Doxorubicin induces phosphorylation of lamin A/C and loss of nuclear membrane integrity: A novel mechanism of cardiotoxicity.

Lamin A/C, essential inner nuclear membrane proteins, have been linked to progeria, a disease of accelerated aging, and many other diseases, which include cardiac disorder. Lamin A/C mutation and its phosphorylation are associated with altering nuclear shape and size. The role of lamin A/C in regulating normal cardiac function was reported earlier. In the present study, we hypothesized that Doxorubicin (Dox) may alter total lamin A/C expression and phosphorylation, thereby taking part in cardiac injury. An in vitro cellular injury model was generated with Dox (0.1-10.0 µM) treatment on cardiomyoblast cells (H9c2) to prove our hypothesis. Increased size and irregular (ameboid) nucleus shape were observed in H9c2 cells after Dox treatment. Similarly, we have observed a significant increase in cell death on increasing the Dox concentration. The expression of lamin A/C and its phosphorylation at serine 22 significantly decreased and increased, respectively in H9c2 cells and rat hearts after Dox exposure. Phosphorylation led to depolymerization of the lamin A/C in the inner nuclear membrane and was evidenced by their presence throughout the nucleoplasm as observed by immunocytochemistry techniques. Thinning and perforation on the walls of the nuclear membrane were observed in Dox-treated H9c2 cells. LMNA-overexpression in H9c2 protected the cells from Dox-induced cell death, reversing all changes described above. Further, improvement of lamin A/C levels was observed in Dox-treated H9c2 cells when treated with Purvalanol A, a CDK1 inhibitor and N-acetylcysteine, an antioxidant. The study provides new insight regarding Dox-induced cardiac injury with the involvement of lamin A/C and alteration of inner nuclear membrane structure.

Molecular tension microscopy of the LINC complex in live cells.

We present a protocol to measure the effect of pharmacological treatments on the mechanical tension experienced by nesprins at the cytoplasmic surface of the nuclear envelope of mammalian cells in culture. We apply this protocol to MDCK epithelial cells exposed to the actin depolymerization agent cytochalasin D. To do so, we perform confocal spectral imaging of transiently expressed molecular tension sensors of mini-nesprin 2G and analyze the FRET signal from the sensors with a custom-made Fiji script. For complete details on the use and execution of this protocol, please refer to Déjardin et al. (2020).

Nuclear envelope budding is a response to cellular stress.

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

The Alkylating Agent Methyl Methanesulfonate Triggers Lipid Alterations at the Inner Nuclear Membrane That Are Independent from Its DNA-Damaging Ability.

In order to tackle the study of DNA repair pathways, the physical and chemical agents creating DNA damage, the genotoxins, are frequently employed. Despite their utility, their effects are rarely restricted to DNA, and therefore simultaneously harm other cell biomolecules. Methyl methanesulfonate (MMS) is an alkylating agent that acts on DNA by preferentially methylating guanine and adenine bases. It is broadly used both in basic genome stability research and as a model for mechanistic studies to understand how alkylating agents work, such as those used in chemotherapy. Nevertheless, MMS exerts additional actions, such as oxidation and acetylation of proteins. In this work, we introduce the important notion that MMS also triggers a lipid stress that stems from and affects the inner nuclear membrane. The inner nuclear membrane plays an essential role in virtually all genome stability maintenance pathways. Thus, we want to raise awareness that the relative contribution of lipid and genotoxic stresses when using MMS may be difficult to dissect and will matter in the conclusions drawn from those studies.

The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone.

Ataxia telangiectasia (AT) is a rare genetic neurodegenerative disease. To date, there is no available cure for the illness, but the use of glucocorticoids has been shown to alleviate the neurological symptoms associated with AT. While studying the effects of dexamethasone (dex) in AT fibroblasts, by chance we observed that the nucleoplasmic Lamin A/C was affected by the drug. In addition to the structural roles of A-type lamins, Lamin A/C has been shown to play a role in the regulation of gene expression and cell cycle progression, and alterations in the LMNA gene is cause of human diseases called laminopathies. Dex was found to improve the nucleoplasmic accumulation of soluble Lamin A/C and was capable of managing the large chromatin Lamin A/C scaffolds contained complex, thus regulating epigenetics in treated cells. In addition, dex modified the interactions of Lamin A/C with its direct partners lamin associated polypeptide (LAP) 2a, Retinoblastoma 1 (pRB) and E2F Transcription Factor 1 (E2F1), regulating local gene expression dependent on E2F1. These effects were differentially observed in both AT and wild type (WT) cells. To our knowledge, this is the first reported evidence of the role of dex in Lamin A/C dynamics in AT cells, and may represent a new area of research regarding the effects of glucocorticoids on AT. Moreover, future investigations could also be extended to healthy subjects or to other pathologies such as laminopathies since glucocorticoids may have other important effects in these contexts as well.

