Stress Granules In Response Research Articles

Dynamic composition of stress granules in Trypanosoma brucei.

Stress granules (SGs) are stress-induced RNA condensates consisting of stalled initiation complexes resulting from translational inhibition. The biochemical composition and function of SGs are highly diverse, and this diversity has been attributed to different stress conditions, signalling pathways involved and specific cell types. Interestingly, mRNA decay components, which are found in ubiquitous cytoplasmic foci known as processing bodies (PB), have also been identified in SG proteomes. A major challenge in current SG studies is to understand the cause of SG diversity, as well as the function of SG under different stress conditions. Trypanosoma brucei is a single-cellular parasite that causes Human African Trypanosomiasis (sleping sickness). In this study, we showed that by varying the supply of extracellular carbon sources during starvation, cellular ATP levels changed rapidly, resulting in SGs of different compositions and dynamics. We identified a subset of SG components, which dissociated from the SGs in response to cellular ATP depletion. Using expansion microscopy, we observed sub-granular compartmentalization of PB- and SG-components within the stress granules. Our results highlight the importance of cellular ATP in SG composition and dynamics, providing functional insight to SGs formed under different stress conditions.

Unravelling stress granules in the deep cold: Characterisation of TIA-1 gene sequence in Antarctic fish species

Stress granules (SGs) are cytoplasmic foci lacking membranes, comprising non-translating messenger ribonucleoproteins, translational initiation factors, and additional proteins. Their formation is crucial for rapidly modulating gene expression in response to adverse environmental conditions, such as pollution and infections. Limited research has focused on investigating the molecular components of SGs in fish, with minimal exploration in Antarctic fish. This study characterises for the first time the transcript sequences of one key protein component of SGs, TIA-1 (T-cell intracellular antigen 1), in two Antarctic endemic fish species, i.e. Trematomus bernacchii and Chionodraco hamatus. The mRNA-binding protein TIA-1 acts as a post-transcriptional regulator of gene expression and its aggregation leads to the formation of SGs in response to cellular damage. The in vitro and bioinformatic analyses of the TIA-1 gene sequences of these two species highlighted interesting peculiarities, which include the transcription of alternatively spliced isoforms unique to the notothenioid lineage, potentially unlocking further insights into their unique adaptations to extreme environmental conditions. This is the first study to analyze tia-1 expression levels in different tissues of Antarctic fish species. Our key findings indicate that the TIA-1 gene is expressed at particularly high levels in the liver and spleen of C. hamatus, as well as in the heart and skeletal muscle of T. bernacchii. This suggests that those tissues play a significant role in the stress response mechanisms of the studied species. This study provides novel insights into the molecular adaptations of Antarctic fish, highlighting the potential importance of TIA-1 in their response to environmental stressors. The unique features of TIA-1 identified in these species may offer broader implications for understanding how Antarctic fish regulate gene transcriptions in their extreme environments.

N6-methyladenosine-mediated feedback regulation of abscisic acid perception via phase-separated ECT8 condensates in Arabidopsis.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNAs, yet how plants recognize this chemical modification to swiftly adjust developmental plasticity under environmental stresses remains unclear. Here we show that m6A mRNA modification and its reader protein EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) act together as a key checkpoint for negative feedback regulation of abscisic acid (ABA) signalling by sequestering the m6A-modified ABA receptor gene PYRABACTIN RESISTANCE 1-LIKE 7 (PYL7) via phase-separated ECT8 condensates in stress granules in response to ABA. This partially depletes PYL7 mRNA from its translation in the cytoplasm, thus reducing PYL7 protein levels and compromising ABA perception. The loss of ECT8 results in defective sequestration of m6A-modified PYL7 in stress granules and permits more PYL7 transcripts for translation. This causes overactivation of ABA-responsive genes and the consequent ABA-hypersensitive phenotypes, including drought tolerance. Overall, our findings reveal that m6A-mediated sequestration of PYL7 by ECT8 in stress granules negatively regulates ABA perception, thereby enabling prompt feedback regulation of ABA signalling to prevent plant cell overreaction to environmental stresses.

Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance.

YPEL2 is a member of the evolutionarily conserved YPEL family involved in cellular proliferation, mobility, differentiation, senescence, and death. However, the mechanism by which YPEL2, or YPEL proteins, mediates its effects is largely unknown. Proteins perform their functions in a network of proteins whose identities, amounts, and compositions change spatiotemporally in a lineage-specific manner in response to internal and external stimuli. Here, we explored interaction partners of YPEL2 by using dynamic TurboID-coupled mass spectrometry analyses to infer a function for the protein. Our results using inducible transgene expressions in COS7 cells indicate that proximity interaction partners of YPEL2 are mainly involved in RNA and mRNA metabolic processes, ribonucleoprotein complex biogenesis, regulation of gene silencing by miRNA, and cellular responses to stress. We showed that YPEL2 interacts with the RNA-binding protein ELAVL1 and the selective autophagy receptor SQSTM1. We also found that YPEL2 localizes stress granules in response to sodium arsenite, an oxidative stress inducer, which suggests that YPEL2 participates in stress granule-related processes. Establishing a point of departure in the delineation of structural/functional features of YPEL2, our results suggest that YPEL2 may be involved in stress surveillance mechanisms.

Translocation of cytosolic hCdc73 to stress granules plays a role in arsenic stress-induced stabilization of p53 mRNA.

Cells trigger the assembly of stress granules (SGs) under various stress conditions. Among the many proteins recruited to SGs are RNA-binding proteins and transcription regulators. Here, we report the translocation of hCdc73, a component of the PAF1 transcription complex, to cytosolic SGs in response to arsenic stress. The hCdc73 protein possesses a long intrinsically disordered region (IDR) from amino acids 256-416, the presence of which is required for the translocation of hCdc73 to cytosolic SGs. The purified hCdc73 IDR formed droplets in vitro, and the light-activated assembly of hCdc73 IDR-mCherry-CRY2 was verified. For translocation of hCdc73 to SGs, physical interactions with the carrier proteins of SGs, such as FMR1, are also needed. Previously, we reported that the cytosolic hCdc73/eEF1Bγ complex controls the stability of p53 mRNA. Under arsenic stress, selective sequestration of cytosolic hCdc73, but not eEF1Bγ or p53 mRNA, was detected. As a result, a transient increase in p53 mRNA at the posttranscriptional level was observed. In conclusion, we propose that the availability of mRNAs for stress-responsive genes can be controlled by restraining their negative regulators within SGs.

The association of UBAP2L and G3BP1 mediated by small nucleolar RNA is essential for stress granule formation

Stress granules (SGs) are dynamic, non-membranous structures composed of non-translating mRNAs and various proteins and play critical roles in cell survival under stressed conditions. Extensive proteomics analyses have been performed to identify proteins in SGs; however, the molecular functions of these components in SG formation remain unclear. In this report, we show that ubiquitin-associated protein 2-like (UBAP2L) is a crucial component of SGs. UBAP2L localized to SGs in response to various stresses, and its depletion significantly suppressed SG organization. Proteomics and RNA sequencing analyses found that UBAP2L formed a protein-RNA complex with Ras-GTP-activating protein SH3 domain binding protein 1 (G3BP1) and small nucleolar RNAs (snoRNAs). In vitro binding analysis demonstrated that snoRNAs were required for UBAP2L association with G3BP1. In addition, decreased expression of snoRNAs reduced the interaction between UBAP2L and G3BP1 and suppressed SG formation. Our results reveal a critical role of SG component, the UBAP2L/snoRNA/G3BP1 protein-RNA complex, and provide new insights into the regulation of SG assembly.

APOBEC3B drives PKR-mediated translation shutdown and protects stress granules in response to viral infection

Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.

