Biology subjects

George, R. E.

Publications and source records attributed to George, R. E..

9 recordsLinked to original sources

bioRxiv2025

Chromatin Perturbation Promotes Susceptibility to Hypomethylating Agents

Cancer-directed drugs are often clinically deployed without definitive understanding of their molecular mechanisms of action (MOA). Hypomethylating agents (HMAs), which result in the degradation of the DNA methyltransferase 1 (DNMT1), have been deployed for decades in the treatment of haematological malignancies1,2. The precise mechanism of action of these drugs, however, has been debated, rendering the design of rational combination therapies challenging. Here, we identified the deubiquitinating enzyme USP48 as a crucial regulator of posttranslational histone modification in the context of DNA demethylation. USP48 loss selectively enhances response to DNMT1 inhibition, leading to a rapid induction of cell death. We demonstrate that USP48 is localized at sites of DNA damage and deubiquitinates H2A variants and proteins important for DNA damage repair. Functionally, loss of USP48 triggers an increase in chromatin accessibility upon HMA treatment, rendering AML cells more susceptible to DNA damage. Our results support USP48 as a posttranslational histone modifier for chromatin stability and DNA damage in response to HMA-related DNA demethylation. These findings propose USP48 as a new target for combination therapy with HMAs for acute myeloid leukaemia (AML).

cancer biology↗

bioRxiv2025

CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

The transcription-associated cyclin dependent kinases (CDKs) 12 and 13 play important roles in the expression of RNAs involved in DNA damage repair and ribosomal proteins, yet their roles if any, in the regulation of ribosomal DNA expression are unclear. Here we show that these CDKs play critical roles in the processing and degradation of noncoding RNAs in the nucleolus and that inhibition of their function leads to the upregulation of unprocessed ribosomal RNA (rRNA) transcripts in the intergenic spacer (IGS) regions between ribosomal subunits. These upregulated RNAs are aberrantly polyadenylated by the TRAMP complex polymerase TENT4, and are represented as polyadenylated rings surrounding the nucleolus. The accumulation of these noncoding RNAs is associated with increased RNA polymerase II (Pol II)-mediated transcription coupled with failure of degradation by the MTREX (SKIV2L2) RNA helicase, a member of the RNA exosome complex whose expression is prematurely terminated by CDK12/13 inhibition. Altogether, these findings provide new insights into Pol II-mediated transcriptional regulation by CDK12/13 within the nucleolus.

cancer biology↗

bioRxiv2024

Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA-modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and BDNF-activating bufalin synergistically arrests proliferation, induces differentiation, and maintains the differentiated state of MES SK-N-AS cells for 8 weeks. RNA-seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained MES neuroblastoma GI-ME-N and SH-EP cell lines, as well as glioblastoma LN-229 and U-251 cell lines, for over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

cancer biology↗

bioRxiv2024

The MYCN 5' UTR as a therapeutic target in neuroblastoma

Tumor cell amplification of the MYCN transcription factor is seen in half of patients with high-risk neuroblastoma, where it functions as an oncogenic driver associated with metastatic disease and poor survival. Yet, direct targeting of MYCN has been met with little success, prompting efforts to inhibit its expression at multiple levels. MYCN-amplified neuroblastoma cells have an increased requirement for protein synthesis to meet the overwhelming transcriptional burden imposed by oncogenic MYCN. Here, we take advantage of this vulnerability to interrogate the therapeutic potential of inhibiting the activity of the eukaryotic translation initiation factor 4A1 (eIF4A1), an RNA-helicase responsible for resolving structural barriers such as polypurine preponderance within 5' untranslated regions (UTRs). We observed that eIF4A1 is a key regulator of transcript-specific mRNA recruitment in MYCN-overexpressing neuroblastomas and MYCN-associated transcripts rank highly in polypurine-rich 5' UTR sequences, the majority of which have critical roles in cell proliferation. Using CMLD012824, a novel synthetic amidino-rocaglate (ADR) derivative, we demonstrate selectively increased eIF4A1 affinity for polypurine-rich 5' UTRs, including the MYCN mRNA, leading to translation inhibition and cytotoxicity in human neuroblastoma cell lines and animal models. Through ribosome profiling and PAR-CLIP analysis, we show that ADR-mediated clamping of eIF4A1 onto mRNA spans the full lengths of target transcripts, whereas translational inhibition is mediated selectively through 5' UTR binding. Both cap-dependent and cap-independent translation of MYCN are disrupted, pointing to the ability of CMLD012824 to disrupt non-canonical translation initiation. Our studies provide insights into the functional role of eIF4A1 in meeting the increased protein synthesis demands of MYCN-amplified neuroblastoma and suggest that its disruption may be therapeutically beneficial in this disease.

