Olivier Destaing

Tumor progression is driven by cancer cell fitness, defined as the capacity of malignant cells to maintain growth, adapt to stress and withstand therapy Cellular fitness is fundamentally governed by nucleolar processes, which act as central regulators by integrating RNA processing with ribosome biogenesis to support protein synthesis and stress adaptation. The nuclear protein CYCLON, containing a large intrinsically disordered region (IDR) could be implicated in mediating biomolecular condensates and regulatory plasticity, which are key elements of nucleolar biology. CYCLON also emerged as a candidate regulator of cancer cell fitness, as it is frequently overexpressed across tumor types. Inducible silencing and cell biology approaches have shown that CYCLON maintains nucleolar integrity, controls nucleoli size and number, nucleolin and Ki-67 distribution and prevents nucleolar stress. CYCLON contributes to ribosome biogenesis by binding ribosomal RNA (rRNA) and regulating 32S and 21S pre-rRNA processing, ultimately influencing ribosomal subunit production and global protein synthesis. Its depletion impairs proliferation and clonogenic capacity by prolonging both interphase and mitosis, leading to slowed cell cycle progression. The impact of CYCLON on cellular fitness has been consistently observed across cancer models, reinforcing its essential role in the regulation of nucleolar biology.

Pancreatic ductal adenocarcinoma (PDAC) is the main and the deadliest form of pancreatic cancer. This is a major problem of public health since it will become the second leading cause of death by cancer in the next few years, mainly due to the lack of efficient therapies. Transient Receptor Potential Cation Channel Subfamily M Member 7 (TRPM7) protein, a cation channel fused with a serine/threonine kinase domain is overexpressed in PDAC and associated with a low survival. In this work, we aim to study the role of kinase domain on pancreatic cell fates by using a model of kinase domain deletion by CRISPR-Cas9. PANC-1 and MIA PaCa-2 PDAC cell lines were used and kinase domain was deleted by CRISPR-Cas9 strategy. Kinase domain deletion (ΔK) was validated by RT-qPCR and western blots. The effect of kinase domain deletion on channel function was studied by patch-clamp and Mn²⁺-quenching. The cell phenotype was studied by MTT and cell migration/invasion assays. Finally, the role of kinase domain was studied in vivo in xenografted mice. Here we show that TRPM7 kinase domain is required to maintain a mesenchymal phenotype in PDAC cells. We also demonstrated that TRPM7 and PAK1 interact in the same protein complexes. Moreover, TRPM7 kinase domain is required for carcinogenesis and cancer cell dissemination in vivo. Intriguingly, the role of TRPM7 kinase is cell specific and may depend on the KRAS oncogene mutation status. In conclusion, TRPM7 kinase domain is required to maintain a mesenchymal and aggressive phenotype in PDAC cells, and it could be a promising target against PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the main and the deadliest form of pancreatic cancer. This is a major problem of public health since it will become the second leading cause of death by cancer in the next few years, mainly due to the lack of efficient therapies. Transient Receptor Potential Cation Channel Subfamily M Member 7 (TRPM7) protein, a cation channel fused with a serine/threonine kinase domain is overexpressed in PDAC and associated with a low survival. In this work, we aim to study the role of kinase domain on pancreatic cell fates by using a model of kinase domain deletion by CRISPR-Cas9. PANC-1 and MIA PaCa-2 PDAC cell lines were used and kinase domain was deleted by CRISPR-Cas9 strategy. Kinase domain deletion (ΔK) was validated by RT-qPCR and western-blots. The effect of kinase domain deletion on channel function was studied by patch-clamp and Mn²⁺-quenching. The cell phenotype was studied by MTT and cell migration/invasion assays. Finally, the role of kinase domain was studied in vivo in xenografted mice. Here we show that TRPM7 kinase domain is required to maintain a mesenchymal phenotype in PDAC cells. We also demonstrated that TRPM7 and PAK1 interact in the same protein complexes. Moreover, TRPM7 kinase domain is required for carcinogenesis and cancer cell dissemination in vivo. Intriguingly, the role of TRPM7 kinase is cell specific and may depend on the KRAS oncogene mutation status. In conclusion, TRPM7 kinase domain is required to maintain a mesenchymal and aggressive phenotype in PDAC cells, and it could be a promising target against PDAC.

Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1β and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck–like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.

Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables β3-integrin-mediated force generation independently of β1 integrin. β3-integrin-mediated forces were associated with a decrease in β3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in β3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.

The spatial organization of cell-surface receptors is fundamental for the coordination of biological responses to physical and biochemical cues of the extracellular matrix. How serine/threonine kinase receptors, ALK3-BMPRII, cooperate with integrins upon BMP2 to drive cell migration is unknown. Whether the dynamics between integrins and BMP receptors intertwine in space and time to guide adhesive processes is yet to be elucidated. We found that BMP2 stimulation controls the spatial organization of BMPRs by segregating ALK3 from BMPRII into β3 integrin-containing focal adhesions. The selective recruitment of ALK3 to focal adhesions requires β3 integrin engagement and ALK3 activation. BMP2 controls the partitioning of immobilized ALK3 within and outside focal adhesions according to single-protein tracking and super-resolution imaging. The spatial control of ALK3 in focal adhesions by optogenetics indicates that ALK3 acts as an adhesive receptor by eliciting cell spreading required for cell migration. ALK3 segregation from BMPRII in integrin-based adhesions is a key aspect of the spatio-temporal control of BMPR signaling.

Mathematical models of biological processes implicated in cancer are built using the knowledge of complex networks of signaling pathways, describing the molecular regulations inside different cell types, such as tumor cells, immune and other stromal cells. If these models mainly focus on intracellular information, they often omit a description of the spatial organization among cells and their interactions, and with the tumoral microenvironment. We present here a model of tumor cell invasion simulated with PhysiBoSS, a multiscale framework which combines agent-based modeling and continuous time Markov processes applied on Boolean network models. With this model, we aim to study the different modes of cell migration by considering both spatial information obtained from the agent-based simulation and intracellular regulation obtained from the Boolean model. Our multiscale model integrates the impact of gene mutations with the perturbation of the environmental conditions and allows the visualization of the results with 2D and 3D representations. The model successfully reproduces single and collective migration processes and is validated on published experiments on cell invasion. In silico experiments are suggested to search for possible targets that can block the more invasive tumoral phenotypes.

Summary: Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.

Cell invasion is associated with numerous patho-physiologic states including cell development and metastatic dissemination. This process couples the activation of cell motility with the capacity to degrade the extracellular matrix, thereby permitting cells to pass through basal membranes. Invasion is sustained by the actions of invadosomes, an ensemble of subcellular structures with high functional homology. Invadosomes are 3D acto-adhesive structures that can also mediate local extracellular matrix degradation through the controlled delivery of proteases. Intracellular RHO GTPases play a central role in the regulation of invadosomes where their complex interplay regulates multiple invadosome functions. This review aims to provide an overview of the synergistic activities of the small GTPases in invadosome biology. This broad-based review also reinforces the importance of the spatiotemporal regulation of small GTPases and the impact of this process on invadosome dynamics.