Nuclear-cytoplasmic Transport Research Articles

Dynamic Single-Cell RNA-Seq reveals mechanism of Selinexor-Resistance in Chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a type of hematologic malignancies caused by BCR-ABL chimeric oncogene. Resistance to tyrosine kinase inhibitors (TKIs) leads to the progression of CML into advanced stages. Selinexor is a small molecule inhibitor that targets a nuclear transporter called Exportin 1. Combined with imatinib, selinexor has been shown to disrupt nuclear-cytoplasmic transport signal of leukemia stem cells, resulting in cell death. The objective of this study was to investigate the mechanism of drug resistance to selinexor in CML. We established K562 cell line resistant to selinexor and conducted single cell dynamic transcriptome sequencing to analyze the heterogeneity within the parental and selinexor resistant cell populations. We identified specific gene expression changes associated with resistance to selinexor. Our results revealed differential expression patterns in genes such as MT2A, TFPI, MTND3, and HMGCS1 in the total RNA, as well as MT-TW, DNAJB1, and HSPB1 in the newly synthesized RNA, between the parental and drug-resistant groups. By applying pseudo-time analysis, we discovered that a specific cluster of cells exhibited characteristics of tumor stem cells. Furthermore, we observed a gradual decrease in the expression of ferroptosis-related molecules as drug resistance developed. In vitro experiments confirmed that the combination of a ferroptosis inducer called RSL3 effectively overcame drug resistance. In conclusion, this study revealed the resistance mechanism of selinexor in CML. In conclusion, we identified a subgroup of CML cells with tumor stem cell properties and demonstrated that ferroptosis inducer improved the efficacy of selinexor in overcoming drug resistance.

The nuclear export protein exportin-1 in solid malignant tumours: From biology to clinical trials.

Exportin-1 (XPO1), a crucial protein regulating nuclear-cytoplasmic transport, is frequently overexpressed in various cancers, driving tumor progression and drug resistance. This makes XPO1 an attractive therapeutic target. Over the past few decades, the number of available nuclear export-selective inhibitors has been increasing. Only KPT-330 (selinexor) has been successfully used for treating haematological malignancies, and KPT-8602 (eltanexor) has been used for treating haematologic tumours in clinical trials. However, the use of nuclear export-selective inhibitors for the inhibition of XPO1 expression has yet to be thoroughly investigated in clinical studies and therapeutic outcomes for solid tumours. We collected numerous literatures to explain the efficacy of XPO1 Inhibitors in preclinical and clinical studies of a wide range of solid tumours. In this review, we focus on the nuclear export function of XPO1 and results from clinical trials of its inhibitors in solid malignant tumours. We summarized the mechanism of action and therapeutic potential of XPO1 inhibitors, as well as adverse effects and response biomarkers. XPO1 inhibition has emerged as a promising therapeutic strategy in the fight against cancer, offering a novel approach to targeting tumorigenic processes and overcoming drug resistance. SINE compounds have demonstrated efficacy in a wide range of solid tumours, and ongoing research is focused on optimizing their use, identifying response biomarkers, and developing effective combination therapies. Exportin-1 (XPO1) plays a critical role in mediating nucleocytoplasmic transport and cell cycle. XPO1 dysfunction promotes tumourigenesis and drug resistance within solid tumours. The therapeutic potential and ongoing researches on XPO1 inhibitors in the treatment of solid tumours. Additional researches are essential to address safety concerns and identify biomarkers for predicting patient response to XPO1 inhibitors.

STAT3 promotes cytoplasmic-nuclear translocation of RNA-binding protein HuR to inhibit IL-1β-induced IL-8 production

Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1β-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1β-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1β induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1β-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1β-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1β-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1β. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1β-induced IL-8 production through this non-transcriptional mechanism.

Examining the molecular mechanisms of topiramate in alleviating insulin resistance: A study on C2C12 myocytes and 3T3L-1 adipocytes.

