Biology subjects

Pommier, Y.

Publications and source records attributed to Pommier, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamic Schlafen 11 (SLFN11) expression revealed by single-cell RNA sequencing of clonal human cancer cells

Schlafen 11 (SLFN11) is a key determinant of sensitivity to DNA-damaging therapies in multiple cancers including leukemia, Ewing sarcoma, head and neck cancers, mesothelioma and small cell lung cancer (SCLC). Here, utilizing immunofluorescence microscopy and single-cell RNA sequencing we characterized SLFN11 expression across eight different human cancer cell lines and found cell-to-cell heterogenous expression both at the protein and RNA levels. Gene set enrichment and pathway analyses show that SLFN11 is coexpressed with a distinct stress-response state and enhanced genome maintenance programs while being inversely correlated with protein translation pathways. Publicly available single-cell RNA-sequencing datasets of human SCLC tumors confirmed the heterogeneous distribution of SLFN11 expression and confirmed its association with replication-related transcriptional programs. These findings indicate that heterogeneous SLFN11 expression reflects dynamic regulation at the single-cell level. They also underscore the limitations of bulk proteomic and transcriptome profiling and point to a potential reservoir of intrinsic chemotherapy resistance.

cancer biology↗

bioRxiv2026

TR-107, a novel mitochondrial ClpP agonist, induces robust antitumor activity against preclinical models of adrenocortical carcinoma

AbstractAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy originating from the adrenal cortex with limited effective treatment options. The underlying pathophysiology of ACC is uniquely characterized by abnormal steroid production and increased metabolic activity, highlighting the critical role of mitochondria in adrenal steroid hormone biosynthesis and tumor metabolism. In this study, we investigated the therapeutic potential of TR-107, a novel and highly selective small-molecule agonist targeting the mitochondrial protease ClpP. Pharmacologic hyperactivation of ClpP disrupts mitochondrial proteostasis and bioenergetics and has shown promising antitumor activity in various preclinical models. Our results demonstrated that TR-107 induces potent dose-dependent cytotoxic effects at nanomolar concentrations in ACC cell lines NCI-H295R and mACC3 as well as short-term ACC patient-derived organoid (PDO) models, markedly reducing cell viability and confluency in vitro. Metabolic analyses revealed that TR-107 significantly impaired oxygen consumption, indicating a disruption of oxidative phosphorylation and substantial attenuation of basal cellular respiration. Mechanistic studies showed dose-dependent increases in reactive oxygen species (ROS) levels and upregulation of proteins involved in mediating the ferroptotic rheostat. Pharmacokinetic assessment uncovered that TR-107 was not a substrate of the ABCB1 (MDR1/P-glycoprotein) efflux transporter, suggesting potential to overcome common multidrug resistance mechanisms. Given the importance of IGF-2 signaling in ACC, we further explored the combinatorial effects of TR-107 with IGF-1 receptor (IGF-1R) inhibitors and discovered that co-treatment produced synergistic reductions in cell viability across NCI-H295R, mACC3, and ACC PDOs. Collectively, these findings support the potential of mitochondrial ClpP hyperactivation as a promising therapeutic strategy for ACC and demonstrate that TR-107 exhibits significant antitumor activity as a monotherapy or in combination with IGF-1R inhibitors. These findings provide a strong rationale for advancing ClpP agonists into clinical development for the management of ACC.

cancer biology↗

bioRxiv2026

Cryo-EM structural analysis of human TOP1 trapping by eight clinical anticancer drugs

