Imaging molecular orientation at the nanoscale in live cells and tissues is fundamental in the understanding of proteins’ organization, which is driven by their structural and conformational properties. Measuring fluorescent molecules’ orientation is an interesting way to approach this problem, providing that the label is rigidly attached to the protein of interest. Despite the great progresses in fluorescence imaging down to nanometric scales, in particular by the use of single molecule (SM) localization-based super resolution imaging, orientations imaging is still only at its early stage. Measuring single molecules’ 3D orientations in addition to their 3D spatial localization is in particular, still today, a challenge, due to the intrinsic coupling of both spatial and orientational parameters in the SM point spread function (PSF) image formation. We present polarized fluorescence microscopy methods that are able to report both orientational and spatial information from single molecules in a non-ambiguous way. These methods, based on polarized splitting imaging or Fourier plane polarization and phase manipulation, give access to orientation parameters including the label wobbling extent, with a few degrees precision, in combination with 10’s nm spatial localization precision. We present the potential of these approaches for the imaging of the nanoscale organization of actin in cells, from very organized stress fiber bundles to complex networks in the cell lamellipodia. 

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Tumors employ complex, multi-scale cellular and molecular interactions that evolve over the course of therapeutic response. The changes in these pathways enables tumors to overcome therapeutic regimens, and ultimately acquire resistance. New molecular profiling technologies, including notably single cell technologies, provide an unprecedented opportunity to characterize these molecular relationships. However, interpreting the specific cellular and molecular pathways in therapeutic response requires complementary computational analysis methods. We developed an unsupervised learning method, CoGAPS, that employs Bayesian non-negative matrix factorization to disentangle distinct biological processes from high-throughput molecular data. Notably, this algorithm discovers dynamic compensatory signaling in acquired therapeutic resistance from time course bulk RNA-seq data and novel NK cell activation in anti-CTLA4 response from post-treatment scRNA-seq data. To further demonstrate that the inferred pathways are biological rather than computational artifacts, we developed a complementary transfer learning method to relate learned patterns between datasets. We demonstrate that this approach identifies robust molecular processes between model systems and human tumors and enables multi-platform data integration to delineate the drivers of therapeutic response and resistance.

STAG2, a cohesin family gene, is among the most recurrently mutated genes in cancer. STAG2 loss-of-function (LOF) is associated with aggressive behavior in Ewing sarcoma, a childhood cancer driven by aberrant transcription induced by the EWSR1-FLI1 fusion oncogene. Here, using isogenic Ewing cells, we show that while STAG2 LOF profoundly changes the transcriptome, it does not significantly impact EWSR1-FLI1, CTCF-cohesin or acetylated H3K27 DNA binding patterns. In contrast, it strongly alters the anchored dynamic loop extrusion process at boundary CTCF sites and dramatically decreases promoter-enhancer interactions, particularly affecting the expression of genes regulated by EWSR1-FLI1 through binding GGAA microsatellite elements. Down-modulation of cis-mediated EWSR1-FLI1 activity, observed in STAG2-LOF conditions, is associated with enhanced migration and invasion properties of Ewing cells previously observed in EWSR1-FLI1low cells. Our study illuminates a process whereby STAG2-LOF fine-tunes the activity of an oncogenic transcription factor through altered CTCF-anchored loop extrusion and cis-mediated enhancer mechanisms.

Chromatin factors such as remodelers, modifiers and histones contribute to the maintenance of genome stability by actively participating to the repair of DNA damage. DNA repair is essential for genome stability. This is particularly evident in highly proliferative cancer cells where it limits damage happening during replication, thus ensuring replication fork (RF) stability. In cancer, RF stability recently came into the spotlight and emerged as a new central mechanism of genome stability maintenance and chemoresistance.

While the overall role of chromatin in the maintenance of genome stability is evident, the contribution of chromatin factors to the maintenance and regulation of RFs stability and repair in human cells remains largely unknown, stressing the need to decipher the complex chromatin network that regulates genome stability at RFs and promotes chemoresistance.

The goal of my research is to provide with a systematic yer functional characterization of chromatin factors that are recruited to RFs upon damage, dissect their function in RF stability and their crosstalk with the repair machinery. Ultimately, I aim at defining how chromatin factors at RFs affect genome stability and promote chemoresistance, with important consequence for patient stratification and tumor treatment. During the seminar, I will present the most recent data that we obtained about this topic and I will propose future research axes. 

In adult, the continuous production of all human blood cells takes place in our bones, through the activity of hematopoietic stem cells (HSCs). The essential functions of HSCs are maintained and tightly regulated in the bone marrow micro-environment, referred to as bone marrow niche. This environment is defined by complex cellular, molecular, structural, and physical cues modulating HSC differentiation, survival and self-renewal. In healthy and disease contexts, the precise cellular and molecular composition of the human bone marrow niche, its functional organization and how these elements interact remains elusive. Gaining fundamental insights appears essential, and directly link to tremendous translational implications; from the identification of factors driving on-demand blood production, to the modelling of cancers and refinement of existing therapies.

