Biology subjects

VanInsberghe, M.

Publications and source records attributed to VanInsberghe, M..

4 recordsLinked to original sources

bioRxiv2026

Oncogenic fusions induce an extensive cancer-restricted cryptic proteome in Ewing sarcoma

How tumors generate a cryptic "dark" proteome absent from healthy tissues, and whether it can be reversibly activated, remains unclear. In Ewing sarcoma, all tumors are driven by EWSR1::ETS fusions, making it a tractable model to study dark proteome activation. Integrating matched transcriptomes, translatomes and proteomes from 48 patient tumors with long-read RNA sequencing, single-cell Ribo-seq, proteomics and immunopeptidomics in cell line models, we show that EWSR1::FLI1 acts as a reversible switch recurrently inducing hundreds of cancer-specific neoproteins. Many arise from canonical coding regions via intragenic transcription start sites, generating truncated or out-of-frame neoproteins. We uncover a fusion-dependent increase in ribosomal readthrough into poly(A) tails, generating a stable 80-amino-acid TRPM4 neoprotein that, despite lacking a stop codon, is the most abundant tumor-specific microprotein across patients. Proteomic, immunopeptidomic, and immunofluorescence analyses validate neoproteins as tumor-restricted antigens, revealing a therapeutically actionable cancer dark proteome controlled by one oncogenic fusion.

cancer biology↗

bioRxiv2025

Dynamic Ribosomal RNA Methylation Regulates Translation in the Hematopoietic System and is Essential for Stem Cell Fitness

Self-renewal and differentiation are at the basis of hematopoiesis. While it is known that tight regulation of translation is vital for hematopoietic stem cells (HSCs) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by ribosomal RNA (rRNA) methylation dynamics. Using ultra-low input ribosome-profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency changes progressively with differentiation and can distinguish between discrete cell populations as well as to define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification - 2-O-methyl (2OMe). We found that, like translation efficiency, 2OMe dynamics followed a distinct trajectory during erythroid differentiation. Genetic perturbation of individual 2OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSCs with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex-vivo, and loss of fitness in-vivo in bone marrow transplantations. Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2OMe profiles during differentiation of non-hematopoietic stem cells. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation. KEY POINTSO_LIHematopoietic differentiation is associated with rRNA methylation dynamics to control cell-type-specific translation. C_LIO_LITranslation efficiency can distinguish discrete cell types and define differentiation trajectories. C_LIO_LIHSC fitness is regulated by a single rRNA methylation. C_LI

molecular biology↗

bioRxiv2025

WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

The WEE1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to WEE1 inhibitors (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of EIF2A, a translation regulator, sensitized to WEE1i. Mechanistically, WEE1i treatment triggers a translational shut-down, which is lethal in combination with the reduced translation of EIF2AKO cells. A genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinases GCN1/2 rescued WEE1i-mediated cytotoxicity. WEE1i induced GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. Loss of the collided ribosome sensor ZNF598 conversely increased sensitivity to WEE1i. Notably, the ISR was not required for WEE1i to induce DNA damage, premature mitotic entry or sensitization to DNA-damaging chemotherapeutics. ISR activation was independent of CDK1/2 activation. Importantly, WEE1i-mediated ISR activation was independent of WEE1 presence, pointing at off-target effects, which are shared by multiple chemically distinct WEE1i. This response was also observed in peripheral blood mononuclear cells. Importantly, low-dose WEE1 inhibition did not induce ISR activation, while it still synergized with PKMYT1 inhibition. Taken together, WEE1i triggers toxic ISR activation and translational shutdown, which can be prevented by low-dose or combination treatments, while retaining the cell cycle checkpoint-perturbing effects.

cancer biology↗

bioRxiv2021

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.

cell biology↗