Human topoisomerase 1 (TOP1) resolves DNA supercoiling during replication and transcription and is a major target for anticancer therapy. TOP1 poisons exert cytotoxicity by stabilizing the TOP1-DNA cleavage complex (TOP1cc), thereby blocking DNA rejoining and generating lethal DNA damage. Several TOP1 poisons have been approved either as conventional therapeutics or as payloads in targeted delivery systems, and many additional candidates are under clinical development. Here, we resent cryo-EM structures of human TOP1cc bound to eight representative and clinically relevant TOP1 poisons: camptothecin (CPT), six CPT derivatives, and an indenoisoquinoline LMP-400. These cryo-EM structures reveal a TOP1cc conformation that differs substantially from canonical crystal structures. These structures also define how specific modifications on the CPT central scaffold and changing to an alternative non-CPT scaffold reshape drug intercalation geometry, molecular interaction networks, and TOP1cc protein architecture. Together with biochemical trapping data, these structural insights establish a foundation for designing next-generation TOP1 poisons with improved pharmacological properties and for their optimization as antibody-drug conjugate (ADC) payloads.

pharmacology and toxicology↗

bioRxiv2026

Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase and RNF168

The molecular mechanisms driving SLFN11 chromatin recruitment remain partially elucidated. Using high-throughput imaging of 162 oncology-focused compounds in U2OS cells with inducible SLFN11 expression, we discovered that deubiquitinase (DUB) inhibitors drive massive SLFN11 recruitment to chromatin, preferentially at promoter regions while concurrently suppressing transcription. DUB inhibitors such as VLX-1570 promote ubiquitin-dependent enrichment of SLFN11 without detectable DNA damage, distinct from the camptothecin-induced RPA-associated SLFN11 foci formed at stressed replication forks. Yet, SLFN11 chromatin recruitment both by DUB inhibitors and DNA damage are suppressed by TAK243 demonstrating their ubiquitylation dependency. RNF168 is required for SLFN11 ubiquitylation and its subsequent chromatin association, and ubiquitylation within SLFN11s middle linker domain (lysines 390, 391, and 429) with K27-linked polyubiquitin chains is essential for the chromatin recruitment of SLFN11. These findings suggest the importance of SLFN11 ubiquitylation by RNF168 for SLFN11 chromatin recruitment and SLFN11 transcriptional regulatory role at promoter regions.

cell biology↗

bioRxiv2026

Schlafen 11 (SLFN11) overexpression and nucleolar localization in response to bortezomib in multiple myeloma

Proteins belonging to the Schlafen family are interferon-inducible and participate in the regulation of antiviral responses, immune signaling and proteotoxic stress. SLFN11 also kills cells with replicative damage, serving as a predictive biomarker for chemotherapeutic response. Here we examined SLFN11 expression and significance in multiple myeloma (MM). The TCGA and MMRF CoMMpass datasets were analyzed for SLFN11 expression. Bone marrow and cell lines samples were analyzed for SLFN11 protein. SLFN11-knockout MM cell lines were used to explore how SLFN11 affects bortezomib response. Retrospective analysis of the HOVON-65/GMMG-HD4 phase III trial (n=327) assessed clinical relevance. SLFN11 is consistently highly expressed across MM subtypes (except CD1 and MAF/MAFB) and in normal plasma cells, and its expression strongly correlates with super-enhancer-driven plasma cell transcriptional programs. CD138-positive normal and myeloma plasma cells retain SLFN11 expression even when proliferative activity (MKI67/Ki-67) increases with disease progression. Bortezomib, a first-line MM treatment, induces SLFN11 nucleolar translocation with suppression of ribosomal RNA synthesis. Knocking out SLFN11 in MM cells enhances bortezomib sensitivity and exatecan resistance, supporting SLFN11s protective role in proteotoxic stress and sensitizing role in replication stress. In the HOVON-65/GMMG-HD4 trial, SLFN11-low patients showed selective benefit from bortezomib-based therapy, suggesting that SLFN11 expression may guide therapeutic stratification in MM. SignificanceSLFN11 is highly expressed in normal and malignant plasma cells. Bortezomib induces SLFN11 nucleolar translocation, suppressing ribosomal RNA synthesis and global translation. SLFN11 confers bortezomib resistance while sensitizing to topoisomerase I inhibitors. Clinical analysis supports that low SLFN11 expression predicts bortezomib benefit, providing a mechanistic basis for SLFN11-guided therapeutic stratification.

