, which may have an expanded therapeutic window compared to pan-lethal dependencies. Many lineage-selective dependencies regulate gene expression and are closely tied to the developmental origins of pediatric tumors. Placing lineage-selective dependencies in a transcriptional network model is helpful for understanding their roles in driving malignant cell behaviors. Here, we discuss the identification of lineage-selective dependencies and how two transcriptional models, core regulatory circuits and gene regulatory networks, can serve as frameworks for understanding their individual and collective actions, particularly in cancers affecting children and young adults., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB., (© 2024. The Author(s).)

Neuroblastoma with MYCN amplification (MNA) is a high-risk disease that has a poor survival rate. Neuroblastoma displays cellular heterogeneity, including more differentiated (adrenergic) and more primitive (mesenchymal) cellular states. Here, we demonstrate that MYCN oncoprotein promotes a cellular state switch in mesenchymal cells to an adrenergic state, accompanied by induction of histone lysine demethylase 4 family members (KDM4A-C) that act in concert to control the expression of MYCN and adrenergic core regulatory circulatory (CRC) transcription factors. Pharmacologic inhibition of KDM4 blocks expression of MYCN and the adrenergic CRC transcriptome with genome-wide induction of transcriptionally repressive H3K9me3, resulting in potent anticancer activity against neuroblastomas with MNA by inducing neuroblastic differentiation and apoptosis. Furthermore, a short-term KDM4 inhibition in combination with conventional, cytotoxic chemotherapy results in complete tumor responses of xenografts with MNA. Thus, KDM4 blockade may serve as a transformative strategy to target the adrenergic CRC dependencies in MNA neuroblastomas., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Anaplastic large cell lymphoma (ALCL) is an aggressive, CD30 + T cell lymphoma of children and adults. ALK fusion transcripts or mutations in the JAK-STAT pathway are observed in most ALCL tumors, but the mechanisms underlying tumorigenesis are not fully understood. Here, we show that dysregulated STAT3 in ALCL cooccupies enhancers with master transcription factors BATF3, IRF4, and IKZF1 to form a core regulatory circuit that establishes and maintains the malignant cell state in ALCL. Critical downstream targets of this network in ALCL cells include the protooncogene MYC, which requires active STAT3 to facilitate high levels of MYC transcription. The core autoregulatory transcriptional circuitry activity is reinforced by MYC binding to the enhancer regions associated with STAT3 and each of the core regulatory transcription factors. Thus, activation of STAT3 provides the crucial link between aberrant tyrosine kinase signaling and the core transcriptional machinery that drives tumorigenesis and creates therapeutic vulnerabilities in ALCL., Competing Interests: Declaration of interests K.S. is a member of the scientific advisory board and has stock options in Auron Therapeutics and received grant funding from Novartis and KronosBio on topics unrelated to this paper. B.J.A. is a shareholder in Syros Pharmaceuticals. N.V.D. is currently an employee of Genentech, and is a stockholder in Roche. A.T.L. is a shareholder in LightHorse Therapeutics and is a consultant/advisory board member for LightHorse Therapeutics and Omega Therapeutics. M.W.Z. is currently an employee and shareholder in Foghorn Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers., (© 2023. The Author(s).)

