Biology subjects

Bellamy, J.

Publications and source records attributed to Bellamy, J..

5 recordsLinked to original sources

bioRxiv2024

PRMT5 inhibition preferentially targets MYCN-amplified neuroblastoma via altered transcriptional and splicing programmes regulating key cancer cell fitness pathways.

About 50% of poor prognosis neuroblastoma arises due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN amplified (MNA) neuroblastoma. Here we evaluate PRMT5 inhibitors GSK3203591/GSK3326593 as targeted therapeutics for MNA neuroblastoma and show MYCN-dependent growth inhibition and apoptosis. RNAseq revealed dysregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Metabolic tracing showed glutamine metabolism was impeded following GSK3203591 treatment, which disrupted the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Glutaminase (GLS) protein was decreased by GSK3203591 despite unchanged transcript levels, suggesting post-transcriptional regulation. We demonstrate the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following splicing alterations; accordingly, we observed hypomethylation of GLS mRNA and decreased GLS following YTHDF3 knockdown. In vivo efficacy of GSK3326593 was confirmed by increased survival of Th-MYCN mice together with splicing events and protein decreases consistent with in vitro data. Our study supports the spliceosome as a key vulnerability of MNA neuroblastoma and rationalises PRMT5 inhibition as a targeted therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/582087v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1ac6b5org.highwire.dtl.DTLVardef@1eb9520org.highwire.dtl.DTLVardef@18b980corg.highwire.dtl.DTLVardef@1ab46e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Identifying targetable metabolic dependencies across colorectal cancer progression

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system - comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

cancer biology↗

bioRxiv2021

Transcriptomic analyses of MYCN-regulated genes in anaplastic Wilms' tumour cell lines reveals oncogenic pathways and potential therapeutic vulnerabilities

The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated over-expression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms tumours with poorer outcome, including tumours with diffuse anaplasia. Surpisingly, however, the expression and functions of the MYCN protein in Wilms tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes included the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of REST target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor prognosis Wilms tumour.

cancer biology↗

bioRxiv2020

A Wnt-BMP4 signalling axis induces MSX and NOTCH proteins and promotes growth suppression and differentiation in neuroblastoma

The Wnt and bone morphogenetic protein (BMP) signalling pathways are known to be crucial in the development of neural crest lineages, including the sympathetic nervous system. Surprisingly, their role in paediatric neuroblastoma, the prototypic tumour arising from this lineage, remains relatively uncharacterised. We previously demonstrated that Wnt/{beta}-catenin signalling can have cell-type specific effects on neuroblastoma phenotypes, including growth inhibition and differentiation, and that BMP4 mRNA and protein were induced by Wnt3a/Rspo2. In this study, we characterise the phenotypic effects of BMP4 on neuroblastoma cells, demonstrating convergent induction of MSX homeobox transcription factors by Wnt and BMP4 signalling and BMP4-induced growth suppression and differentiation. Immunohistochemical analysis of BMP4 expression in primary neuroblastomas confirms a striking absence of BMP4 in poorly differentiated tumours, in contrast to high expression in ganglion cells. These results are consistent with a tumour suppressive role for BMP4 in neuroblastoma. RNA sequencing following BMP4 treatment revealed induction of Notch signalling, verified by increases of Notch3 and Hes1 proteins. Together, our data demonstrate for the first time Wnt-BMP-Notch signalling crosstalk associated with growth suppression of neuroblastoma.

cancer biology↗

bioRxiv2019

Increased efficacy of histone methyltransferase G9a inhibitors against MYCN-amplified Neuroblastoma

Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a paediatric solid tumour with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumours is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638 and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumour suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2 and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumours.

cancer biology↗