Migraine, a severely debilitating condition, may be effectively managed with topiramate, known for its migraine prevention and weight loss properties due to changes in body muscle and fat composition and improved insulin sensitivity. However, the mechanism of topiramate in modulating insulin response in adipocytes and myocytes remains elusive. This study aims to elucidate these molecular mechanisms, offering insights into its role in weight management for migraine sufferers and underpinning its clinical application. Insulin resistance improvements were evaluated through glucose uptake measurements in C2C12 muscle cells and 3T3L-1 adipocytes, with Oil red O staining conducted on adipocytes. RNA-seq transcriptome analysis was used to identify the regulatory target genes of topiramate in these cells. The involvement of key genes and pathways was further validated through western blot analysis. Topiramate effectively reduced insulin resistance in C2C12 and 3T3L-1 cells. In C2C12 cells, it significantly lowered SORBS1 gene and protein levels. In 3T3L-1 cells, topiramate upregulated CTGF and downregulated MAPK8 and KPNA1 genes. Changes were notable in nuclear cytoplasmic transport and circadian signaling pathways. Furthermore, it caused downregulation of MKK7, pJNK1/ JNK1, BMAL1, and CLOCK proteins compared to the insulin-resistant model. This study provides preliminary insights into the mechanisms through which topiramate modulates insulin resistance in C2C12 myocytes and 3T3L-1 adipocytes, enhancing our understanding of its therapeutic potential in managing weight and insulin sensitivity in migraine patients.

The Functions of N6-Methyladenosine in Nuclear RNAs.

N6-methyladenosine (m6A) is one of the most common modifications in both eukaryotic and prokaryotic mRNAs. It has been experimentally confirmed that m6A methylation is involved in the regulation of stability and translation of various mRNAs. Until recently, the majority of m6A-related studies have been focused on the cytoplasmic functions of this modification. Here, we review new data on the role of m6A in several key biological processes taking place in the cell nucleus, such as transcription, chromatin organization, splicing, nuclear-cytoplasmic transport, and R-loop metabolism. Based on analysis of these data, we suggest that m6A methylation of nuclear RNAs is another level of gene expression regulation which, together with DNA methylation and histone modifications, controls chromatin structure and functioning in various biological contexts.

Dynamic Single-Cell RNA-Seq Reveals Mechanism of Selinexor-Resistance in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm caused by BCR-ABL chimeric oncogene. The administration of tyrosine kinase inhibitors (TKI) has been shown to significantly improve the prognosis of the CML cases. TKI-resistance may lead to progression of the disease into accelerated and blast phases. To tackle this problem, there are currently several strategies that can be employed to manage or overcome the difficulties associated with TKI-resistance. Selinexor is a small molecule inhibitor that targets the nuclear transporter Exportin 1 (XPO1). This inhibition leads to disruptions in various signaling pathways involved in tumor cell cycle regulation, DNA repair, and apoptosis. In CML, Selinexor can increase the nuclear-cytoplasmic distribution ratio of BCR/ABL, cause cell cycle arrest, inhibit the proliferation of CML cell line K562, and exhibit anti-tumor properties in mouse model. When combined with imatinib, Selinexor can effectively disrupt the nuclear-cytoplasmic transport signal of leukemia stem cells, resulting in reduced formation of clones and increased apoptosis. In this work, we were aiming at investigating the mechanism of drug-resistance of Selinexor in CML cell lines K562. We performed single cell dynamic transcriptome sequence to analysis intratumor heterogeneity of subpopulation of CML and identified key gene expression changes that occur as Selinexor-resistance. We generated Selinexor-resistant (SR) lines by culturing K562 cell lines with increasing concentrations of Selinexor. At first, we conducted an analysis of differential gene expression between the parental group and various levels of SR groups. Our findings revealed differential expression patterns in MT2A, TFPI, MTND3, and HMGCS1 in total RNA, as well as MT-TW, DNAJB1, and HSPB1 in newly synthesized RNA, within both the parental and drug-resistant groups. These results suggest that mitochondrial metabolism and heat shock proteins play a significant role in the regulation of cellular drug resistance. Furthermore, we found hallmark signaling pathways of epithelial mesenchymal transition and protein secretion upregulated in SR groups. Through the application of pseudo-time analysis, it was observed that cluster 6 persisted in both the parental and drug-resistant groups, and displayed characteristics indicative of tumor stem cells. Notably, a set of distinct genes were identified that distinguish cluster 6 from the other clusters, these genes are associated with lysosomes and ferroptosis pathways by KEGG enrichment analysis. Next, it was determined that the gene set in the newly synthesized RNA exhibits a consistent upregulation by the expression pattern clustering analysis. Within this gene set, we revealed an enrichment of the endoplasmic reticulum protein processing and ferroptosis signaling pathway. Finally, to provide additional evidence for the ferroptosis-related pathway in the SR group, we observed that the expression of ferroptosis-inhibitory molecules FTH1 and SLC7A11 increased gradually with the development of drug resistance. Conversely, the expression of ferroptosis-driving molecules HMGB1 and MTDH decreased gradually. Subsequently, in vitro experiments were conducted to validate that the combination of the ferroptosis inducer RSL3 can effectively overcome Selinexor resistance. In summary, this study identified a subgroup exhibiting tumor stem cell properties. Additionally, it demonstrated that the combination of ferroptosis inducer can improve resistance to Selinexor. Through single-cell dynamic transcriptome RNA-seq technology, this investigation elucidated the mechanism of resistance to Selinexor in the K562 cell line, highlighting the potential involvement ferroptosis in drug resistance. These findings offer novel insights for the treatment of CML.