In my laboratory, we ambition to decipher the functional diversity of the human bone marrow niche at the cellular and molecular levels, both during homeostasis and cancer settings. To do so, we exploit two complementary biotechnological platforms, offering the reconstitution of the human bone marrow environment. These consist in: (i) an advanced 3D in vitro system allowing the generation of functional bone marrow organoid in perfusion bioreactor, and (ii) the in vivo formation of humanized bone organs, aka ossicles. These technologies can be exploited to study hematopoietic malignancies (Myelodysplastic syndromes, Acute Myeloid Leukemia) but also solid tumors metastasizing to bones (Breast cancer, Neuroblastoma). Ultimately, we target the validation of our systems for the personalized modelling of cancer progression, as well as the testing of novel therapeutics.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in
infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas
are proposed to be epigenetically driven tumors for which model systems are
lacking. Here we demonstrate that PFA ependymomas are maintained under
hypoxia, associated with restricted availability of specific metabolites to diminish
histone methylation, and increase histone demethylation and acetylation at histone
3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first
trimester of human development that resides in restricted oxygen. Unlike other
ependymomas, transient exposure of PFA cells to ambient oxygen induces
irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and,
paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor
growth. Targeting metabolism and/or the epigenome presents a unique opportunity
for rational therapy for infants with PFA ependymoma.

Small cell lung cancer (SCLC) accounts for approximately 15% of all lung malignancies. It is a highly aggressive neuroendocrine tumour type characterized by rapid growth and widespread metastasis. The mechanisms underlying SCLC initiation and progression remain poorly understood and to date, there are no effective, long lasting treatments for SCLC patients. Using CRISPR-Cas9 genome-wide screens and genetically engineered mouse models, we uncovered and characterized novel SCLC tumour suppressive networks. Notably, we identified an unexpected role for MAX, the obligate heterodimer partner for MYC oncoproteins, as a context specific tumour suppressor gene. Mechanistically, we found that MAX loss leads to a metabolic rewiring that involves one carbon (1C) metabolism. This discovery sheds new light on the regulation of the fine-tuned MYC network. Next, in an attempt to identify tailored treatment options for SCLC patients, we explored several pharmacological approaches. SCLC tumours frequently harbor inactivating mutations in epigenetic regulators suggesting broad epigenetic and transcriptional alterations. We therefore investigated a series of epigenetic inhibitors and identified LSD1 inhibition as a viable therapeutic strategy. Altogether, this work has revealed novel tumour suppressive networks and therapeutic avenues for the most aggressive form of lung cancer.

University College London, UMC Utrecht et l'Institut Curie s'associent pour organiser un événement virtuel le 25 mai dans le cadre de la série d'événements ADAPT to Thrive sur l'échec et la résilience.

Vous voulez savoir comment des universitaires et des chercheurs accomplis ont échoué et ont survécu pour raconter leur histoire ? Obtenir des conseils sur la façon de construire une carrière de chercheur lorsque l'échec et la prise de risques en font partie intégrante ? Alors inscrivez-vous pour participer au prochain événement ADAPT to Thrive !

En partageant les expériences de chercheurs à différents stades de leur carrière, la série d'événements "ADAPT to Thrive" apporte un soutien aux chercheurs en début de carrière en montrant comment l'échec et le succès vont de pair.

"ADAPT to Thrive" va :

Intervenants :

Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis, however its effects on cancer progression remain poorly understood. To address this question we used ribosome profiling to compare translation efficiencies genome-wide in poorly and highly metastatic breast cancer cells and in breast cancer patient-derived xenografts. We identified HNRNPC (heterogeneous nuclear ribonucleoprotein C), as a translational controller of a specific mRNA regulon. Mechanistically, HNRNPC, in concert with PABPC4 (poly(A) binding protein cytoplasmic 4), binds near to poly(A) signals, thereby governing the alternative polyadenylation of a set of mRNAs. We found that HNRNPC and PABPC4 are downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3’ UTR lengthening and subsequently, translational repression. We showed that modulating HNRNPC expression impacted the metastatic capacity of breast cancer cells in in vivo mouse models. We also found that a small molecule, previously shown to induce a distal-to-proximal poly(A) site switch (i.e. 3’ UTR shortening), counteracts the HNRNPC-PABPC4 driven translational deregulation and reduces the metastatic lung colonization of breast cancer cells in vivo.

