MYCN and SNRPD3 cooperate to maintain a balance of alternative splicing events that drives neuroblastoma progression.

Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms remain unknown. We found that MYCN up-regulated the expression of the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. High mRNA expression of SNRPD3 in human neuroblastoma tissues was a strong, independent prognostic factor for poor patient outcome. Repression of SNRPD3 expression correlated with loss of colony formation in vitro and reduced tumorigenicity in vivo. The effect of SNRPD3 on cell viability was in part dependent on MYCN as an oncogenic co-factor. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed. Surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increased differential splicing, particularly of cell cycle regulators, such as BIRC5 and CDK10. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5, consequently increasing SNRPD3 methylation. Indeed, the PRMT5 inhibitor, JNJ-64619178, reduced cell viability and SNRPD3 methylation in neuroblastoma cells with high SNRPD3 and MYCN expression. Our findings demonstrate a functional relationship between MYCN and SNRPD3, which maintains the fidelity of MYCN-driven alternative splicing in the narrow range required for neuroblastoma cell growth. SNRPD3 methylation and its protein-protein interface with MYCN represent novel therapeutic targets. Hypothetical model for SNRPD3 as a co-factor for MYCN oncogenesis. SNRPD3 and MYCN participate in a regulatory loop to balance splicing fidelity in neuroblastoma cells. First MYCN transactivates SNRPD3 to lead to high-level expression. Second, SNRPD3 and MYCN form a protein complex involving PRMT5. Third, this leads to balanced alterative splicing (AS) activitiy that is favorable to neuroblastoma. Together this forms as a therapeutic vulnerability where SNRPD3 perturbation or PRMT5 inhibitors are selectively toxic to neuroblastoma by conditionally disturbing splicing activity.

NONO enhances mRNA processing of super-enhancer-associated GATA2 and HAND2 genes in neuroblastoma.

High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.

Development of siRNA-Loaded Lipid Nanoparticles Targeting Long Non-Coding RNA LINC01257 as a Novel and Safe Therapeutic Approach for t(8;21) Pediatric Acute Myeloid Leukemia.

Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered 'undruggable' targets because they do not encode for a protein product. Here, we describe the development of a new siRNA-loaded lipid nanoparticle for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Transcriptomic analysis of children with AML identified LINC01257 as specifically expressed in t(8;21) AML and absent in healthy patients. Using NxGen microfluidic technology, we efficiently and reproducibly packaged anti-LINC01257 siRNA (LNP-si-LINC01257) into lipid nanoparticles based on the FDA-approved Patisiran (Onpattro®) formulation. LNP-si-LINC01257 size and ζ-potential were determined by dynamic light scattering using a Malvern Zetasizer Ultra. LNP-si-LINC01257 internalization and siRNA delivery were verified by fluorescence microscopy and flow cytometry analysis. lncRNA knockdown was determined by RT-qPCR and cell viability was characterized by flow cytometry-based apoptosis assay. LNP-siRNA production yielded a mean LNP size of ~65 nm with PDI ≤ 0.22 along with a >85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment was able to successfully ablate LINC01257 expression which was accompanied by a significant 55% reduction in total cell count following 48 h of treatment. In contrast, healthy peripheral blood mononuclear cells (PBMCs), which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. This is the first report demonstrating the use of LNP-assisted RNA interference modalities for the silencing of cancer-driving lncRNAs as a therapeutically viable and non-toxic approach in the management of AML.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma.

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.