cancer biology↗

bioRxiv2022

Treg cells drive MYCN-mediated immunosuppression and tumor aggressiveness in high-risk neuroblastoma

Solid tumors, especially those with aberrant MYCN activation, harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance1,2, yet the underlying mechanisms are elusive and effective strategies to tackle this challenge are lacking. Here we demonstrated the crucial role of T regulatory (Treg) cells in MYCN-mediated immune repression and tumor aggression using high-risk neuroblastoma (NB) as a model system. Human MYCN-activated NB attracts CD4+ Treg cells, which are also found enriched in MYCN-high primary patient samples. Zebrafish MYCN-overexpressing neural crests recruit Cd4+ cells before tumor formation and induce an immunosuppressive microenvironment, thereby promoting tumor onset and progression. Strikingly, disruption of Treg cells through depletion of forkhead box protein 3a restores anti-tumor immunity and impairs NB development. Together, our studies establish Treg cells as the key driver of MYCN-mediated immunosuppression and tumor aggressiveness, providing mechanistic insights and therapeutic implications.

cancer biology↗

bioRxiv2021

Cell lineage as a predictor of immune response in neuroblastoma

Immunotherapy for patients with neuroblastoma has met with limited success, partly due to an incomplete understanding of the mechanisms underlying immune responsiveness in this clinically and genetically heterogenic tumor. Here, we undertook an unbiased analysis using dimension reduction and UMAP visualization of transcriptional signatures derived from 498 primary neuroblastoma tumors. Four distinct clusters based on differentially expressed genes emerged, of which one, representing about 30% and comprising mainly of MYCN-nonamplified tumors, was notable for the high expression of genes associated with both immune response activation and suppression. This capacity to elicit a productive immune response resided exclusively in tumors with dominant populations of undifferentiated, neural crest-like or mesenchymal cells; by contrast, tumors comprising primarily of committed, adrenergic neuron-like cells were less immunogenic. Mesenchymal neuroblastoma cells were enriched for innate and adaptive immune gene signatures, demonstrated engagement with cytotoxic T and natural killer cells, and induced immune cell infiltration in an immunocompetent mouse model. Transcriptional or targeted therapy-induced reprogramming of adrenergic cells to the mesenchymal state led to reactivation of tumor cell-intrinsic immune genes. Key immune response genes in adrenergic tumor cells were found to be epigenetically silenced by the PRC2 complex, and such repression could be relieved by either mesenchymal cell state reprogramming or EZH2 inhibition, leading to increased activation of natural killer cells by the tumor cells. These data identify cell lineage as a major determinant of the immunogenic potential in neuroblastoma that could be used to stratify patients who are most likely to benefit from immunotherapy.

cancer biology↗

bioRxiv2019

Giotto, a pipeline for integrative analysis and visualization of single-cell spatial transcriptomic data

The rapid development of novel spatial transcriptomic and proteomic technologies has provided new opportunities to investigate the interactions between cells and their native microenvironment. However, effective use of such technologies requires the development of innovative computational tools that are easily accessible and intuitive to use. Here we present Giotto, a comprehensive, flexible, robust, and open-source toolbox for spatial transcriptomic and proteomic data analysis and visualization. The data analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing cell-type distribution, spatially coherent gene expression patterns, and interactions between each cell and its surrounding neighbors. Furthermore, Giotto can also be used in conjunction with external single-cell RNAseq data to infer the spatial enrichment of cell types from data that do not have single-cell resolution. The data visualization module allows users to interactively visualize the gene expression data, analysis outputs, and additional imaging features, thereby providing a user-friendly workspace to explore multiple modalities of information for biological investigation. These two modules can be used iteratively for refined analysis and hypothesis development. We applied Giotto to a wide range of public datasets encompassing diverse technologies and platforms, thereby demonstrating its general applicability for spatial transcriptomic and proteomic data analysis and visualization.

bioinformatics↗

bioRxiv2019

Development of Human Neuroblastomas in Mouse-Human Neural Crest Chimeras

Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here we establish a platform that allows studying human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in-utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that closely resembled that seen in primary NBs of patients but were significantly different from homogenous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune checkpoints similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows studying human tumor initiation, progression, manifestation and tumor - immune-system interactions in an animal model system.

cancer biology↗

bioRxiv2018

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and appears to selectively affect the expression of DNA damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition due primarily to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

cancer biology↗