The mitotic machinery segregates an equal genomic complement to daughter cells during cell division. Variation in the mitotic machinery between cell types and due to genomic alterations in specific cancer types offers an opportunity to identify cancer-specific mitotic vulnerabilities that can be leveraged in cancer therapy. My recent work identified a synthetic lethality between the inhibition of the PLK4 kinase, which is required for centrosome assembly, and the cancer-specific amplification of a chromosomal region that includes the gene encoding the ubiquitin ligase TRIM37 (50-60% of neuroblastoma and ~10% of breast cancer). This sensitivity is explained by TRIM37 promoting the degradation of a protein component required for the backup pathway that enables cells to assemble acentrosomal mitotic spindles following PLK4 inhibition. In addition to the identification of cancer-specific mitotic vulnerabilities, a second focus of my work has been understanding the tumor suppressive pathways that monitor mitosis to prevent the proliferation of cells that have undergone chromosome missegregation and the implications of these pathways for cancer development and treatment. ~ 90% of all cancers are aneuploid, indicating that they experienced mitotic errors during oncogenesis. My work identified the first two molecular components, 53BP1 and the ubiquitin protease USP28, of a p53-based mitotic surveillance mechanism (‘the mitotic stopwatch’) that monitors the quality of chromosome segregation by precisely measuring the time it takes for a cell to complete mitosis. Even a 1.5-fold increase in the duration of mitosis, if experienced in two sequential generations, results in p53 stabilization by the mitotic stopwatch, marking the resulting cells for arrest or death. p53-negative cancers lack a functional mitotic stopwatch, however about 50% of cancers are p53-positive. My analysis indicates that the mitotic stopwatch is inactivated in many p53-positive cancers, consistent with the idea that inactivation of the stopwatch may contribute to oncogenic transformation and/or cancer progression. p53-positive cancers with a perturbed mitotic stopwatch often have mutations in the genes encoding USP28 or 53BP1. However, in many p53-positive cancers the cause of the inactivation of the stopwatch is unclear, indicating the existence of as-yet unknown mechanisms­. Pediatric cancers like neuroblastoma, with a low mutational burden, frequently retain a functional mitotic stopwatch and are consequently highly susceptible to mitotic inhibitors that prolong mitosis. In neuroblastoma, TRIM37 amplification and the presence of an intact mitotic stopwatch synergize to render them highly susceptible to PLK4 inhibition. My future goals are to understand mitotic surveillance mechanisms and their relationship to cancer and to identify novel cancer-specific mitotic vulnerabilities that stimulate new avenues in cancer therapy.

Antigen-specific T lymphocytes are key orchestrators and effectors of nearly all adaptive immune responses. Upon antigen exposure, naive T cells proliferate and undergo differentiation into distinct classes of effector cells. After antigen clearance, a small proportion of long-lived antigen-specific memory T cells remain with the capacity to give rise to recall responses in the event of antigen re-exposure. Functional memory T cells can be detected in humans for several decades following acute viral infections or vaccinations. However, in case of antigen persistence during chronic viral infections or cancer, T cells develop a dysfunctional phenotype toward a state named “T cell exhaustion” that is thought to limit immunopathology but ultimately leads to disease progression and/or persistence.

Here, through a specifically designed clinical trial, we investigated whether termination of antigen exposure enables human exhausted T cell populations to re-differentiate into effective T cell memory. In another study, we use a systematic experimental approach of challenging co-cultures of ex vivo purified cell populations to interrogate the cellular mechanisms involved in antigen-specific CD8+ T cell recall responses in human.

The results discussed in this seminar identify several novel fundamental mechanisms that have potential implications for the design of next-generation vaccines and immunotherapies targeting T cell responses.

TBD

Due to the advent of high-throughput technologies, high-dimensional “omics” data are produced at an increasing pace. In cancer biology, national and international consortia have profiled thousands of tumors at multiple molecular levels (“multi-omics”) allowing to gather a comprehensive molecular picture of this disease. Moreover, multi-omics profiling approaches are currently being transposed at single-cell resolution, further increasing the information accessible from cancer samples.  The current main challenge is to design appropriate methods to integrate this wealth of information and translate it into actionable biological knowledge.

In this talk, I will discuss two main computational directions for multi-omics integration: (i) multiplex networks, to integrate a large range of interactions and (ii) joint Dimensionality Reduction (jDR), to extract biological knowledge simultaneously from multiple omics. First, I will present their application to bulk data and the biological results that we derived. Then I will discuss our ongoing research in single-cell.

TBD

TBD

Epigene2Sys is a CNRS GDRi, coordinated by G. Almouzni (CNRS, Paris) and M-E Torres Padilla (HGMU, Munich), was launched in 2018 in continuation to the EpiGeneSys Network of Excellence,  with the aim to stimulate discoveries and discussions in epigenetics research and to encourage equal opportunity and career evolution of young researchers. Following the success of the “Happygene2sys” online version of the annual network meeting last October, a second online version will take place starting in the afternoon of December 13th, 2021 and ending in the evening of December 14th, 2021.

The days will be comprised of : 

KEYNOTE SPEAKERS   |   Suzana Hadjur – UCL, Timothee Lionnet – NYU, Karen Miga – UCSC, Didier Trono - EPFL

SHORT TALKS   |   Young researchers selected from submitted abstracts 

POSTERS/FLASH TALKS (pre-recorded)   |   PhDs & Postdocs selected from submitted abstracts

SOCIAL TIMES, MIXERS & DISCUSSION SESSIONS

Registration is free but mandatory.

Talks will be selected from submitted abstracts so please register early and submit an abstract!

Looking forward to "seeing" you in December!

Séminaire proposé par shauna katz