Nuclear pore complex and nucleocytoplasmic transport disruption in neurodegeneration.

Nuclear pore complexes (NPCs) play a critical role in maintaining the equilibrium between the nucleus and cytoplasm, enabling bidirectional transport across the nuclear envelope, and are essential for proper nuclear organization and gene regulation. Perturbations in the regulatory mechanisms governing NPCs and nuclear envelope homeostasis have been implicated in the pathogenesis of several neurodegenerative diseases. The ESCRT-III pathway emerges as a critical player in the surveillance and preservation of well-assembled, functional NPCs, as well as nuclear envelope sealing. Recent studies have provided insights into the involvement of nuclear ESCRT-III in the selective reduction of specific nucleoporins associated with neurodegenerative pathologies. Thus, maintaining quality control of the nuclear envelope and NPCs represents a pivotal element in the pathological cascade leading to neurodegenerative diseases. This review describes the constituents of the nuclear-cytoplasmic transport machinery, encompassing the nuclear envelope, NPC, and ESCRT proteins, and how their structural and functional alterations contribute to the development of neurodegenerative diseases.

Effects of ionizing radiation on radio sensitivity of cell nuclei of wheat seedlings

Effects of the 50 Gy and 100 Gy doses of Y-irradiation on radiosensitivity of cell nuclei of soft dwarf wheat seedlings were studied. Changes in the biochemical composition of the nuclear fractions of seedlings of wheat seeds exposed to gamma-irradiation were determined. After exposure to ionizing radiation, total protein and nucleic acids levels appeared to be greater in the soluble nuclear fraction and the nuclear membrane-associated DNA content and proteins increased. It was also found that the value of the dzeta-potential of isolated nuclei of seedlings of irradiated seeds decreased in a static electric field. Changes in the surface charge of the nucleus lead to a violation of its functional activity, since the surface charge of the nuclei is necessary for the normal functioning and regulation of the nuclear-cytoplasmic transport of macromolecules. Exposure to radiation causes destruction of ionogenic groups of molecules of the nuclear membrane, leading to a decrease in the electronegativity of the nuclei, which, in turn, entails a change in the functional activity of the nucleus. The results obtained show that changes in electronegativity and in the content of nuclear fractions are directly dependent on the dose of ionizing radiation. It can be concluded that Y-irradiation in a range from 50 to 100 Gy had beneficial effects on metabolic and proliferative activities of soft wheat cells.

Working a second job: Cell adhesion proteins that moonlight in the nucleus.