Deuterium Depletion Inhibits Cell Proliferation, RNA and Nuclear Membrane Turnover to Enhance Survival in Pancreatic Cancer.

The effects of deuterium-depleted water (DDW) containing deuterium (D) at a concentration of 25 parts per million (ppm), 50 ppm, 105 ppm and the control at 150 ppm were monitored in MIA-PaCa-2 pancreatic cancer cells by the real-time cell impedance detection xCELLigence method. The data revealed that lower deuterium concentrations corresponded to lower MiA PaCa-2 growth rate. Nuclear membrane turnover and nucleic acid synthesis rate at different D-concentrations were determined by targeted [1,2-13C2]-D-glucose fate associations. The data showed severely decreased oxidative pentose cycling, RNA ribose 13C labeling from [1,2-13C2]-D-glucose and nuclear membrane lignoceric (C24:0) acid turnover. Here, we treated advanced pancreatic cancer patients with DDW as an extra-mitochondrial deuterium-depleting strategy and evaluated overall patient survival. Eighty-six (36 male and 50 female) pancreatic adenocarcinoma patients were treated with conventional chemotherapy and natural water (control, 30 patients) or 85 ppm DDW (56 patients), which was gradually decreased to preparations with 65 ppm and 45 ppm deuterium content for each 1 to 3 months treatment period. Patient survival curves were calculated by the Kaplan-Meier method and Pearson correlation was taken between medial survival time (MST) and DDW treatment in pancreatic cancer patients. The MST for patients consuming DDW treatment (n = 56) was 19.6 months in comparison with the 6.36 months' MST achieved with chemotherapy alone (n = 30). There was a strong, statistically significant Pearson correlation (r = 0.504, p < 0.001) between survival time and length and frequency of DDW treatment.

Insulin-promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10.

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.

Spatial control of nucleoporin condensation by fragile X-related proteins.

Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation.

Ultrastructural Characteristics of DHA-Induced Pyroptosis.

Microglial cells are resident macrophages of the central nervous system (CNS) that respond to bioactive lipids such as docosahexaenoic acid (DHA). Low micromolar concentrations of DHA typically promote anti-inflammatory functions of microglia, but higher concentrations result in a form of pro-inflammatory programmed cell death known as pyroptosis. This study used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to investigate the morphological characteristics of pyroptosis in BV-2 microglial cells following exposure to 200 µM DHA. Vehicle-treated cells are characterized by extended processes, spine-like projections or 0.4 to 5.2 µm in length, and numerous extracellular vesicles (EVs) tethered to the surface of the plasma membrane. In contrast to vehicle-treated cells, gross abnormalities are observed after treating cells with 200 µM DHA for 4 h. These include the appearance of numerous pits or pores of varying sizes across the cell surface, structural collapse and flattening of the cell shape. Moreover, EVs and spines were lost following DHA treatment, possibly due to release from the cell surface. The membrane pores appear after DHA treatment initially measured ~ 30 nm, consistent with the previously reported gasdermin D (GSDMD) pore complexes. Complete collapse of cytoplasmic organization and loss of nuclear envelope integrity were also observed in DHA-treated cells. These processes are morphologically distinct from the changes that occur during cisplatin-induced apoptosis, such as the appearance of apoptotic bodies and tightly packed organelles, and the maintenance of EVs and nuclear envelope integrity. Cumulatively, this study provides a systematic description of the ultrastructural characteristics of DHA-induced pyroptosis, including distinguishing features that differentiate this process from apoptosis.

Marine Oomycetes (Halophytophthora and Salispina): A Potential Source of Fatty Acids with Cytotoxic Activity Against Breast Adenocarcinoma Cells (MCF7).