Role of Stress Granules in the Pathogenesis of Pulmonary Hypertension Kosmas Kosmas 1,2, Fotios Spyropoulos1,2,Helen Christou 1,21Department of Pediatric Newborn Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA 2Harvard Medical School, Boston, MA 02215, USA

BackgroundPAH is a devastating and incurable disease characterized by abnormal pulmonary vascular phenotypes and progressive right ventricular failure, but the underlying mechanisms are incompletely understood. There is an urgent need of new therapeutic approaches, and a better understanding of disease pathogenesis is essential in identifying novel pathways that can be therapeutically targeted. Stress granules (SGs) are small intracellular structures that form in response to stress and contain mRNA molecules and mRNA‐binding proteins. These granules allow cells to temporarily halt translation by “parking” or sequestering certain mRNAs in a protected zone (the stress granule) and this allows the cells to survive in conditions of stress. The role of SGs in abnormal vascular and cardiac homeostasis in PAH is not known.HypothesesWe hypothesized that increased SG formation may contribute to both abnormal vascular cellular phenotypes and cardiac failure in human and experimental PAH. Further, we examined whether treatment with the carbonic anhydrase Acetazolamide (ACTZ) which is known to ameliorate experimental PAH, would alter SG tissue abundance in the lungs and heart.MethodsWe used the adult rat (Sprague Dawley) sugen/hypoxia (Su/Hx) model of severe pulmonary hypertension and studied tissue sections from lungs, heart and skeletal muscle as well as isolated primary pulmonary artery smooth muscle cells (PASMCs). We also used PASMCs from patients with PAH. We applied a combination of immunofluorescence and protein expression studies as well as drug (ACTZ) treatment and in vitro induction of oxidative stress (arsenite) to assess SG formation and SG related markers in experimental PAH and the effect of ACTZ.ResultsWe have discovered that PASMCs from patients with PAH have a higher number of stress granules in response to oxidative stress than cells from normal individuals. We also found that PASMCs from a preclinical model of PAH (Sugen/Hypoxia) have more stress granules and increased SG components in response to oxidative stress compared to normal animals. Further, we found that treatment of PASMCs with ACTZ resulted in lower number of stress granules after cell stress. In addition, we found increased G3BP1 (key component of SG assembly) puncta in alveoli and bronchi and sarcoplasmic G3BP1 puncta in cardiac and soleus muscles from the rat sugen/hypoxia (Su/Hx) model compared to controls and with ACTZ treatment.ConclusionsWe conclude that there is increased SG formation in PASMCs and tissues in experimental PAH and acetazolamide inhibits stress granule assembly in response to oxidative stress. Further studies are needed to define the role of SG in disease pathogenesis, and evaluate whether targeting stress granules may be effective in restoring normal cellular function and improving PAH.

TDP-43 Mutation Affects Stress Granule Dynamics in Differentiated NSC-34 Motoneuron-Like Cells.

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of motor neurons in the brain and spinal cord. Cytoplasmic inclusions of TDP-43 are frequently reported in motor neurons of ALS patients. TDP-43 has also been shown to associate with stress granules (SGs), a complex of proteins and mRNAs formed in response to stress stimuli that temporarily sequester mRNA translation. The effect of pathogenic TDP-43 mutations within glycine-rich regions (where the majority of ALS-causing TDP-43 mutations occur) on SG dynamics in motor neurons is poorly understood. To address this issue, we generated murine NSC-34 cell lines that stably over-express wild type TDP-43 (TDP-43WT) or mutant forms (ALS-causing TDP-43 mutations TDP-43A315T or TDP-43M337V). We then differentiated these NSC-34 lines into motoneuron-like cells and evaluated SG formation and disassembly kinetics in response to oxidative or osmotic stress treatment. Wild type and mutant TDP-43 appeared to be largely retained in the nucleus following exposure to arsenite-induced oxidative stress. Upon arsenite removal, mutant TDP-43 clearly accumulated within HuR positive SGs in the cytoplasm, whereas TDP-43WT remained mostly within the nucleus. 24 h following arsenite removal, all SGs were disassembled in both wild type and mutant TDP-43 expressing cells. By contrast, we observed significant differences in the dynamics of mutant TDP-43 association with SGs in response to hyperosmotic stress. Specifically, in response to sorbitol treatment, TDP-43WT remained in the nucleus, whereas mutant TDP-43 relocalized to HuR positive SGs in the cytoplasm following exposure to sorbitol stress, resulting in a significant increase in TDP-43 SG numbers. These SGs remained assembled for 24 h following removal of sorbitol. Our data reveal that under certain stress conditions the rates of SG formation and disassembly is modulated by TDP-43 mutations associated with ALS, and suggest that this may be an early event in the seeding of insoluble cytoplasmic inclusions observed in ALS.