cancer biology↗

bioRxiv2025

SLFN11 puts the brakes on Alternative lengthening of telomeres

Alternative lengthening of telomeres (ALT) is a homologous recombination-dependent mechanism maintaining telomere length in approximately 10-15% of all cancers that are telomerase (TERT) negative. ALT is most prominent in osteosarcoma. Although many ALT cells feature loss of the ATRX/DAXX chromatin remodeling complex, ATRX/DAXX deficiency alone is insufficient to trigger ALT. Here, we provide evidence that Schlafen 11 (SLFN11) acts as a suppressor of the telomeric ALT pathway. TERT-negative osteosarcoma U2-OS (ALT) cells, that normally lack SLFN11 expression, show SLFN11 localization to telomeres upon doxycycline-induced SLFN11 expression. This re-expression markedly suppresses ALT activity, as evidenced by reduced ALT-associated PML bodies (APBs) and decreased levels of Telomeric Repeat-containing RNA (TERRA). SLFN11 re-expression also attenuates the telomeric DNA damage response (DDR) and induces telomere destabilization in ALT cells. Furthermore, SLFN11 suppresses ALT induction in ATRX-depleted prostate carcinoma DU145 cells. Collectively, our findings identify SLFN11 as a negative telomeric regulator of the ALT pathway, indicating that its loss, together with ATRX/DAXX inactivation, contributes to ALT activation.

cell biology↗

bioRxiv2025

Peroxiredoxin 1 safeguards the nucleolar genome from oxidative damage

Peroxiredoxin 1 (PRDX1) is a highly conserved, thiol-dependent peroxidase that rapidly scavenges reactive oxygen species to modulate redox signaling. PRDX1-null mice exhibited genomic instability, shortened lifespan, and accelerated tumorigenesis, including development of lymphomas, sarcomas, and carcinomas. Despite extensive characterization of these phenotypes, the molecular mechanism by which PRDX1 loss causes genomic instability remains poorly understood. Here we show that PRDX1 deficiency alters nucleolar morphology, impairs RNA Polymerase I (POL-I)-dependent transcription of pre-ribosomal RNAs and triggers nucleolar genomic instability. This oxidative stress-induced nucleolar dysfunction promotes the stability of secondary DNA structures, such as RNA-DNA hybrids and G-quadruplex DNA, contributing to nucleolar genomic instability. We demonstrate that PRDX1 loss reduces nascent rRNA levels and impairs rRNA processing, further affecting ribosome biogenesis. Mechanistically, we established that PRDX1 loss triggers activation of the nucleolar DNA damage response including activation of DNA repair kinase ATM and the nucleolar factor TCOF1 within the nucleolus, and recruitment of the MRE11-RAD50-NBS1 (MRN) complex subunit NBS1 to ribosomal DNA (rDNA) loci. NBS1 accumulation correlates with the repression of rDNA transcription by POL-I, potentially delaying rRNA synthesis, and safeguarding the nucleolar genome from further oxidative damage. Collectively, these findings uncover a previously unrecognized, but critical role, for PRDX1 in maintaining nucleolar integrity and ribosomal biogenesis through redox-dependent regulation of rDNA transcription and processing machinery.

molecular biology↗

bioRxiv2025

SCLC-TumorMiner: A Directly Accessible Genomics Resource for Precision Oncology: Big Data for Small Cells

Small cell lung cancer (SCLC) is among the most aggressive malignancies. Unlike many other cancers, it is not represented in The Cancer Genome Atlas, and available datasets are fragmented across institutions, disease stages, and treatment settings. RNA sequencing provides a powerful and cost-effective approach, but the high dimensionality of transcriptomic data and the heterogeneity of patient cohorts pose significant challenges. To address such challenges, we developed SCLC TumorMiner (https://discover.nci.nih.gov/SclcTumorMinerCDB/), which includes 50 tumor samples from relapse patients at the National Cancer Institute (NCI) and 154 samples from untreated patients at the University of Cologne and Tongji University. SCLC TumorMiner enables molecular classification, genomic pathway analyses, risk stratification, identification of predictive cell-surface biomarkers such as DLL3 or TROP2, and drug-response biomarkers such as SLFN11. SCLC TumorMiner illustrates profound differences between untreated and relapse patient samples. Additionally, "MyPatient", one of SCLC TumorMiners modules, is presented as a medical assistant application prototype.