Transcriptional co-regulators have been widely pursued as targets for disrupting oncogenic gene regulatory programs. However, many proteins in this target class are universally essential for cell survival, which limits their therapeutic window. Here we unveil a genetic interaction between histone deacetylase 1 (HDAC1) and HDAC2, wherein each paralog is synthetically lethal with hemizygous deletion of the other. This collateral synthetic lethality is caused by recurrent chromosomal deletions that occur in diverse solid and hematological malignancies, including neuroblastoma and multiple myeloma. Using genetic disruption or dTAG-mediated degradation, we show that targeting HDAC2 suppresses the growth of HDAC1-deficient neuroblastoma in vitro and in vivo. Mechanistically, we find that targeted degradation of HDAC2 in these cells prompts the degradation of several members of the nucleosome remodeling and deacetylase (NuRD) complex, leading to diminished chromatin accessibility at HDAC2-NuRD-bound sites of the genome and impaired control of enhancer-associated transcription. Furthermore, we reveal that several of the degraded NuRD complex subunits are dependencies in neuroblastoma and multiple myeloma, providing motivation to develop paralog-selective HDAC1 or HDAC2 degraders that could leverage HDAC1/2 synthetic lethality to target NuRD vulnerabilities. Altogether, we identify HDAC1/2 collateral synthetic lethality as a potential therapeutic target and reveal an unexplored mechanism for targeting NuRD-associated cancer dependencies., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. Here, we report an improved MYC-driven hepatoblastoma-like murine model that recapitulates the pathological features of embryonal type of hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identify distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we map cancer dependency genes using CRISPR-Cas9 screening and identify druggable targets shared with human hepatoblastoma (e.g., CDK7, CDK9, PRMT1, PRMT5). Our screen also reveals oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identifies modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies provide a set of resources including disease models suitable for identifying and validating potential therapeutic targets in human high-risk hepatoblastoma., (© 2023. The Author(s).)

Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans.

Cell state is controlled by master transcription factors (mTFs) that determine the cellular gene expression program. Cancer cells acquire dysregulated gene expression programs by mutational and non-mutational processes. Intratumoral heterogeneity can result from cells displaying distinct mTF-regulated cell states, which co-exist within the tumor. One archetypal tumor associated with transcriptionally regulated heterogeneity is high-risk neuroblastoma (NB). Patients with NB have poor overall survival despite intensive therapies, and relapsed patients are commonly refractory to treatment. The cellular populations that comprise NB are marked by different cohorts of mTFs and differential sensitivity to conventional therapies. Recent studies have highlighted mechanisms by which NB cells dynamically shift the cell state with treatment, revealing new opportunities to control the cellular response to treatment by manipulating cell-state-defining transcriptional programs. Here, we review recent advances in understanding transcriptionally defined cancer heterogeneity. We offer challenges to the field to encourage translation of basic science into clinical benefit., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells., Significance: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587., (©2021 The Authors; Published by the American Association for Cancer Research.)

Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription., Competing Interests: Declaration of interests J.C.C. is a co-founder of PrecisionProfile. H.L.F. is a co-founder of Sieyax, LLC., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroblastoma cell identity depends on a core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive the oncogenic gene expression program. For neuroblastomas dependent on the adrenergic CRC, treatment with retinoids can inhibit cell growth and induce differentiation. Here, we show that when MYCN -amplified neuroblastoma cells are treated with retinoic acid, histone H3K27 acetylation and methylation become redistributed to decommission super-enhancers driving the expression of PHOX2B and GATA3 , together with the activation of new super-enhancers that drive high levels of MEIS1 and SOX4 expression. These findings indicate that treatment with retinoids can reprogram the enhancer landscape, resulting in down-regulation of MYCN expression, while establishing a new retino-sympathetic CRC that causes proliferative arrest and sympathetic differentiation. Thus, we provide mechanisms that account for the beneficial effects of retinoids in high-risk neuroblastoma and explain the rapid down-regulation of expression of MYCN despite massive levels of amplification of this gene.

Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member ( gm6890 ), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin ( CLSPN ) and three chromosome 17q loci CBX2 , GJC1 and LIMD2 . Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo . Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM., Competing Interests: DECLARATION OF INTERESTS J.Q. is scientific co-founder and consultant of Epiphanes. Other authors declare no potential conflicts of interest.

Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.