Marine oomycetes are ubiquitous, fungus-like eukaryotes known to produce fatty acids with potential anticancer activity. The long chain omega-3 and omega-6 fatty acids are currently popular and considered as safe when used as nutraceuticals in cancer treatment. In this study, crude fatty acids from three marine oomycetes, Halophytophthora spp. (T12GP1 and T12YBP2) and Salispina hoi (USTCMS 1611), were explored for their cytotoxic and apoptotic potentials against human breast adenocarcinoma cells (MCF7) and normal human dermal fibroblasts (HDFn). Extracts from mycelia mats consisted of diverse saturated, monounsaturated, and polyunsaturated fatty acids such as linoleic, α-linolenic, γ-linolenic, eicosatrienoic and eicosapentaenoic acids. The crude fatty acids from all three oomycetes in in vitro assays for cytotoxicity showed no toxicity (30% toxicity values) on HDFn cells. On MCF7 cells, however, IC50 values of 23.44, 15.63, and 26.15 µg/mL were obtained with extracts from Halophytophthora T12GP1 and T12YBP2 and S. hoi, respectively. Treated MCF7 cells exhibited deformed cell membrane in MTT assay and also aggregation of DNA and disruption of nuclear membrane aggregation in nuclear staining; further, green signals indicative of apoptosis was recorded in caspase 3/7 assay.

De novo lipogenesis at the mitotic exit is used for nuclear envelope reassembly/expansion. Implications for combined chemotherapy.

Mitosis has been traditionally considered a metabolically inactive phase. We have previously shown, however, that extensive alterations in lipids occur as the cells traverse mitosis, including increased de novo fatty acid (FA) and phosphatidylcholine (PtdCho) synthesis and decreased lysophospholipid content. Given the diverse structural and functional properties of these lipids, we sought to study their metabolic fate and their importance for cell cycle completion. Here we show that FA and PtdCho synthesized at the mitotic exit are destined to the nuclear envelope. Importantly, FA and PtdCho synthesis, but not the decrease in lysophospholipid content, are necessary for cell cycle completion beyond G2/M. Moreover, the presence of alternative pathways for PtdCho synthesis renders the cells less sensitive to its inhibition than to the impairment of FA synthesis. FA synthesis, thus, represents a cell cycle-related metabolic vulnerability that could be exploited for combined chemotherapy. We explored the combination of fatty acid synthase (FASN) inhibition with agents that act at different phases of the cell cycle. Our results show that the effect of FASN inhibition may be enhanced under some drug combinations.

The organization of leukotriene biosynthesis on the nuclear envelope revealed by single molecule localization microscopy and computational analyses.

The initial steps in the synthesis of leukotrienes are the translocation of 5-lipoxygenase (5-LO) to the nuclear envelope and its subsequent association with its scaffold protein 5-lipoxygenase-activating protein (FLAP). A major gap in our understanding of this process is the knowledge of how the organization of 5-LO and FLAP on the nuclear envelope regulates leukotriene synthesis. We combined single molecule localization microscopy with Clus-DoC cluster analysis, and also a novel unbiased cluster analysis to analyze changes in the relationships between 5-LO and FLAP in response to activation of RBL-2H3 cells to generate leukotriene C4. We identified the time-dependent reorganization of both 5-LO and FLAP into higher-order assemblies or clusters in response to cell activation via the IgE receptor. Clus-DoC analysis identified a subset of these clusters with a high degree of interaction between 5-LO and FLAP that specifically correlates with the time course of LTC4 synthesis, strongly suggesting their role in the initiation of leukotriene biosynthesis.

SILAC-based quantitative proteomics identifies size-dependent molecular mechanisms involved in silver nanoparticles-induced toxicity.

Silver nanoparticles are currently one of the most widely used metallic nanoparticles. Due to their antibacterial properties, they are applied in textiles, house-holds items, and medical devices, among many other products. Understanding the potential toxicity associated with silver nanoparticles and the differential effect that nanoparticles of different size might induce is crucial, due to the increasing human and environmental exposure to this type of nanoparticles. In this work, we explored the different biomolecular mechanisms underlying the toxicity of silver nanoparticles in a size-dependent manner. Quantitative proteomic analysis of hepatic cells exposed to 10 and 60 nm silver nanoparticles demonstrated the alteration of a different set of proteins depending on the particle size. We demonstrated that while 10 nm silver nanoparticles induce nucleolar stress and ribosome biogenesis halt, both types of nanoparticles induce DNA damage and oxidative stress but through different pathways. In addition, both types of nanoparticles also affected cell proliferation, disrupted the cell cycle and ultimately, induced apoptosis. The alteration of different cellular mechanisms in a size-dependent manner, have relevant implications not only from a toxicity point of view, but also for the potential applications of silver nanoparticles.

Nuclear envelope expansion in budding yeast is independent of cell growth and does not determine nuclear volume.