cancer biology↗

bioRxiv2025

CRISPR-Based Gene Dependency Screens reveal Mechanism of BRAF Inhibitor Resistance in Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by the WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors both in vitro and in vivo. Using gene essentiality score across a large panel of cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.

cancer biology↗

bioRxiv2025

Lamin A/C Deficiency Drives Genomic Instability and Poor Survival in Small-Cell Lung Cancer through Increased R-loop Accumulation

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [1]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [2]. Here, we investigate LMNAs role in small-cell lung cancer (SCLC), a highly aggressive malignancy characterized by extreme genomic instability [3, 4]. We demonstrate that LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA loss disrupts nuclear pore complex distribution, reducing phenylalanine-glycine (FG)-nucleoporin incorporation and impairing RNA export efficiency. Furthermore, we show that LMNA expression is epigenetically repressed by EZH2 during SCLC differentiation from neuroendocrine (NE) to non-NE states. Clinically, low LMNA levels correlate with significantly worse survival in SCLC patients. These findings uncover a novel role for LMNA in safeguarding genome integrity and shaping tumor heterogeneity, with broad implications for cancer and aging. Significance StatementLamin A/C, a key structural component of the nuclear envelope, is frequently lost or mutated in cancer, laminopathies, and aging-related disorders. Lamin A/C loss is associated with genomic instability, but the underlying mechanisms remain incompletely understood. We demonstrate that LMNA loss drives genomic instability by promoting R-loop accumulation through disrupted nuclear pore dynamics and impaired RNA export. These findings reveal a previously unrecognized link between LMNA loss, nuclear envelope dysfunction, and genome instability. Targeting this pathway could help mitigate genomic instability in aging and laminopathies, while leveraging R-loop accumulation may enhance the efficacy of DNA-damaging therapies in cancer.

cancer biology↗

bioRxiv2025

Bimodal genomic approach predicting Semaphorin 7A (SEMA7A) as prognostic biomarker in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy with high mortality and poor prognosis. To elucidate the genetic underpinnings of ACCs, we have analyzed the transcriptome data of 112 ACC tumor samples from patients enrolled in the TCGA and NCI. Among 72 bimodally expressed genes stratifying patients into prognostic groups, we focused on SEMA7A, as it encodes a glycosylphosphatidylinositol-anchored membrane glycoprotein (Semaphorin 7a) regulating integrin-mediated signaling, cell migration and immune responses. We find that high SEMA7A gene expression is associated with poor prognosis (hazard ratio = 4.27; p-value < 0.001). In hormone-producing ACCs, SEMA7A expression is elevated and positively correlated with genes driving steroidogenesis, aldosterone and cortisol synthesis, including CYP17A1, CYP11A1, INHA, DLK1, NR5A1 and MC2R. Correlation analyses show that SEMA7A is co-expressed with the integrin-{beta}1, FAK (focal adhesion kinase) and MAPK/ERK (mitogen-activated protein kinase/extracellular signal regulated kinases) signaling pathways. Immunohistochemistry (IHC) staining demonstrates the feasibility of evaluating SEMA7A in ACC tissues and shows significant correlation between gene expression (RNA-Seq) and protein expression (IHC). These findings suggest SEMA7A as a candidate for further research in ACC biology, a candidate for cancer therapy, as well as a potential prognosis biomarker for ACC patients. Translational relevanceAdrenocortical cancer (ACC) remains a challenging disease primarily due to the scarcity of reliable biomarkers for predicting patient outcomes and informing innovative therapeutic strategies, as well as its rarity, which restricts the scope of clinical trials. In our study, we performed RNAseq and IHC analyses of ACC samples sourced from The Cancer Genome Atlas (TCGA), tissue microarray slide, and National Cancer Institute (NCI) cancer patient samples. Our findings indicate that a substantial proportion of ACC tumors exhibit expression of SEMA7A, a glycoprotein involved in Semaphorin cell surface signaling. Notably, elevated levels of SEMA7A were identified as a poor prognostic biomarker and were associated with activation of the integrin-ERK-MAPK kinase signaling pathways. These results suggest that ACC tumors with high SEMA7A expression should be considered at elevated risk, and SEMA7A may serve as a potential target for immunotherapeutic strategies, including antibody-drug conjugates, T-cell engagers, and/or small molecule inhibitors targeting the MAPK pathway.

