Abstract Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50–60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs ( lnc-THADA-4 , lnc-ACOT9-1 and NRIR ) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4 , lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.
<div>Abstract<p>Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, <i>SYNCRIP</i> (encoding hnRNP-Q) and <i>SNHG5</i> (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a <i>Tal1/Lmo1/Notch1</i>-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of <i>SYNCRIP</i> and <i>SNHG5.</i> Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression.</p>Significance:<p>The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome–mitochondria axis, suggesting the potential for therapeutic intervention.</p><p><i>This article is highlighted in the In This Issue feature, p. 1494</i></p></div>
Abstract Introduction: Neuroblastoma (NB) is a pediatric malignancy arising from peripheral neuronal sympathoblasts and exhibiting remarkable clinical and genetic heterogeneity. Patients older than 18 months have a poor prognosis with tumors presenting with highly recurrent segmental copy number alterations and MYCN amplification in half of these high-risk cases. The mechanism by which MYCN contributes to the development of neuroblastoma is unresolved and direct targeting of this key oncogene is not currently possible. Experimental Procedures: Our discovery efforts focused on identifying cooperating interactors and vulnerabilities in the MYCN regulatory network. MYCN-driven NBs can be modeled in mice with morphologic and genomic features that recapitulate human MYCN amplified NBs. Thus, this model serves as a valid tool for cross-species genomic analysis. Using this model, we performed a time-resolved analysis of the dynamic transcriptional changes of protein coding genes during murine TH-MYCN driven neuroblastoma development, focusing on timepoints representing tumor initiation and early tumor growth. We triangulated expression changes of key genes with publicly available exome-wide CRISPR-cas9 knockout analyses on a panel of human neuroblastoma cell lines and patient survival data. This unique data resource uncovered the relevance of MEIS2 as putative early cooperating initiating factor for neuroblastoma. Analysis of the genome-wide binding profile of MEIS2 in MYCN-amplified NB cell lines showed a striking overlap with enhancer-driven gene expression in regions of open chromatin, providing evidence that MEIS2 is a novel member of the adrenergic neuroblastoma core-regulatory circuitry. CRISPR-Cas9 mediated deletion of MEIS2 in animal models suppresses establishment of neuroblastoma tumors, indicating its putative requirement for tumor initiation. MEIS2, as a member of the CRC binds to several master regulators of gene expression, including the FOXM1 locus. Summary and conclusion: In conclusion, we present an in-depth characterization of the dynamic transcriptome profiles of TH-MYCN driven murine premalignant and established tumors and integrate with both primary human neuroblastoma tumor expression data, epigenetic and functional genomics data to identify and validate candidate cooperating dependencies suitable for targeting as a precision medicine approach in neuroblastoma. Citation Format: Kaat Durinck, Mark Zimmerman, Nina Weichert-Leahey, Jolien Dewyn, Wouter Van Loocke, Carolina Nunes, Anneleen Beckers, Bieke Decaesteker, Pieter-Jan Volders, Christophe Van Neste, Belamy Cheung, Daniel Carter, Thomas A. Look, Glenn Marshall, Katleen De Preter, Adam Durbin, Franki Speleman. Time-resolved transcriptome analysis of murine TH-MYCN driven neuroblastoma identifies MEIS2 as early initiating factor and novel core gene regulatory circuitry constituent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2481.