RNase L promotes the formation of unique ribonucleoprotein granules distinct from stress granules

Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response since many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L-dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, as RLBs did not require protein kinase R (PKR), phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L-mediated antiviral response.

Tellurite Promotes Stress Granules and Nuclear SG-Like Assembly in Response to Oxidative Stress and DNA Damage

Tellurium oxyanion, tellurite (TeO3–2), is a highly toxic compound for many organisms. Its presence in the environment has increased over the past years due to industrial manufacturing processes and has been associated with adverse effects on human health. Although tellurite induces the phosphorylation of eIF2α, DNA damage and oxidative stress, the molecular mechanisms related to the cellular responses to tellurite-induced stress are poorly understood. In this work, we evaluated the ability of tellurite to induce phosphorylation of eIF2α, stress granules (SGs) assembly and their relationship with DNA damage in U2OS cells. We demonstrate that tellurite promotes the assembly of bona fide cytoplasmic SGs. Unexpectedly, tellurite also induces the assembly of nuclear SGs. Interestingly, we observed that the presence of tellurite-induced nuclear SGs correlates with γH2AX foci. However, although H2O2 also induce DNA damage, no nuclear SGs were observed. Our data show that tellurite promotes the assembly of cytoplasmic and nuclear SGs in response to oxidative stress and DNA damage, revealing a new aspect of cellular stress response mediated by the assembly of nuclear stress granules.

NUDT21 Links Mitochondrial IPS-1 to RLR-Containing Stress Granules and Activates Host Antiviral Defense.

Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid-inducible protein-I-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms, including type I IFN induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. In this study, we show that NUDT21, an RNA-binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of viral dsRNA. We found that despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection in both human HeLa cells and mouse macrophage cell line RAW264.7 cells. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.

Autophagy and heat-shock response impair stress granule assembly during cellular senescence

Stress granules (SGs) are membraneless organelles formed in response to insult. These granules are related to pathological granules found in age-related neurogenerative diseases such as Parkinson’s and Alzheimer’s. Previously, we demonstrated that senescent cells, which accumulate with age, exposed to chronic oxidative stress, are unable to form SGs. Here, we show that the senescent cells’ inability to form SGs correlates with an upregulation in both the heat-shock response and autophagy pathways, both of which are well-established promoters of SG disassembly. Our data also reveals that the knockdown of HSP70 and ATG5, important components of the heat-shock response and autophagy pathways, respectively, restores the number of SGs formed in senescent cells exposed to chronic oxidative stress. Surprisingly, under these conditions, the depletion of HSP70 or ATG5 did not affect the clearance of these SGs during their recovery from chronic stress. These data reveal that senescent cells possess a unique heat-shock and autophagy-dependent ability to impair the formation of SGs in response to chronic stress, thereby expanding the existing understanding of SG dynamics in senescent cells and their potential contribution to age-related neurodegenerative diseases.

