Virginie Petrilli

Ninjurin-1 (NINJ1) executes cell lysis by polymerizing into filaments, yet how these form membrane lesions is unknown. Integrating cell-based assays with molecular dynamics simulations, we demonstrate that NINJ1-driven membrane rupture starts with the lateral association of NINJ1 dimers into double filaments. Subsequently, swelling of necrotic cells increases plasma membrane tension, leading to separation of the filament interface and NINJ1 lesion opening. Inhibiting cell swelling prevents this increase in tension, blocking NINJ1-mediated plasma membrane rupture without disrupting NINJ1 oligomerization, implicating membrane tension as key for opening NINJ1 lesions. The NINJ1 homolog NINJ2 oligomerizes but fails to form membrane lesions during cell death due to the greater stability of its dimer. Finally, using atomic force microscopy of NINJ1-containing proteoliposomes, we show that NINJ1 forms large pores by stabilizing membrane edges. In summary, our findings support a model whereby membrane tension drives a zipper-like opening of NINJ1 double filaments, initiating necrotic cell lysis.

Secondary resistance to the amino-acid-depleting agent L-asparaginase (ASNase) remains poorly understood. Using ASNase-sensitive B-cell lymphoma (BCL) models, we investigate tumor relapse during treatment. Through in vitro and in vivo metabolic profiling, here we show that ASNase triggers a metabolic reprogramming characterized by increased de novo serine biosynthesis driven by phosphoglycerate dehydrogenase (PHGDH). This response mitigates treatment-induced oxidative stress and associated DNA damage, enabling malignant cells to survive. We evidence that ASNase-treated malignant cells exhibit features of replication stress and increase activity of poly(ADP-ribose) polymerase (PARP), revealing a dependence on DNA repair. Combining ASNase with the clinically approved PARP inhibitor Olaparib enhances the antineoplastic effect of each monotherapy in vitro and in vivo. Moreover, this combination shows effectiveness in homologous recombination-proficient colorectal cancer cells, suggesting broader therapeutic potential. Overall, our study identifies tumor metabolic and genomic vulnerabilities induced by ASNase and supports a rational combination strategy using clinically approved drugs.

Pyroptosis, an inflammatory form of cell death, is characterized by massive cell swelling and plasma membrane rupture. Although swelling was recently shown to occur in two steps, the molecular and biophysical mechanisms driving this process remained unclear. Using fast quantitative microscopy, we reveal that between the two swelling phases, cell volume transiently stabilizes despite sustained plasma membrane permeability to ions and small molecules. From a biophysical perspective, the existence of such a plateau is puzzling, as ion pumps should not be able to regulate cell volume under these conditions. To address this, we developed a physical model based on an ion pump and leak framework that incorporates the dynamics of nonselective pore formation. Experimentally, we demonstrate that the plateau phase is controlled by the dynamics of the gasdermin D (GSDMD) pore enlargement, which is modulated by ninjurin-1 (Ninj1) activation, possibly through intracellular calcium. Ninj1-mediated lesions are also required for the second swelling phase. We further show that fully opened GSDMD pores display an effective hydrodynamic radius slightly above 1.9 nm, providing an in situ upper bound for pore size. Together, our findings demonstrate that pyroptotic volume dysregulation emerges from the successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity due to pore opening. These insights bridge molecular and biophysical perspectives on lytic cell death and may inform the broader understanding of membrane rupture in inflammatory and pathological contexts.

Intrinsic mechanisms driving secondary resistance to L-asparaginase (ASNase) treatment remain poorly understood. Using a preclinical model of B-cell lymphomas (BCL), sensitive to ASNase, we observed tumor relapse during ASNase treatment despite an initial antineoplastic response. Through in vivo metabolic profiling of BCL and stable isotope resolved metabolomics in vitro and in vivo , we show that ASNase induces a flexible metabolic reprogramming marked by increased de novo serine biosynthesis. This adaptive response is driven in part by phosphoglycerate dehydrogenase (PHGDH), which mitigates ASNase-induced reactive oxygen species (ROS) and ROS-mediated DNA damage, enabling tumor cell survival under therapeutic pressure. ASNase-treated malignant B cells exhibit features of replication stress, with activation of the ATR-dependent DNA damage response and increased poly(ADP-ribose) polymerase 1/2 (PARP) activity. Targeting initiation of DNA repair mechanism using the PARP inhibitor Olaparib, enhances the antineoplastic effect of each individual mono-chemotherapy, both in vitro and in vivo. Importantly, we showed that this therapeutic combination was also effective in homologous recombination (HR)-proficient colorectal cancer cells, supporting its broader therapeutic relevance beyond lymphoid malignancies. Altogether, our findings reveal redox- and DNA repair-dependent metabolic vulnerabilities in ASNase-treated B-cell lymphomas and provide proof-of-concept for a rational combination therapy using clinically approved agents, ASNase and Olaparib.

