mTOR inhibition suppresses salinomycin-induced ferroptosis in breast cancer stem cells by ironing out mitochondrial dysfunctions.

Ferroptosis constitutes a promising therapeutic strategy against cancer by efficiently targeting the highly tumorigenic and treatment-resistant cancer stem cells (CSCs). We previously showed that the lysosomal iron-targeting drug Salinomycin (Sal) was able to eliminate CSCs by triggering ferroptosis. Here, in a well-established breast CSCs model (human mammary epithelial HMLER CD24low/CD44high), we identified that pharmacological inhibition of the mechanistic target of rapamycin (mTOR), suppresses Sal-induced ferroptosis. Mechanistically, mTOR inhibition modulates iron cellular flux and thereby limits iron-mediated oxidative stress. Furthermore, integration of multi-omics data identified mitochondria as a key target of Sal action, leading to profound functional and structural alteration prevented by mTOR inhibition. On top of that, we found that Sal-induced metabolic plasticity is mainly dependent on the mTOR pathway. Overall, our findings provide experimental evidence for the mechanisms of mTOR as a crucial effector of Sal-induced ferroptosis pointing not only that metabolic reprogramming regulates ferroptosis, but also providing proof-of-concept that careful evaluation of such combination therapy (here mTOR and ferroptosis co-targeting) is required in the development of an effective treatment.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Patient-derived organoids identify an apico-basolateral polarity switch associated with survival in colorectal cancer.

The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies - MUC CRCs either display a conventional 'apical-in' polarity or, more frequently, harbor an inverted 'apical-out' topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFß and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

Paradoxical implication of BAX/BAK in the persistence of tetraploid cells.

Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of ß-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.

ATG4D is the main ATG8 delipidating enzyme in mammalian cells and protects against cerebellar neurodegeneration.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

Melanoma Persister Cells Are Tolerant to BRAF/MEK Inhibitors via ACOX1-Mediated Fatty Acid Oxidation.

Emerging evidence indicates that non-mutational drug tolerance mechanisms underlie the survival of residual cancer "persister" cells. Here, we find that BRAF(V600E) mutant melanoma persister cells tolerant to BRAF/MEK inhibitors switch their metabolism from glycolysis to oxidative respiration supported by peroxisomal fatty acid ß-oxidation (FAO) that is transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARα). Knockdown of the key peroxisomal FAO enzyme, acyl-CoA oxidase 1 (ACOX1), as well as treatment with the peroxisomal FAO inhibitor thioridazine, specifically suppresses the oxidative respiration of persister cells and significantly decreases their emergence. Consistently, a combination treatment of BRAF/MEK inhibitors with thioridazine in human-melanoma-bearing mice results in a durable anti-tumor response. In BRAF(V600E) melanoma samples from patients treated with BRAF/MEK inhibitors, higher baseline expression of FAO-related genes and PPARα correlates with patients' outcomes. These results pave the way for a metabolic strategy to overcome drug resistance.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Lethal Poisoning of Cancer Cells by Respiratory Chain Inhibition plus Dimethyl α-Ketoglutarate.

Inhibition of oxidative phosphorylation (OXPHOS) by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells in vitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combined with B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both in vitro and in vivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneous inhibition of OXPHOS and glycolysis provoked a bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens.

Biology and prognostic impact of clonal plasmacytoid dendritic cells in chronic myelomonocytic leukemia.

Islands of CD123high cells have been commonly described in the bone marrow of patients with chronic myelomonocytic leukemia (CMML). Using a multiparameter flow cytometry assay, we detected an excess of CD123+ mononucleated cells that are lineage-negative, CD45+, CD11c-, CD33-, HLA-DR+, BDCA-2+, BDCA-4+ in the bone marrow of 32/159 (20%) patients. Conventional and electron microscopy, flow cytometry detection of cell surface markers, gene expression analyses, and the ability to synthesize interferon alpha in response to Toll-like receptor agonists identified these cells as bona fide plasmacytoid dendritic cells (pDCs). Whole-exome sequencing of sorted monocytes and pDCs identified somatic mutations in genes of the oncogenic RAS pathway in the two cell types of every patient. CD34+ cells could generate high amount of pDCs in the absence of FMS-like tyrosine kinase 3-ligand (FLT3L). Finally, an excess of pDCs correlates with regulatory T cell accumulation and an increased risk of acute leukemia transformation. These results demonstrate the FLT3L-independent accumulation of clonal pDCs in the bone marrow of CMML patients with mutations affecting the RAS pathway, which is associated with a higher risk of disease progression.

Mutations in the SRP54 gene cause severe congenital neutropenia as well as Shwachman-Diamond-like syndrome.