Nephronophthisis gene products display RNA-binding properties and are recruited to stress granules

Mutations of cilia-associated molecules cause multiple developmental defects that are collectively termed ciliopathies. However, several ciliary proteins, involved in gating access to the cilium, also assume localizations at other cellular sites including the nucleus, where they participate in DNA damage responses to maintain tissue integrity. Molecular insight into how these molecules execute such diverse functions remains limited. A mass spectrometry screen for ANKS6-interacting proteins suggested an involvement of ANKS6 in RNA processing and/or binding. Comparing the RNA-binding properties of the known RNA-binding protein BICC1 with the three ankyrin-repeat proteins ANKS3, ANKS6 (NPHP16) and INVERSIN (NPHP2) confirmed that certain nephronophthisis (NPH) family members can interact with RNA molecules. We also observed that BICC1 and INVERSIN associate with stress granules in response to translational inhibition. Furthermore, BICC1 recruits ANKS3 and ANKS6 into TIA-1-positive stress granules after exposure to hippuristanol. Our findings uncover a novel function of NPH family members, and provide further evidence that NPH family members together with BICC1 are involved in stress responses to maintain tissue and organ integrity.

Conserved metabolite regulation of stress granule assembly via AdoMet.

Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5' end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43-positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.

RNase L promotes the formation of unique ribonucleoprotein granules distinct from stress granules

Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response because many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L–dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, because RLBs did not require protein kinase R, phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties, they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L–mediated antiviral response.

Mutant KRAS Enhances Stress Granules and Resistance to Proteasome Inhibition Via 15-d-PGJ2 in Multiple Myeloma

Introduction: Mutant KRAS leads to activation of the MEK/ERK pathway in approximately 20% of multiple myeloma (MM) patients. Stress granules (SGs) are cytosolic non-membranous structures composed of translational mRNAs, ribosomal proteins, and RNA-binding proteins, which form in response to different stress stimuli and chemotherapeutic treatment. We have previously shown that mutant KRAS upregulates SG formation. The molecular mechanisms underlying this process are unclear. The purpose of this study was to elucidate a) the mechanism by which mutant KRAS upregulates SG formation and b) to provide a molecular rationale for the targeting the cascades for SG formation in MM patients. Methods. We determined whether mutant KRAS is necessary for SG upregulation. To this purpose a panel of MM cell lines including mutant KRAS (KRASM), mutant NRAS (NRASm) and wildtype KRAS (KRASWT) was used. The gain- and loss-of-function of mutant KRAS was generated to suppress (KRASM/KO) in KRASM or switch on by expression of mutant KRAS G12A (KRASG12A) in KRASWT cells by a lentiviral vector system. SG formation was assessed by measuring the cellular distribution of SG resident proteins by immunofluorescence using anti-G3BP and anti-eIF4G antibodies. The quantitative readout for SG formation was