cancer biology↗

bioRxiv2025

Topoisomerase IIIb protects from tumorigenesis and immune dysregulation

Topoisomerase III-beta (Top3b) reduces nucleic acid torsional stress and intertwining generated during RNA and DNA metabolism while protecting the genome from pathological R-loops, which otherwise result in DNA breakage and genome instability. By studying Top3b knockout mice (Top3b-KO), we find that the loss of Top3b accelerates the development of spontaneous lymphoid tumors arising in spleens and lymph nodes, the organs with prominent Top3b expression. Aging Top3b-KO mice also display splenomegaly and systemic immune alterations including neutrophilia and lymphopenia suggestive of chronic inflammation. At the molecular level, Top3b deficiency causes genome-wide R-loop accumulation in splenocytes as measured by CUT&Tag sequencing. Increased R-loops is associated with genomic DNA breaks and activation of immune signaling pathways including the IL-6 signaling, interleukin-7 signaling and cGAS-STING. Moreover, knocking-out Top3b promotes the rapid development of syngeneic EL4 T-cell lymphomas. In conclusion, our work implies that, in addition to its role in preserving the nervous system, Top3b protects from tumorigenesis and immune dysregulations. SignificanceTopoisomerase III-beta (TOP3B) is the only dual RNA-DNA topoisomerase. Its inactivation has previously been shown to be critical for neurodevelopmental disorders in humans and R-loop suppression in cell line models. Here, we demonstrate that loss of Top3b accelerates spontaneous lymphomagenesis and promotes syngeneic lymphoma growth in association with innate immune defects and R-loop accumulation in Top3b-KO mice. These findings provide evidence for a previously unappreciated role of Top3b as a tumor suppressor gene and immune regulator.

cancer biology↗

bioRxiv2024

Identification of DLK1, a Notch ligand, as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

Immunotherapeutic targeting of cell surface proteins is an increasingly effective cancer therapy. However, given the limited number of current targets, the identification of new surface proteins, particularly those with biological importance, is critical. Here, we uncover delta-like non-canonical Notch ligand 1 (DLK1) as a cell surface protein with limited normal tissue expression and high expression in multiple refractory adult metastatic cancers including small cell lung cancer (SCLC) and adrenocortical carcinoma (ACC), a rare cancer with few effective therapies. In ACC, ADCT-701, a DLK1 targeting antibody-drug conjugate (ADC), shows potent in vitro activity among established cell lines and a new cohort of patient-derived organoids as well as robust in vivo anti-tumor responses in cell line-derived and patient-derived xenografts. However, ADCT-701 efficacy is overall limited in ACC due to high expression and activity of the drug efflux protein ABCB1 (MDR1, P-glycoprotein). In contrast, ADCT-701 is extremely potent and induces complete responses in DLK1+ ACC and SCLC in vivo models with low or no ABCB1 expression. Genetic deletion of DLK1 in ACC dramatically downregulates ABCB1 and increases ADC payload and chemotherapy sensitivity through NOTCH1-mediated adrenocortical de-differentiation. Single cell RNA-seq of ACC metastatic tumors reveals significantly decreased adrenocortical differentiation in DLK low or negative cells compared to DLK1 positive cells. This works identifies DLK1 as a novel immunotherapeutic target that regulates tumor cell plasticity and chemoresistance in ACC. Our data support targeting DLK1 with an ADC in ACC and neuroendocrine neoplasms in an active first-in-human phase I clinical trial (NCT06041516).

