Biology subjects

Draetta, G.

Publications and source records attributed to Draetta, G..

7 recordsLinked to original sources

bioRxiv2025

RF-SIRF defining reversed DNA replication forks with single-cell and spatio-temporal resolution reveals a replication stress specific epigenetic code

DNA replication stress responses are key genomic stability guardians critical during development, aging, hematopoiesis, disease suppression and cancer therapy response. Reversed forks (RF) form at stalled DNA replication forks as a distinct four-way DNA structure to protect against formation and exposure to toxic DNA lesions. So far, prevailing methods to measure RFs involve specialized electron microscopy precluding studies within their cellular context. Here we describe an in-situ method to quantitatively measure RFs by harnessing intrinsic bio-physical properties of this distinct DNA structure (RF-SIRF). RF-SIRF reveals that RFs accumulate at the nuclear periphery and form predominantly during early-mid S-phase of the cell cycle. Importantly, RFs are chromatinized and utilize an epigenetic replication stress code distinct from transcription that explains how DNA stress response proteins are recruited to RFs. Collectively, RF-SIRF enables robust, quantitative, temporal and spatial analyses of RFs and associated proteomics, empowering advanced cellular investigations of DNA replication stress responses.

molecular biology↗

bioRxiv2023

Evolutionary fingerprints of EMT in pancreatic cancers

Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.

cancer biology↗

bioRxiv2020

Targeting mitochondrial respiration and the BCL2 family in MYC-associated B-cell lymphoma

Multiple molecular features, such as activation of specific oncogenes (e. g. MYC, BCL2) or a variety of gene expression signatures, have been associated with disease course in diffuse large B-cell lymphoma (DLBCL). Understanding the relationships between these features and their possible exploitation toward disease classification and therapy remains a major priority in the field. Here, we report that MYC activity in DLBCL is closely correlated with - and most likely a driver of - gene signatures related to Oxidative Phosphorylation (OxPhos). On this basis, we hypothesized that enzymes involved in Oxidative Phosphorylation, and in particular electron-transport chain (ETC) complexes, might constitute tractable therapeutic targets in MYC-associated lymphoma. Indeed, our data show that MYC sensitizes B-cells to IACS-010759, a selective inhibitor of ETC complex I. Mechanistically, IACS-010759 activates an ATF4-driven Integrated Stress Response (ISR), engaging the intrinsic apoptosis pathway through the transcription factor CHOP. In line with these findings, IACS-010759 shows synergy with the BCL2 inhibitor venetoclax against double-hit lymphoma (DHL), a high-grade form of DLBCL with concurrent activation of MYC and BCL2. Similarly, in BCL2-negative lymphoma cell lines, inhibition of the BCL2-related protein Mcl-1 potentiates killing by IACS-010759. Altogether, ETC complex I inhibition engages the ISR to lower the apoptotic threshold in MYC-driven lymphomas and, in combination with select BCL2-family inhibitors, provides a novel therapeutic principle against this aggressive DLBCL subset. Statement of significanceThis work points to OxPhos as a key MYC-activated process and a tractable therapeutic target toward personalized treatment of high-grade DLBCL, providing strong context-dependent cooperation with BH3-mimetic compounds.

cancer biology↗

bioRxiv2020

Medium-chain acyl-CoA dehydrogenase, a gatekeeper of mitochondrial function in glioblastoma multiforme

Glioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain. In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

cancer biology↗

bioRxiv2020

Immune landscape of a genetically-engineered murine model of glioma relative to human glioma by single-cell sequencing

BackgroundThis study focuses on the evaluation of intratumoral immune infiltrates of the Qk/trp53/Pten (QPP) triple-knockout mouse model of glioblastoma with the purpose of establishing its relevance compared to the human disease. This included an analysis at the single cell level, of immune cells composition in both spontaneous and implanted mouse tumors and of samples obtained from human tumor resections. MethodsWe analyzed a cohort of fifteen spontaneous QPP mice, nine implanted QPP mice and 10 glioma patients by standard immune profiling methods such as IHC. Of those, we analyzed three spontaneous QPP mice, three implanted QPP mice, and 10 glioma patients by single cell RNA sequencing. ResultsIn the QPP samples we identified a predominantly myeloid cell population of monocytes, macrophages, and microglia, with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found that there were more neutrophil, T and NKT cells in the implanted model. In human samples, most of the lymphoid and myeloid compartments were immunosuppressive. While the complexity of the myeloid cell populations is preserved between the QPP model and the human disease, we observed significantly more immune-stimulatory populations in the implanted mouse model. ConclusionsWe found that, despite differences at the subpopulation level, the immunosuppressive nature of the immune system in glioma, is recapitulated in our mouse model. Although we observed differences in the proportions of immune infiltrates derived from the spontaneous and implanted mouse models and in LGG compared to GBM, the major constituents are present in all cases and these differences may be caused by various etiological or pathogenic influences on tumor-immune interactions. Key PointsO_LINew GBM mouse model for immunotherapy C_LIO_LISingle cell profiling of the immune systems in human and mouse GBM C_LI

cancer biology↗

bioRxiv2020

Inhibition of the av integrin-TGF-b axis improves natural killer cell function against glioblastoma stem cells

Glioblastoma, the most aggressive brain cancer, often recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. Here, we show that patient-derived GSCs, but not normal astrocytes, are highly sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. In contrast, single cell analysis of autologous, tissue infiltrating NK cells isolated from surgical samples of high-grade glioblastoma patient tumors using mass cytometry and single cell RNA sequencing revealed an abnormal phenotype associated with impaired lytic function compared with peripheral blood NK cells from GBM patients or healthy donors. This immunosuppression was attributed to an integrin-TGF-{beta} mechanism, activated by direct cell-cell contact between GSCs and NK cells. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-{beta} signaling, or with TGF-{beta} receptor 2 gene-edited NK cells prevented GSC-induced NK cell dysfunction and tumor growth. Collectively, our findings reveal a novel mechanism of NK cell immune evasion by GSCs and implicate the integrin-TGF-{beta} axis as a useful therapeutic target to eliminate GSCs in this devastating tumor.

immunology↗

bioRxiv2018

Multiplexed pooled library screening with Cpf1

RNA interference and CRISPR/Cas9-based pooled library screens have revolutionized the field of functional genomics. However, currently available pooled library screens face a trade-off between library effectiveness and library complexity. We developed a multiplexed, high-throughput screening strategy based on an optimized AsCpf1 nuclease that minimizes library size without sacrificing gene targeting efficiency. Our AsCpf1-based multiplexed library performed similarly well compared to currently available CRISPR/Cas9 libraries, but with a single polycistronic crRNA clone targeting each gene. With this strategy, we constructed the smallest whole-genome knock-out library available, \"Mini-human\" for the human genome, which is one-fourth the size of the smallest CRISPR library currently available.

systems biology↗