We recently identified activated protein kinase B (PKB/AKT) as a tumorigenic driver in childhood ganglioneuroma. Inhibition of the mechanistic target of rapamycin (mTOR), a serine/threonine kinase downstream of AKT, effectively reduced the tumor burden in zebrafish with ganglioneuroma. We propose a clinical trial of mTOR inhibitors as a means to shrink large ganglioneuromas prior to surgical resection., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss., Competing Interests: Declaration of Interests F.V. and B.R.P. have received research support for this work from Novo Ventures. A.J.A. has consulted for Oncorus, Inc., Arrakis Therapeutics, and Merck & Co., Inc. and has research funding from Mirati Therapeutics and Deerfield, Inc. that is unrelated to this work. B.M.W. consults for BioLineRx, Celgene, G1 Therapeutics, and GRAIL and receives unrelated funding from Celgene and Eli Lilly. A.T. consults for Tango Therapeutics. W.C.H. consults for ThermoFisher, Solvasta Ventures, MPM Capital, KSQ Therapeutics, iTeos, Tyra Biosciences, Frontier Medicine, and Paraxel. K.S. has previously consulted for Novartis and Rigel Pharmaceuticals and receives funding through the Novartis/Dana-Farber Cancer Institute Drug Discovery Program unrelated to this project. T.R.G. is a consultant to GlaxoSmithKline, a founder of Sherlock Biosciences, and was formerly a consultant and equity holder in Foundation Medicine, acquired by Roche. T.R.G. receives research funding unrelated to this project from Bayer Healthcare. J.E.N., B.R.P., F.V., and A.J.A. are inventors on pending patent applications related to this work that are owned by the Broad Institute of MIT and Harvard and the Dana-Farber Cancer Institute., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peripheral sympathetic nervous system tumors are the most common extracranial solid tumors of childhood and include neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Surgery is the only effective therapy for ganglioneuroma, which may be challenging due to the location of the tumor and involvement of surrounding structures. Thus, there is a need for well-tolerated presurgical therapies that could reduce the size and extent of ganglioneuroma and therefore limit surgical morbidity. Here, we found that an AKT-mTOR-S6 pathway was active in human ganglioneuroma but not neuroblastoma samples. Zebrafish transgenic for constitutively activated myr-Akt2 in the sympathetic nervous system were found to develop ganglioneuroma without progression to neuroblastoma. Inhibition of the downstream AKT target, mTOR, in zebrafish with ganglioneuroma effectively reduced the tumor burden. Our results implicate activated AKT as a tumorigenic driver in ganglioneuroma. We propose a clinical trial of mTOR inhibitors as a means to shrink large ganglioneuromas before resection in order to reduce surgical morbidity., Competing Interests: Disclosures: T. Tao reported grants from Pediatric Cancer Research Foundation and grants from Rally Foundation for Childhood Cancer Research and the Open Hands Overflowing Hearts during the conduct of the study. H. Shi reported grants from Alex's Lemonade Stand Foundation during the conduct of the study. A.T. Look reported grants from National Institutes of Health during the conduct of the study. No other disclosures were reported., (© 2020 Tao et al.)