cancer biology↗

bioRxiv2024

Metastatic organotropism in small cell lung cancer

Metastasis is the leading cause of cancer-related deaths, yet its regulatory mechanisms are not fully understood. Small-cell lung cancer (SCLC) is the most metastatic form of lung cancer, with most patients presenting with widespread disease, making it an ideal model for studying metastasis. However, the lack of suitable preclinical models has limited such studies. We utilized rapid autopsy-derived tumors to develop xenograft models that mimic key features of SCLC, including histopathology, rapid and widespread development of metastasis to the liver, brain, adrenal, bone marrow, and kidneys within weeks, and response to chemotherapy. By integrating in vivo lineage selection with comprehensive bulk and single cell multiomic profiling of transcriptomes and chromatin accessibility, we identified critical cellular programs driving metastatic organotropism to the liver and brain, the most common sites of SCLC metastasis. Our findings reveal the key role of nuclear-cytoskeletal interactions in SCLC liver metastasis. Specifically, the loss of the nuclear envelope protein lamin A/C, encoded by the LMNA gene, increased nuclear deformability and significantly increased the incidence of liver metastasis. Human liver metastases exhibited reduced LMNA expression compared to other metastatic sites, correlating with poorer patient outcomes and increased mortality. This study introduces novel preclinical models for SCLC metastasis and highlights pathways critical for organ-specific metastasis, offering new avenues for the development of targeted therapies to prevent or treat metastatic disease.

cancer biology↗

bioRxiv2024

Mitochondrial topoisomerase I (Top1MT) prevents the onset of metabolic dysfunction-associated steatohepatitis (MASH) in mice

High fat (HF) diet is a major factor in the development of metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatis (MASH), and mitochondria have been proposed to play a role in the pathogenesis of HF diet-induced MASH. Because Mitochondrial topoisomerase I (Top1MT) is exclusively present in mitochondria and Top1MT knock-out mice are viable, we were able to assess the role of Top1MT in the development of MASH. We show that after 16 weeks of HF diet, mice lacking Top1MT are prone to the development of severe MASH characterized by liver steatosis, lobular inflammation and hepatocyte damage. Mice lacking Top1MT also show prominent mitochondrial dysfunction, ROS production and mitochondrial DNA (mtDNA) release, accompanied by hepatic inflammation and fibrosis. In summary, our study demonstrates the importance of Top1MT in sustaining hepatocyte functions and suppressing MASH.

molecular biology↗

bioRxiv2024

Genome-wide Mapping of Topoisomerase Binding Sites Suggests Topoisomerase 3α(TOP3A) as a Reader of Transcription-Replication Conflicts (TRC)

Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and topological problems. Here we asked which topoisomerase(s) is/are the best candidate(s) for sensing TRC. Genome-wide topoisomerase binding sites were mapped in parallel for all the nuclear topoisomerases (TOP1, TOP2A, TOP2B, TOP3A and TOP3B). To increase the signal to noise ratio (SNR), we used ectopic expression of those topoisomerases in H293 cells followed by a modified CUT&Tag method. Although each topoisomerase showed distinct binding patterns, all topoisomerase binding signals positively correlated with gene transcription. TOP3A binding signals were suppressed by DNA replication inhibition. This was also observed but to a lesser extent for TOP2A and TOP2B. Hence, we propose the involvement of TOP3A in sensing both head-on TRCs (HO-TRCs) and codirectional TRCs (CD-TRCs). In which case, the TOP3A signals appear concentrated within the promoters and first 20 kb regions of the 5 -end of genes, suggesting the prevalence of TRCs and the recruitment of TOP3A in the 5-regions of transcribed and replicated genes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/599352v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1d1afdeorg.highwire.dtl.DTLVardef@17146eaorg.highwire.dtl.DTLVardef@1e6bed3org.highwire.dtl.DTLVardef@1fa40db_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Metabolism-focused CRISPR screen unveils Mitochondrial Pyruvate Carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (PARP1/2) is a proven target with several PARP inhibitors (PARPi) currently in clinical use. Resistance to PARP inhibitors often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. PARP1/2 utilize NAD+ (nicotinamide adenine dinucleotide), an essential substrate not only for PARPs but also for multiple pathways in cellular metabolism, TCA cycle and mitochondrial functions. Thus, NAD+ is central to many key cellular functions. Both activation of PARPs by DNA damage and their inhibition by drugs such as olaparib affect NAD+ consumption. We surmised that alterations in NAD+ metabolism by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused CRISPR knockout (KO) screen to identify genes which undergo alterations during treatment of tumor cells with PARP inhibitors. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing NSCLC lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the TCA cycle and oxidative phosphorylation pathways to overcome PARP inhibitor treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.