analyzed by ImageJ analyze-particle tool as the SG index. Immunoblotting was used to detect inactivation of eIF2a or activity of MAPK. qPCR analysis was employed to detect the mRNA of COX2. MTT assays were used to detect the effect of SGs on the survival of MM cells alone or in combination with diclofenac sodium (DS), or bortezomib (Bzb). Results: We compared the induction of SGs in response to oxidative stress across a panel of MM cells with different Ras mutation status (KRASM, NRASM and KRASWT)to investigate the role mutant KRAS in GS formation. The level of SGs were significantly higher (p<0.01) in six KRASM, compared with four KRASWT cell lines.We assessed SG formation in KRASM, KRASM/KO and KRAS/G12A cells to determine whether mutant KRAS is a causative factor leading to elevated SG levels. SG formation in KRASM/KO cells was significantly attenuated, by approximately 5-fold (p<0.001), compared to KRASM in response to oxidative stress. Conversely, SG levels were 3.5-fold elevated in KRAS/G12A cells (P<0.01), compared to KRASWT cells. Upregulation of SGs by mutant KRAS resulted in a larger average size and number of SGs per cells. Together, these results indicate that mutant KRAS is necessary and sufficient for SG upregulation under oxidative stress condition. We investigated whether 15-d-PGJ2-depedent inactivation of elF4A is involved in SG upregulation to determine the mechanism through which mutant KRAS promotes SG formation. To do this, we assessed the effect of 15-d-PGJ2 on SG formation because that elF4A inactivation is controlled by 15-d-PGJ2, which is one of COX2-derived products. Treatment of KRASM cells with 15-d-PGJ2 significantly induced accumulation of SGs compared with untreated cells (40-fold increase vs control cells, p<0.001). To determine whether COX2, which controls the rate of biosynthesis of 15-d-PGJ2, is regulated by mutant KRAS, we first compared COX2 mRNA of KRASM/KO with KRASM and showed that a decrease in COX2 expression in KRASM/KO (2-fould decrease, P< 0.01). Conversely, increased COX2 mRNA was seen in KRAS/G12A, compared with KRASWT cells. Furthermore, treatment of KRASM cells with DS led to a decrease in SG levels and this effect was rescued by the addition of 15-d-PGJ2. These results indicate that increase in prostaglandin levels by upregulating prostaglandin biosynthesis is essential for mutant KRAS-induced up-regulation of SGs. Finally, we determined if inhibition of COX2 activity by DS was able to overcome Bzb resistance in KRASM or in KRASM co-occurring t(14:16)/MAF. A significant decrease of cell viability (P<0.001) was observed when treatment of the cells with Bzb and DS were combined, compared with DS treatment alone. Taken together, these results indicate that inhibition of COX2 activity enhances mutant KRAS MM cell sensitivity to proteasome inhibition. Conclusion: Our results suggest that mutant KRAS mutant upregulates SG formation by 15-d-PGJ2 and inhibition of COX2 activity enhances KRAS sensitivity to proteasome inhibition and targeting SG formation might be represent an effective strategy to treatment of KRAS mutant myeloma. Disclosures Davies: Janssen, Celgene: Other: Research Grant, Research Funding; Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding. Walker:Celgene: Research Funding. van Rhee:Karyopharm Therapeutics: Consultancy; Kite Pharma: Consultancy; Adicet Bio: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Sanofi Genzyme: Consultancy; Takeda: Consultancy.

