Biology subjects

Tilleman, L.

Publications and source records attributed to Tilleman, L..

6 recordsLinked to original sources

bioRxiv2023

The chromatin reader PHF6 at the crossroad of the replication stress and DNA damage responses in neuroblastoma through interaction with RRM2

The PHF6 protein is a presumed chromatin reader implicated in disease through germline loss-of-function mutations causing cognitive disability syndromes and somatic mutations are predominantly observed in acute T-cell leukemia. Previous reports support a role for PHF6 in DNA damage repair, replication fork restart as well as hematopoietic precursor cell self-renewal capacity and lineage commitment. To explore better how PHF6 mediates these functions, we mapped the PHF6 interactome and identified RRM2 as a consistent binding partner across different normal and malignant cell types. Next, PHF6 knockdown imposed increased replicative stress/DNA damage and suggested possible binding of PHF6 to H3K56ac, a marker for nascent DNA at sites of DNA damage repair. Genome-wide mapping of PHF6 chromatin binding indeed revealed overlap with sites of active DNA damage, binding sites of replication fork proteins and functional crosstalk with the neuroblastoma transcription core regulatory circuitry. Altogether, we show a canonical PHF6-RRM2 interaction enabling active transport of RRM2 to genomic sites of PHF6 mediated fork restart and PHF6 localization to H3K56ac at highly transcribed genes facilitating fork restart following replication-transcription conflicts.

cancer biology↗

bioRxiv2022

Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping

CYP2D6 is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants. Author SummaryDuring the last decades, the usefulness of personalized medicine has become increasingly apparent. Directly linked to that is the need for accurate genotyping assays to determine the pharmacogenetic profile of patients. Continuing research has led to the development of genotyping assays that perform quite robustly. However, complex genes remain an issue when it comes to determining the complete sequence correctly. An example of such a complex but very important pharmacogene is CYP2D6. Therefore, we developed a genotyping assay in an attempt to generate complete allele-specific consensus sequences of CYP2D6, by optimizing a targeted amplification-free long-read sequencing method and developing a new analysis pipeline. In reference samples, we showed that our genotyping assay performed accurately and confirmed the presence of variants that go undetected by most current assays. However, the implementation of this assay in practice is still hampered as the selected enrichment strategies inherently lead to a low percentage of on-target reads, resulting in low on-target sequencing depths. Further optimization and validation of the assay is thus needed, but definitely worth considering for follow-up research as we already demonstrated the added value for generating more complete genotypes, which on its turn will result in more accurate gene function predictions.

genetics↗

bioRxiv2021

MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as beta-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation. HighlightsO_LIActivation of MYCN attenuates proliferation in RPE1 cells C_LIO_LIGrowth arrest is associated with an early senescence-like transcriptional signature C_LIO_LITranscriptional and phenotypic evidence of nucleolar stress C_LIO_LICDKN1A upregulation in G2 phase primes cells for faltering in subsequent G1 C_LI

cancer biology↗

bioRxiv2020

RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma

Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10-15 percent of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease at diagnosis. Despite intensive multi-modal therapies nearly 50 percent of high-risk cases relapse and die of their disease. In contrast to the overall paucity of mutations, high-risk neuroblastoma nearly invariably present with recurrent somatic segmental chromosome copy number variants. For several focal aberrations (e.g. MYCN and LIN28B amplification), the direct role in tumor formation has been established. However, for recurrent aberrations, such as chromosome 2p and 17q gains, the identification of genes contributing to tumor initiation or progression has been challenging due to the scarcity of small segmental gains or amplifications. In this study, we identified and functionally evaluated the ribonucleotide reductase regulatory subunit 2 (RRM2) as a top-ranked 2p putative co-driver and therapeutic target in high-risk neuroblastoma enforcing replicative stress resistance. In vitro knock down and pharmacological RRM2 inhibition highlight RRM2 dependency in neuroblastoma cells, further supported by the finding that co-overexpression of RRM2 in a d{beta}h-MYCN transgenic zebrafish line increased tumor penetrance with 80% and accelerated tumor formation. Given the critical role of RRM2 in replication fork progression and regulation of RRM2 through ATR/CHK1 signaling, we tested combined RRM2 and ATR/CHK1 small molecule inhibition with triapine and BAY1895344/prexasertib respectively, and observed strong synergism, in particular for combined RRM2 and CHK1 inhibition. Transcriptome analysis following combinatorial drugging revealed HEXIM1 as one of the strongest upregulated genes. Using programmable DNA binding of dCas9 with a promiscuous biotin ligase, RRM2 promotor bound proteins were identified including HEXIM1 and NurRD complex members, supporting a cooperative role for HEXIM1 upregulation together with CHK1 inhibition in further attenuating RRM2 expression levels. We evaluated the impact of combined RRM2/CHK1 inhibition in vivo, with treatment of a murine xenograft model showing rapid and complete tumor regression, without tumor regrowth upon treatment arrest. In conclusion, we identified RRM2 as a novel dependency gene in neuroblastoma and promising target for synergistic drug combinations with small compounds targeting DNA checkpoint regulators.

cancer biology↗

bioRxiv2020

Endogenous suppression of WNT signalling in human embryonic stem cells leads to low differentiation propensity towards definitive endoderm

SummaryLow differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2018

Positive human health effects of sea spray aerosols: molecular evidence from exposed lung cell lines.

Sea spray aerosols (SSAs) have profound effects on climate and ecosystems. Furthermore, the presence of microbiota and biogenic molecules, produced by among others marine phytoplankton, in SSAs could lead to potential human health effects. Yet the exposure and effects of SSAs on human health remain poorly studied. Here, we exposed human epithelial lung cells to different concentrations of extracts of a natural sea spray aerosol (SSA), a laboratory-generated SSA, the marine algal toxin homoyessotoxin and a chemical mTOR inhibitor. The mTOR inhibitor was included as it has been hypothesized that natural SSAs may influence the mTOR cell signaling pathway. We observed significant effects on the mTOR pathway and PCSK9 in all exposures. Based on these expression patterns, a clear dose response relationship was observed. Our results indicate a potential for positive health effects when lung cells are exposed to environmentally relevant concentrations of natural SSAs, whereas potential negative effects were observed at high levels of the laboratory SSA and the marine algal toxin. Overall, these results provide a substantial molecular evidence base for potential positive health effects of SSAs at environmentally relevant concentrations through the mTOR pathway. The results provided here suggest that SSAs contain biomolecules with significant pharmaceutical potential in targeting PCSK9.

pharmacology and toxicology↗