Mutations in genes encoding SWI/SNF chromatin remodeling complexes are found in approximately 20% of all human cancers, with ARID1A being the most frequently mutated subunit. Here, we show that disruption of ARID1A homologs in a zebrafish model accelerates the onset and increases the penetrance of MYCN-driven neuroblastoma by increasing cell proliferation in the sympathoadrenal lineage. Depletion of ARID1A in human NGP neuroblastoma cells promoted the adrenergic-to-mesenchymal transition with changes in enhancer-mediated gene expression due to alterations in the genomic occupancies of distinct SWI/SNF assemblies, BAF and PBAF. Our findings indicate that ARID1A is a haploinsufficient tumor suppressor in MYCN-driven neuroblastoma, whose depletion enhances tumor development and promotes the emergence of the more drug-resistant mesenchymal cell state., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7 -independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis., Competing Interests: Competing interest statement: B.J.A. is a shareholder in Syros Pharmaceuticals, Inc. The other authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Transcription is epigenetically regulated by the orchestrated function of chromatin-binding proteins that tightly control the expression of master transcription factors, effectors, and supportive housekeeping genes required for establishing and propagating the normal and malignant cell state. Rapid advances in chemical biology and functional genomics have facilitated exploration of targeting epigenetic proteins, yielding effective strategies to target transcription while reducing toxicities to untransformed cells. Here, we review recent developments in conventional active site and allosteric inhibitors, peptidomimetics, and novel proteolysis-targeted chimera (PROTAC) technology that have deepened our understanding of transcriptional processes and led to promising preclinical compounds for therapeutic translation, particularly in cancer., Competing Interests: Declaration of Interests J.Q. is scientific co-founder and consultant for Epiphanes and shareholder of Zentalis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates., Competing Interests: Declaration of Interests E.S.F. is a member of the scientific advisory board of C4 Therapeutics and a consultant to Novartis and Deerfield. He is a science advisory board member, founder, and shareholder for Civetta Therapeutics. E.S.F. received research funding from Novartis, Deerfield, and Astellas not related to this work. N.S.G. is a founder, science advisory board member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. A patent application related to this work has been submitted to the U.S. Patent and Trademark Office entitled “Compositions and Methods of Treating Cancers by Administering a Phenothiazine-Related Drug that Activates Protein Phosphatase 2A (PP2A) with Reduced Inhibitory Activity Targeted to the Dopamine D2 Receptor and Accompanying Toxicity” (application no. PCT/US19/67508)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1-all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that frequently arise in patients with neurofibromatosis type 1 (NF1). Most of these tumors are unresectable at diagnosis and minimally responsive to conventional treatment, lending urgency to the identification of new pathway dependencies and drugs with potent antitumor activities. We therefore examined a series of candidate agents for their ability to induce apoptosis in MPNST cells arising in nf1/tp53-deficient zebrafish. In this study, we found that DNA topoisomerase I-targeted drugs and mTOR kinase inhibitors were the most effective single agents in eliminating MPNST cells without prohibitive toxicity. In addition, three members of these classes of drugs, either AZD2014 or INK128 in combination with irinotecan, acted synergistically to induce apoptosis both in vitro and in vivo. In mechanistic studies, irinotecan not only induces apoptosis by eliciting a DNA damage response, but also acts synergistically with AZD2014 to potentiate the hypophosphorylation of 4E-BP1, a downstream target of mTORC1. Profound hypophosphorylation of 4E-BP1 induced by this drug combination causes an arrest of protein synthesis, which potently induces tumor cell apoptosis. Our findings provide a compelling rationale for further in vivo evaluation of the combination of DNA topoisomerase I-targeted drugs and mTOR kinase inhibitors against these aggressive nerve sheath tumors.

Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively 1 . This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival 2-6 , we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential transcription factors-MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2-are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.

Background: Posterior ischemic optic neuropathy results from ischemia of the retrobulbar aspect of the optic nerve. It presents as acute loss of vision without optic disc swelling. This is rare in children, with only seven cases reported to date. Neuroimaging is frequently used to aid in the diagnosis of acute visual complaints in children; however, none of the cases described to date delineate the neuroimaging findings of this entity in children., Methods: We retrospectively reviewed the electronic medical record., Results: We describe the MRI findings in a 10-month-old boy with posterior ischemic optic neuropathy after intraophthalmic artery injection of chemotherapy for retinoblastoma., Conclusions: As targeted therapies for retinoblastoma and other diseases amenable to intravascular treatment delivery are more frequently used, the risk of grave vision-related side effects increases. Posterior ischemic optic neuropathy should be considered in the differential diagnosis of any child presenting with acute loss of vision. Dedicated imaging of the orbits can elucidate specific findings that may aid in the diagnosis of this entity in children., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here, we show that the family member MYC 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 and 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. Significance: Amplification of the MYCN oncogene is a recognized hallmark of high-risk pediatric neuroblastoma. Here, we demonstrate that MYC is also activated as a potent oncogene in a distinct subset of neuroblastoma cases through either focal amplification of distal enhancers or enhancer hijacking mediated by chromosomal translocation. Cancer Discov; 8(3); 320-35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 253 ., (©2017 American Association for Cancer Research.)