DNA double-strand break (DSB) is a highly toxic lesion that can generate genome instability, a major source of tumorigenesis. DSBs are mainly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The selection of the DSB repair pathway primarily depends on the DNA resection of the DSB ends. Indeed, HR is initiated by resection at the DSB, generating 3′ single-stranded overhang. The shieldin complex prevents resection fostering DSB repair toward NHEJ. Here, we reveal that the inflammasome sensor NLRP3 facilitates DNA end resection to promote the HR pathway in an inflammasome-independent manner. Strikingly, NLRP3 silencing decreases HR efficiency, as evidenced by RAD51 foci and functional HR assays. Mechanistically, we describe that NLRP3 interacts with REV7, a subunit of the shieldin complex, and its depletion increases REV7 recruitment to IR-induced DSBs. Similar to cancer cells harboring HR-mutated genes, we find that NLRP3-deficient cells are sensitive to Poly-ADP-ribose polymerase (PARP) inhibitors (PARPi) and exhibit an epistatic relationship with BRCA1 deficiency. Remarkably, loss of REV7 in NLRP3-depleted cells induces PARPi resistance by restoring HR. This study unravels the crucial role of the innate immune receptor NLRP3 in regulating the selection of DSB repair pathways to maintain genome integrity.

Pyroptosis, an inflammatory form of cell death, is characterized by massive cell swelling and plasma membrane rupture. Although swelling was recently shown to occur in two steps, the molecular and biophysical mechanisms driving this process remained unclear. Using fast quantitative microscopy, we reveal that between the two swelling phases, cell volume transiently stabilizes despite sustained plasma membrane permeability to ions and small molecules. From a biophysical perspective, the existence of such a plateau is puzzling, as ion pumps should not be able to regulate cell volume under these conditions. To address this, we developed a physical model based on an ion pump and leak framework that incorporates the dynamics of non-selective pore formation. Experimentally, we demonstrate that the plateau phase is controlled by the dynamics of the GSDMD pore enlargement, which is modulated by Ninj1 activation, possibly through intracellular calcium. Ninj1-mediated lesions are also required for the second swelling phase. We further show that fully opened GSDMD pores display an effective hydrodynamic radius slightly above 1.9 nm, providing an insitu upper bound for pore size. Together, our findings demonstrate that pyroptotic volume dysregulation emerges from the successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity due to pore opening. These insights bridge molecular and biophysical perspectives on lytic cell death and may inform the broader understanding of membrane rupture in inflammatory and pathological contexts. Significance Statement: Among programmed modes of lytic cell death, pyroptosis mediated by gasdermin D (GSDMD) and ninjurin-1 (Ninj1) involves dramatic changes in cell shape and large volume fluctuations, fundamentally altering the cell’s physical properties. By combining optogenetics, quantitative microscopy, and modeling, we show that a progressive increase in plasma membrane pore size drives cell swelling and membrane lysis through successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity. A deeper understanding of these dynamic cell modifications will shed light on the molecular and biophysical mechanisms driving different forms of cell death.

Médecine/sciences (M/S), revue internationale dans le domaine de la recherche biologique, médicale et en santé

DNA double strand break (DSB) is a highly toxic lesion that can generate genome instability, a major source of tumorigenesis. DSBs are mainly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The selection of the DSB repair pathway primarily depends on the DNA resection of the DSB ends. Indeed, HR is initiated by resection at the DSB generating 3’ single stranded extension. The shieldin complex prevents resection fostering DSB repair toward NHEJ. Here, we reveal that the inflammasome sensor NLRP3 facilitates DNA end resection to promote the HR pathway in an inflammasome-independent manner. Strikingly, NLRP3 silencing decreases HR efficiency, as evidenced by RAD51 foci and functional HR assays. Mechanistically, we describe that NLRP3 interacts with REV7, a subunit of the shieldin complex, and its depletion increases REV7 recruitment to IR-induced DSBs. Similar to cancer cells harboring HR mutated genes, we find that NLRP3 deficient cells are sensitive to PARP inhibitors (PARPi) and exhibit an epistatic relationship with BRCA1 deficiency. Remarkably, loss of REV7 in NLRP3-depleted cells induces PARPi resistance by restoring HR. This study unravels the crucial role of the innate immune receptor NLRP3 in regulating the selection of DSB repair pathways to maintain genome integrity.

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.

Eukaryotic cells encounter diverse threats jeopardizing their integrity, prompting the development of defense mechanisms against these stressors. Among these mechanisms, inflammasomes are well‐known for their roles in coordinating the inflammatory response against infections. Extensive research has unveiled their multifaceted involvement in cellular processes beyond inflammation. Recent studies emphasize the intricate relationship between the inflammasome and the DNA damage response (DDR). They highlight how the DDR participates in inflammasome activation and the reciprocal impact of inflammasome on DDR and genome integrity preservation. Moreover, novel functions of inflammasome sensors in DDR pathways have emerged, broadening our understanding of their roles. Finally, this review delves into identifying common signals that drive the activation of inflammasome sensors alongside activation cues for the DNA damage response, offering potential insights into shared regulatory pathways between these critical cellular processes.