The TRAPP complex mediates secretion arrest induced by stress granule assembly.

The TRAnsport Protein Particle (TRAPP) complex controls multiple membrane trafficking steps and is strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified the TRAPP complex as a component of a branch of the integrated stress response that impinges on the early secretory pathway. The TRAPP complex associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and arrest of ER export. The relocation of the TRAPP complex and COPII to SGs only occurs in cycling cells and is CDK1/2-dependent, being driven by the interaction of TRAPP with hnRNPK, a CDK substrate that associates with SGs when phosphorylated. In addition, CDK1/2 inhibition impairs TRAPP complex/COPII relocation to SGs while stabilizing them at ER exit sites. Importantly, the TRAPP complex controls the maturation of SGs. SGs that assemble in TRAPP-depleted cells are smaller and are no longer able to recruit RACK1 and Raptor, two TRAPP-interactive signaling proteins, sensitizing cells to stress-induced apoptosis.

Mutant KRAS and Brafs Upregulate Stress Granules and Mediate Drug Resistance, Which Can be Modulated By Cox2 Inhibition in Multiple Myeloma

Introduction: Mutant RAS leads to activation of the RAS/RAF/MEK/ERK pathway in approximately 50% of multiple myeloma (MM). Stress granules (SGs) are non-membranous structures composed of translational mRNAs, ribosomal proteins, and RNA-binding proteins, which form in response to different stress stimuli and chemotherapeutic treatment. TheKRAS effector pathway mediates the upregulation of SGs conferring resistance to chemotherapeutic agents in solid tumors. However, it is unclear if SGs are upregulated in KRAS mutant MM and if this is associated with chemotherapeutic resistance. The purpose of this study was to characterize the role of RAS/RAF mutants in SG formation and to provide a molecular rationale for the clinical testing of combinations targeting RAS mutation with inhibition of SG formation.Methods. To investigate the association of RAS mutation status with SG formation, we used two K-RAS mutant positive (KRASM), two BRAF mutant positive (BRAFM),and 2 wildtype KRAS (KRASWT) and wild type BRAF (BRAFWT) MM cell lines as models.We assessed the cellular distribution of SG proteins by immunofluorescence using anti-G3BP and anti-eIF4G antibodies. To establish a quantitative readout for SG formation (SG index), we applied the ImageJ analyze-particle tool to calculate the fraction of the SG area in the total cell area based on G3BP and eIF4G immunofluorescence (SG area/cell area). MTT assays were used to detect the effect of SGs on the survival of MM cells alone or in combination with diclofenac sodium (DS),or bortezomib.Results:Immunofluorescence analysis showed that exposure of KRASM MM cells lines to oxidative stress via treatment with sodium arsenite (SA))was associated with the induction of SGs as indicated by accumulation of cytoplasmic puncta of G3BP and elF4G at high level, compared with RASWTMM cells. Quantitative analysis of SG formation by Image J analyze-particle tool revealed that the SG index for G3BP (13.225 ±3.2) in KRASM was significantly higher compared with KRASWT(2.936 ±1.2,P =0.0004) in response to SA. The SG index for elF4G(32.89 ±6.2) in KRASM was significantly higher than that in KRASWT(4.96, ±2.3, p<5.03E-5). These results indicate that mutant KRAS may play a regulatory role in SG formation.Given that BRAF mutation leads to activation of MAPK pathway, we next determined if BRAF mutants regulate SGs in response to SA. MM cells harboring K601N mutation, showed a high level of induction of SG formation following treatment with SA, compared with BRAFWTcells. The SG index for G3BP (11.52 ±0.89) in BRAFM was significant higher than that in BRAFWTcells (4.02 ±1.92; P=1.03E-6). Similarly, the SG index for elF4G (19.2±1.3) in BRAFMwas significant upregulated compared with BRAFWT(6.3 ±2.8, p=5.3E-5). MM cells harboring BRAF mutation at D4594N displayed intermediate positive of SG formation. These results suggest that the presence of the BRAF mutation promotes SG formation in response to oxidative stress.Since SGs have been shown to contribute to mutant KRAS-mediated resistance to chemotherapeutic reagents in solid tumors and mutant KRAS enhances SG formation by up-regulating cox biosynthesis, we next determine if inhibition of COX2 activity by diclofenac sodium (DS), a pharmacological inhibitor of COX2, was able to overcome Bzb resistance in KRAS and BRAF mutant MM cells. MTT assay showed that the viability index (0.8±0.16) reduced to (0.49 ±0.015) in KRASMMM cells treated by 10 nM of Bortezomib (Bzb) alone or to (0.46 ±0.016) with 200 mM of DS alone. A significant decrease of cell viability (0.22±0.01, P<0.001) was observed when treatment with Bzb and DS were combined. Similar synergistic effects of Bzb and DS were observed in BRAFM cells, compared with BRAFWT. Taken together, these results indicate that inhibition of COX2 activity enhance KRAS and BRAF mutant MM cell sensitivity to proteasome inhibition.Conclusion: Our results suggest that KRAS mutant and BRAF mutant MM cells significantly upregulate SG formation and inhibition of COX2 activity enhances KRAS and BRAF mutant MM cell sensitivity to proteasome inhibition. Further experiments are ongoing to determine the molecular mechanism by which KRAS and BRAF mutant MM cells upregulate SG formation and their role in MM cell resistance to chemotherapeutic reagents. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding. Davies:TRM Oncology: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Abbvie: Consultancy; TRM Oncology: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; ASH: Honoraria; Janssen: Consultancy, Honoraria.

The Atypical Dual Specificity Phosphatase hYVH1 Associates with Multiple Ribonucleoprotein Particles

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.