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Vitamin B12 deficiency is classically encountered in the adult Caucasian population and manifests as a subacute combined degeneration in the presence or absence of macrocytic anemia. However, B12 deficiency is extremely rare in children in developed countries, and pernicious anemia is even rarer etiology of this deficiency. The clinical presentation of B12 deficiency in children is not as easily recognizable or well-characterized and may result in missed or delayed diagnosis., Patient Description: We describe a previously healthy adolescent girl who presented with emotional lability, mental status changes, hyperreflexia, and ataxia. She was found to have megaloblastic anemia and subacute combined degeneration secondary to B12 deficiency caused by pernicious anemia., Conclusion: Though B12 deficiency is uncommon in pediatrics, it is important to maintain a high index of suspicion because early recognition and treatment of B12 deficiency are key to preventing long-term neurological sequelae., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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 < 0.0001). We demonstrate allelic imbalance favouring the G-containing strand in tumours heterozygous for this SNP, as demonstrated both by RNA sequencing (P < 0.0001) and reporter assays (P = 0.002). These findings indicate that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumour cells.

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., (Copyright © 2014, American Association for the Advancement of Science.)

While most reports detail an oncogenic function for the integrin-linked kinase (ILK) in human cancer, few describe a contradictory growth-suppressive function. We previously reported that ILK functions as either a tumor suppressor or an oncogene in rhabdomyosarcoma (RMS), in a manner linked to expression of the c-jun amino terminal kinase-1 (JNK1). However, studies in other tumors are lacking. With the advent of bioavailable small molecule inhibitors of ILK, defining both the function of ILK and biomarkers to predict its behaviour are of critical importance. Here, we studied the role of ILK in a panel of tumor cell lines. We demonstrate that ILK functions as either a growth-promoter or suppressor in numerous tumor cell lines. Further, cell lines in which ILK functioned as a growth suppressor displayed elevated JNK1 expression relative to cells in which ILK functioned as an oncogene. Comparison of endogenous JNK1 and JNK1β isoform expression levels to the cellular response to ILK overexpression demonstrated that JNK1β isoforms represent biomarkers differentiating the two functions of ILK. Moreover, RNAi and overexpression-based alteration of JNK1 expression levels was sufficient to switch the function of ILK in both transformed and untransformed cells. These results indicate widespread oncogenic and growth-suppressive functions for ILK in multiple human malignancies and suggest that JNK1 isoforms represent biomarkers for ILK neoplastic activity. These results provide a rationale for stratifying patients to receive ILK kinase inhibitors based on individualized tumor-specific ILK function.

Osteosarcomas are copy number alteration (CNA)-rich malignant bone tumors. Using microarrays, fluorescence in situ hybridization, and quantitative PCR, we characterize a focal region of chr3q13.31 (osteo3q13.31) harboring CNAs in 80% of osteosarcomas. As such, osteo3q13.31 is the most altered region in osteosarcoma and contests the view that CNAs in osteosarcoma are nonrecurrent. Most (67%) osteo3q13.31 CNAs are deletions, with 75% of these monoallelic and frequently accompanied by loss of heterozygosity (LOH) in flanking DNA. Notably, these CNAs often involve the noncoding RNAs LOC285194 and BC040587 and, in some cases, a tumor suppressor gene that encodes the limbic system-associated membrane protein (LSAMP). Ubiquitous changes occur in these genes in osteosarcoma, usually involving loss of expression. Underscoring their functional significance, expression of these genes is correlated with the presence of osteo3q13.31 CNAs. Focal osteo3q13.31 CNAs and LOH are also common in cell lines from other cancers, identifying osteo3q13.31 as a generalized candidate region for tumor suppressor genes. Osteo3q13.31 genes may function as a unit, given significant correlation in their expression despite the great genetic distances between them. In support of this notion, depleting either LSAMP or LOC285194 promoted proliferation of normal osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1. Moreover, genetic deletions of LOC285194 or BC040587 were also associated with poor survival of osteosarcoma patients. Our findings identify osteo3q13.31 as a novel region of cooperatively acting tumor suppressor genes.