Congenital neutropenias (CNs) are rare heterogeneous genetic disorders, with about 25% of patients without known genetic defects. Using whole-exome sequencing, we identified a heterozygous mutation in the SRP54 gene, encoding the signal recognition particle (SRP) 54 GTPase protein, in 3 sporadic cases and 1 autosomal dominant family. We subsequently sequenced the SRP54 gene in 66 probands from the French CN registry. In total, we identified 23 mutated cases (16 sporadic, 7 familial) with 7 distinct germ line SRP54 mutations including a recurrent in-frame deletion (Thr117del) in 14 cases. In nearly all patients, neutropenia was chronic and profound with promyelocytic maturation arrest, occurring within the first months of life, and required long-term granulocyte colony-stimulating factor therapy with a poor response. Neutropenia was sometimes associated with a severe neurodevelopmental delay (n = 5) and/or an exocrine pancreatic insufficiency requiring enzyme supplementation (n = 3). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). We showed that SRP54 was specifically upregulated during the in vitro granulocytic differentiation, and that SRP54 mutations or knockdown led to a drastically reduced proliferation of granulocytic cells associated with an enhanced P53-dependent apoptosis. Bone marrow examination of SRP54-mutated patients revealed a major dysgranulopoiesis and features of cellular ER stress and autophagy that were confirmed using SRP54-mutated primary cells and SRP54 knockdown cells. In conclusion, we characterized a pathological pathway, which represents the second most common cause of CN with maturation arrest in the French CN registry.

DNA repair deficiency sensitizes lung cancer cells to NAD+ biosynthesis blockade.

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non-small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house-generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro - ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells - and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.

Tumour spheres with inverted polarity drive the formation of peritoneal metastases in patients with hypermethylated colorectal carcinomas.

Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-ß signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

Critical role of the HDAC6-cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect.

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6-CTTN axis as a positive regulator of human but not mouse MK maturation.

Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis.

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

PrP(c) deficiency and dasatinib protect mouse intestines against radiation injury by inhibiting of c-Src.

BACKGROUND & AIM: Despite extensive study of the contribution of cell death and apoptosis to radiation-induced acute intestinal injury, our knowledge of the signaling mechanisms involved in epithelial barrier dysfunction remains inadequate. Because PrP(c) plays a key role in intestinal homeostasis by renewing epithelia, we sought to study its role in epithelial barrier function after irradiation. DESIGN: Histology, morphometry and plasma FD-4 levels were used to examine ileal architecture, wound healing, and intestinal leakage in PrP(c)-deficient (KO) and wild-type (WT) mice after total-body irradiation. Impairment of the PrP(c) Src pathway after irradiation was explored by immunofluorescence and confocal microscopy, with Caco-2/Tc7 cells. Lastly, dasatinib treatment was used to switch off the Src pathway in vitro and in vivo. RESULTS: The decrease in radiation-induced lethality, improved intestinal wound healing, and reduced intestinal leakage promoted by PrP(c) deficiency demonstrate its involvement in acute intestinal damage. Irradiation of Cacao2/Tc7 cells induced PrP(c) to target the nuclei associated with Src activation. Finally, the protective effect triggered by dasatinib confirmed Src involvement in radiation-induced acute intestinal toxicity. CONCLUSION: Our data are the first to show a role for the PrP(c)-Src pathway in acute intestinal response to radiation injury and offer a novel therapeutic opportunity.

The oncolytic peptide LTX-315 triggers necrotic cell death.

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation.

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease.

SWI/SNF chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear bodies.

Paraspeckles are subnuclear structures that form around nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA (lncRNA). Recently, paraspeckles were shown to be functional nuclear bodies involved in stress responses and the development of specific organs. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >40 paraspeckle proteins (PSPs). In this study, subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA. EM observations revealed that SWI/SNF complexes were enriched in paraspeckle subdomains depleted of chromatin. Knockdown of SWI/SNF components resulted in paraspeckle disintegration, but mutation of the ATPase domain of the catalytic subunit BRG1 did not affect paraspeckle integrity, indicating that the essential role of SWI/SNF complexes in paraspeckle formation does not require their canonical activity. Knockdown of SWI/SNF complexes barely affected the levels of known essential paraspeckle components, but markedly diminished the interactions between essential PSPs, suggesting that SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. The interactions between SWI/SNF components and essential PSPs were maintained in NEAT1-depleted cells, suggesting that SWI/SNF complexes not only facilitate interactions between PSPs, but also recruit PSPs during paraspeckle assembly. SWI/SNF complexes were also required for Satellite III lncRNA-dependent formation of nuclear stress bodies under heat-shock conditions. Our data suggest the existence of a common mechanism underlying the formation of lncRNA-dependent nuclear body architectures in mammalian cells.