Biology subjects

Sturla, S. J.

Publications and source records attributed to Sturla, S. J..

3 recordsLinked to original sources

bioRxiv2023

Single-nucleotide-resolution genomic maps of O6-methylguanine from the glioblastoma drug temozolomide

Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to apoptosis due to mismatch-repair overload. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Characterizing genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT. O6-MeG signatures were highly similar to mutational signatures from patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes, suggesting an additional transcription-associated contribution to its repair. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and regulation. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/571283v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@357353org.highwire.dtl.DTLVardef@12688b9org.highwire.dtl.DTLVardef@da3381org.highwire.dtl.DTLVardef@16b7e7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, a unique agent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides new therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, a full mechanistic understanding of the drugs TC-NER-dependent toxicity is needed. Here, we determined that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We mapped the 3-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. We showed that trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs were also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use trabectedin for precision oncology and for studying TC-NER and transcription.

molecular biology↗

bioRxiv2022

Chemical screening of food-related chemicals for human fatty liver risk: Combining high content imaging of cellular responses with in vitro to in vivo extrapolation

1Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent human disease with accumulating evidence linking its pathophysiology and co-morbidities to chemical exposures. The complex pathophysiology of NAFLD has limited the elucidation of potential chemical etiologies. In this study we generated a high-content imaging analysis method for the simultaneous quantification of sentinel steatosis cellular markers in chemically exposed human liver cells in vitro combined with a computational model for the extrapolation of human oral equivalent doses (OED). First, the in vitro test method was generated using 14 reference chemicals with known capacities to induce cellular alterations in nuclear morphology, lipid accumulation, mitochondrial membrane potential and oxidative stress. These effects were quantified on a single cell- and population-level, and then, using physiologically based pharmacokinetic modelling and reverse dosimetry, OEDs were extrapolated from these in vitro data. The extrapolated OEDs were confirmed to be within biologically relevant exposure ranges for the reference chemicals. Next, we tested 14 chemicals found in food, selected from thousands of putative chemicals on the basis of structure-based prediction for nuclear receptor activation. Amongst these, orotic acid had an extrapolated OED overlapping with realistic exposure ranges. By the strategy developed in this study, we were able to characterize known NAFLD-inducing chemicals and translate to data scarce food-related chemicals, amongst which we identified orotic acid to induce steatosis. This strategy addresses needs of next generation risk assessment, and can be used as a first chemical prioritization hazard screening step in a tiered approach to identify chemical risk factors for NAFLD.

pharmacology and toxicology↗