In neoplastic cells, proteins exert either pro or anti-tumorigenic functions. However, some proteins exhibit both properties, commonly dependent on specific aberrations occurring in a tumor-specific context. Recently, we demonstrated that the integrin-linked kinase (ILK), generally characterized as an oncogenic protein kinase, functions as a tumor suppressor protein in vitro and in vivo in the aggressive pediatric tumor, rhabdomyosarcoma (RMS). Other studies have similarly demonstrated both growth and tumor suppressive functions for ILK in normal and transformed tissues. The mechanism of ILK tumor suppression in RMS relies on expression levels of another kinase, the c-jun amino terminal kinase-1 (JNK1). These findings support a model in which ILK tumor suppression is mediated in part by elevated JNK1 expression, and indicate both a rationale for stratification of patients to receive anti-ILK therapies, and a need to better understand the context in which ILK displays its seemingly contradictory functions. This review discusses the complex roles of ILK in tumorigenesis, and offers arguments to harness ILK and JNK signaling as novel targets for anti-cancer therapy.

Although most reports describe the protein kinase integrin-linked kinase (ILK) as a proto-oncogene, occasional studies detail opposing functions in the regulation of normal and transformed cell proliferation, differentiation, and apoptosis. Here, we demonstrated that ILK functions as an oncogene in the highly aggressive pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) and as a tumor suppressor in the related embryonal rhabdomyosarcoma (ERMS). These opposing functions hinge on signaling through a noncanonical ILK target, JNK1, to the proto-oncogene c-Jun. RNAi-mediated depletion of ILK induced activation of JNK and its target, c-Jun, resulting in growth of ERMS cells, whereas in ARMS cells, it led to loss of JNK/c-Jun signaling and suppression of growth both in vitro and in vivo. Ectopic expression of the fusion gene characteristic of ARMS (paired box 3-forkhead homolog in rhabdomyosarcoma [PAX3-FKHR]) in ERMS cells was sufficient to convert them to an ARMS signaling phenotype and render ILK activity oncogenic. Furthermore, restoration of JNK1 in ARMS reestablished a tumor-suppressive function for ILK. These findings indicate what we believe to be a novel effector pathway regulated by ILK, provide a mechanism for interconversion of oncogenic and tumor-suppressor functions of a single regulatory protein based on the genetic background of the tumor cells, and suggest a rationale for tailored therapy of rhabdomyosarcoma based on the different activities of ILK.

Recent studies have shown a significant involvement of insulin-like growth factor (IGF) signaling components in the pathogenesis of rhabdomyosarcoma (RMS). Furthermore, there has been some evidence to indicate that differential expression of IGF pathway genes can distinguish RMS subtypes. The present study utilized immunohistochemistry to determine the expression patterns of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGF receptor 1 (IGF1R), and IGF receptor 2 (IGF2R) in 24 embryonal RMS (ERMS) and 8 alveolar RMS (ARMS). A majority of tumors were positive for IGF2, IGFBP2, IGF1R, and IGF2R and negative for IGF1 expression. However, only IGF2 showed a significant difference in expression between the ERMS and ARMS subtypes, with higher levels of expression in ERMS (P = 0.0003). Within the ARMS subtype, IGF2 positivity was limited to PAX/FKHR translocation-negative tumors. The staining pattern for all 5 proteins was diffuse cytoplasmic in the majority of tumors. Analysis of RMS cell lines by real-time reverse transcriptase-polymerase chain reaction for IGF2 expression revealed significantly higher mean expression levels in ERMS and translocation-negative ARMS cell lines when compared to translocation-positive ARMS cell lines (P = 0.0027). Stable introduction of PAX3/FKHR into an ERMS cell line also demonstrated a significant reduction in IGF2 expression. The results of this study show that expression of the IGF2 ligand is associated with translocation-negative tumors and may serve as a diagnostic aid in distinguishing RMS subtypes. Furthermore, the in vitro results are supportive of a role for the PAX3/FKHR fusion gene in the inhibition of IGF2 expression.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Highly malignant, RMS frequently fails to respond to conventional aggressive multimodal radiation, surgery, and chemotherapy treatment protocols that also cause significant sequelae in the growing child. Other tumors of mesenchymal origin, such as locally aggressive fibromatoses and desmoid tumors, have been successfully treated with a selective estrogen receptor (ER) modulator, tamoxifen. In an effort to identify new targets for RMS therapy, our group investigated the previously uncharacterized ER pathway in RMS cell culture and primary tumors. We detected ER isoform beta (ER beta), but not isoform alpha, RNA, and protein in five RMS cell lines. Immunohistochemical staining of primary RMS tumor sections confirmed high levels of ER beta but not ER alpha protein. RMS cell growth was dramatically inhibited in steroid-free conditions, and this growth inhibition was rescued with 17-beta-estradiol (E2) supplementation. Exposure of RMS cells to 4'OH-tamoxifen (4OHT) decreased cell viability and inhibited colony formation as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony-forming assays. 4OHT also induced apoptotic signaling in RMS cells as detected by cleavage of caspase-3 and poly(ADP)ribose polymerase. This effect increased 3- to 8-fold in steroid-deprived conditions but was rescued by supplementation with E2. Immunofluorescence studies detected a change in the subcellular localization of ER beta in response to 4OHT. Together, these data suggest an active ER beta-mediated signal transduction pathway in RMS. The ability of 4OHT to induce apoptotic signaling and disrupt estradiol-mediated proliferation provides a rationale to explore a role for selective ER modulators in the treatment of RMS.