cancer biology↗

bioRxiv2023

Flap endonuclease 1 repairs DNA-protein crosslinks via ADP-ribosylation

DNA-protein crosslinks (DPCs) are among the most ubiquitous and detrimental DNA lesions which arise from exposure to metabolic stresses, drugs, or crosslinking agents such as formaldehyde (FA). FA is a cellular by-product of methanol metabolism, histone demethylation, lipid peroxidation as well as environmental pollutants. Failure to repair FA-induced DPCs blocks nearly all chromatin-based processes including replication and transcription, leading to immunodeficiencies, neurodegeneration, and cancer. Yet, it remains largely unknown how the cell repairs DPCs. The study of DPC repair is impeded by our incomprehension of the types of proteins crosslinked by FA due to the lack of techniques to identify the DPCs. Here, we designed a novel bioassay to profile FA-induced DPCs by coupling cesium chloride differential ultracentrifugation with HPLC-mass spectrometry (MS). Using the method, we revealed the proteome of FA-induced DPCs in human cells and found that the most abundant proteins that form DPCs are PARP1, topoisomerases I and II, methyltransferases, DNA and RNA polymerases, histones, as well as ribosomal proteins. To identify enzymes that repair DPCs, we carried out RNA interference screening and found that downregulation of flap endonuclease 1 (FEN1) rendered cells hypersensitive to FA. Since FEN1 possesses 5-flap endonuclease activity, we hypothesized that FA induces DPC-conjugated 5-flap DNA fragments that can be processed by FEN1. Indeed, we demonstrate that FA damages DNA bases that are converted into 5-flap via the base excision pathway (BER). We also observed that the damaged DNA bases were colocalized with DPCs and FEN1. Mechanistically, we showed that FEN1 repairs FA-induced DPCs in vivo and cleaves 5-flap DNA substrate harboring DPC mimetic in vitro. We also found that FEN1 repairs enzymatic topoisomerase II (TOP2)-DPCs induced by their inhibitors etoposide and doxorubicin independently of the BER pathway, and that FEN1 and the DPC-targeting protease SPRTN act as parallel pathways for the repair of both FA-induced non-enzymatic DPCs and etoposide-induced enzymatic TOP2-DPCs. Notably, we found that FA-induced non-enzymatic DPCs and enzymatic TOP2-DPCs are promptly modified by poly-ADP-ribosylation (PARylation), a post-translational modification catalyzed by PARP1, a key DNA damage response effector that acts by PARylating both DNA damage sites and DNA repair proteins. We performed immunoprecipitation (IP) assays with anti-PAR antibody for HPLC-MS and identified FEN1 as a PARylation substrate. Next, we showed that PARylation of DPC substrates signaled FEN1 whereas PARylation of FEN1 drove FEN1 to DPC sites. Finally, using the enzymatic labeling of the terminal ADP-ribose-MS method, we identified the E285 residue of FEN1 as a dominant PARylation site, which appeared to be required for FEN1 relocation to DPCs. Taken together, our work not only unveiled the identities of FA-induced DPCs but also discovered an unprecedented PARP1-FEN1 nuclease pathway as a universal and imperative mechanism to repair the miscellaneous DPCs and prevent DPC-induced genomic instability.

molecular biology↗