Most cells exhibit a constant ratio between nuclear and cell volume. The mechanism dictating this constant ratio and the nuclear component(s) that scale with cell size are not known. To address this, we examined the consequences to the size and shape of the budding yeast nucleus when cell expansion is inhibited by down-regulating components of the secretory pathway. We find that under conditions where cell size increase is restrained, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. The formation of bilobed nuclei is dependent on fatty acid and phospholipid synthesis, suggesting that it is associated with nuclear membrane expansion. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell-cycle cues rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei. Therefore, the bilobed nucleus could be a consequence of continued NE expansion as cells traverse the cell cycle without an accompanying increase in nuclear volume due to the inhibition of cell growth. Our data suggest that nuclear volume is not determined by nuclear envelope availability but by one or more nucleoplasmic factors.

Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells.

Preventing loss of mechanosensation by the nuclear membranes of alveolar cells reduces lung injury in mice during mechanical ventilation.

The nuclear membrane acts as a mechanosensor that drives cellular responses following changes in the extracellular environment. Mechanically ventilated lungs are exposed to an abnormally high mechanical load that may result in clinically relevant alveolar damage. We report that mechanical ventilation in mice increased the expression of Lamin-A, a major determinant of nuclear membrane stiffness, in alveolar epithelial cells. Lamin-A expression increased and nuclear membrane compliance decreased in human bronchial epithelial cells after a mechanical stretch stimulus and in a murine model of lung injury after positive-pressure ventilation. Reducing Lamin-A maturation by depletion of the protease-encoding gene Zmpste24 preserved alveolar nuclear membrane compliance after mechanical ventilation in mice. Ventilator-induced proapoptotic gene expression changes and lung injury were reduced in mice lacking Zmpste24 compared to wild-type control animals. Similarly, treatment with the human immunodeficiency virus protease inhibitors lopinavir and ritonavir reduced the accumulation of Lamin-A at nuclear membranes and preserved nuclear membrane compliance after mechanical ventilation, mimicking the protective phenotype of Zmpste24-/- animals. These results show that the pathophysiological response to lung mechanical stretch is sensed by the nuclear membranes of lung alveolar cells, and suggest that protease inhibitors might be effective in preventing ventilator-induced lung injury.

Influence of acute promyelocytic leukemia therapeutic drugs on nuclear pore complex density and integrity.

During cell division, a large number of nuclear proteins are released into the cytoplasm due to nuclear envelope breakdown. Timely nuclear import of these proteins following exit from mitosis is critical for establishment of the G1 nuclear environment. Dysregulation of post-mitotic nuclear import may affect the fate of newly divided stem or progenitor cells and may lead to cancer. Acute promyelocytic leukemia (APL) is a malignant disorder that involves a defect in blood cell differentiation at the promyelocytic stage. Recent studies suggest that pharmacological concentrations of the APL therapeutic drugs, all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), affect post-mitotic nuclear import of the APL-associated oncoprotein PML/RARA. In the present study, we have investigated the possibility that ATRA and ATO affect post-mitotic nuclear import through interference with components of the nuclear import machinery. We observe reduced density and impaired integrity of nuclear pore complexes after ATRA and/or ATO exposure. Using a post-mitotic nuclear import assay, we demonstrate distinct import kinetics among different nuclear import pathways while nuclear import rates were similar in the presence or absence of APL therapeutic drugs.

Biological effects of adipocytes in sulfur mustard induced toxicity.

Sulphur mustard (2,2'-dichloroethyl sulfide; SM) is a vesicant chemical warfare agent whose mechanism of acute or chronic action is not known with any certainty and to date there is no effective antidote. SM accumulation in adipose tissue (AT) has been originally verified in our previous study. To evaluate the biological effect caused by the presence of abundant SM in adipocyte and assess the biological role of AT in SM poisoning, in vitro and in vivo experiments were performed. High content analysis revealed multi-cytotoxicity in SM exposed cells in a time and dose dependent manner, and adipocytes showed a relative moderate damage compared with non-adipocytes. Cell co-culture model was established and revealed the adverse effect of SM-exposed adipocyte supernatant on the growth of co-cultured cells. The pathological changes in AT from 10mg/kg SM percutaneously exposed rats were checked and inflammation phenomena were observed. The mRNA and protein levels of inflammation-related adipokines secreted from AT in rats exposed to 1, 3 and 10mg/kg doses of SM were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. The expressions of proinflammatory and anti-inflammatory adipokines together promoted the inflammation development in the body. The positive correlations between AT and serum adipokine levels were explored, which demonstrated a substantial role of AT in systemic inflammation responding to SM exposure. Thus, AT is not only a target of SM but also a modulator in the SM toxicity.

Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins.

Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed "blebs" are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson-Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity.

Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.