Rhabdomyosarcoma (RMS) is the most common malignant soft-tissue tumor of childhood. Nearly 15% of children present with metastatic disease, frequently involving the lungs and bone marrow. The prognosis for patients with metastatic RMS is dismal, with an estimated 3-year overall survival of 30%. Stromal-cell derived factor 1-alpha (SDF1alpha, CXCL12) is a chemokine that plays a crucial role in the metastatic attraction of tumor cells expressing its receptor, CXCR4. We investigated the role of the bone marrow microenvironment on RMS signaling through the CXCR4/SDF1alpha pathway in cell lines and primary tumors. Conditioned media (CM) isolated from cultured patient-derived bone marrow stromal cells (BMS) induced migration and proliferation in multiple RMS cell lines. CXCR4 was expressed in RMS cell lines and primary tumors, with higher expression in alveolar subtype RMS. Further, SDF1alpha was secreted by all BMS cultures and potently induced the migration and proliferation of RMS cells. Small molecule or blocking antibody-mediated inhibition of CXCR4 or SDF1alpha suppressed RMS cell migration towards BMS-CM, confirming the activity of this axis. Our study provides strong evidence for the involvement of the bone marrow microenvironment and CXCR4/SDF1alpha signaling in metastasis of RMS. These results form the basis for future studies to delineate the mechanisms of bone marrow metastasis in RMS.

Background: Composed of endocardial endothelial, valvular interstitial, cardiac muscle, and smooth muscle cells (SMC), heart valves are prone to various pathologic conditions the morphology of which has been well described. The morphology of diseased valves suggest that the "response to injury" process occurs in these valves, and is associated with an accumulation of interstitial cells and matrix, valvular inflammation and calcification, conditions that lead to dysfunction. The purpose of this study is to describe the current knowledge of the regulation of the valvular "response to injury" process, since we feel that this paradigm is essential to understanding valve disease., Methods: The pertinent literature relating to the cell and molecular biology of valvular repair, and specifically interstitial cell function in valve repair, is reviewed., Results: The cell and molecular biology of valve interstitial cells are poorly understood. Molecules regulating some of the aspects of the "response to injury" process have been studied, however, the signal transduction pathways, gene activation, and interactions of bioactive molecules with each other, with cells, and with the matrix have not been characterized. Initial studies identify the cell and molecular biology of interstitial cells to be an important area of research. Agents that have been studied include nitric oxide (NO) and FGF-2 and several matrix-related proteins including osteopontin. The present review suggests several directions for future study and a working model of valvular repair is presented., Discussion: The regulation of the "response to injury" process in the human heart valve is still largely unknown. The cell and molecular events and processes that occur in heart valve function and repair remain poorly understood. These events and processes are vital to our understanding of the pathobiology of heart valve disease, and to the successful design of tissue engineered replacement valves.