Cells are adept at sensing changes in their environment, transmitting signals internally to coordinate responses to external stimuli, and thereby influencing adaptive changes in cell states and behavior. Often, this response involves modulation of gene expression in the nucleus, which is seen largely as a physically separated process from the rest of the cell. Mechanosensing, whereby a cell senses physical stimuli, and integrates and converts these inputs into downstream responses including signaling cascades and gene regulatory changes, involves the participation of several macromolecular structures. Of note, the extracellular matrix (ECM) and its constituent macromolecules comprise an essential part of the cellular microenvironment, allowing cells to interact with each other, and providing both structural and biochemical stimuli sensed by adhesion transmembrane receptors. This highway of information between the ECM, cell adhesion proteins, and the cytoskeleton regulates cellular behavior, the disruption of which results in disease. Emerging evidence suggests a more direct role for some of these adhesion proteins in chromatin structure and gene regulation, RNA maturation and other non-canonical functions. While many of these discoveries were previously limited to observations of cytoplasmic-nuclear transport, recent advances in microscopy, and biochemical, proteomic and genomic technologies have begun to significantly enhance our understanding of the impact of nuclear localization of these proteins. This review will briefly cover known cell adhesion proteins that migrate to the nucleus, and their downstream functions. We will outline recent advances in this very exciting yet still emerging field, with impact ranging from basic biology to disease states like cancer.

Changes in nuclear pore numbers control nuclear import and stress response of mouse hearts.

Nuclear pores are essential for nuclear-cytoplasmic transport. Whether and how cells change nuclear pores to alter nuclear transport and cellular function is unknown. Here, we show that rat heart muscle cells (cardiomyocytes) undergo a 63% decrease in nuclear pore numbers during maturation, and this changes their responses to extracellular signals. The maturation-associated decline in nuclear pore numbers is associated with lower nuclear import of signaling proteins such as mitogen-activated protein kinase (MAPK). Experimental reduction of nuclear pore numbers decreased nuclear import of signaling proteins, resulting in decreased expression of immediate-early genes. In a mouse model of high blood pressure, reduction of nuclear pore numbers improved adverse heart remodeling and reduced progression to lethal heart failure. The decrease in nuclear pore numbers in cardiomyocyte maturation and resulting functional changes demonstrate how terminally differentiated cells permanently alter their handling of information flux across the nuclear envelope and, with that, their behavior.

Loss-of-function mutation survey revealed that genes with background-dependent fitness are rare and functionally related in yeast

In natural populations, the same mutation can lead to different phenotypic outcomes due to the genetic variation that exists among individuals. Such genetic background effects are commonly observed, including in the context of many human diseases. However, systematic characterization of these effects at the species level is still lacking to date. Here, we sought to comprehensively survey background-dependent traits associated with gene loss-of-function (LoF) mutations in 39 natural isolates of Saccharomyces cerevisiae using a transposon saturation strategy. By analyzing the modeled fitness variability of a total of 4,469 genes, we found that 15% of them, when impacted by a LoF mutation, exhibited a significant gain- or loss-of-fitness phenotype in certain natural isolates compared with the reference strain S288C. Out of these 632 genes with predicted background-dependent fitness effects, around 2/3 impact multiple backgrounds with a gradient of predicted fitness change while 1/3 are specific to a single genetic background. Genes related to mitochondrial function are significantly overrepresented in the set of genes showing a continuous variation and display a potential functional rewiring with other genes involved in transcription and chromatin remodeling as well as in nuclear-cytoplasmic transport. Such rewiring effects are likely modulated by both the genetic background and the environment. While background-specific cases are rare and span diverse cellular processes, they can be functionally related at the individual level. All genes with background-dependent fitness effects tend to have an intermediate connectivity in the global genetic interaction network and have shown relaxed selection pressure at the population level, highlighting their potential evolutionary characteristics.

Allergen protease-activated stress granule assembly and gasdermin D fragmentation control interleukin-33 secretion.

Interleukin-33 (IL-33), an epithelial cell-derived cytokine that responds rapidly to environmental insult, has a critical role in initiating airway inflammatory diseases. However, the molecular mechanism underlying IL-33 secretion following allergen exposure is not clear. Here, we found that two cell events were fundamental for IL-33 secretion after exposure to allergens. First, stress granule assembly activated by allergens licensed the nuclear-cytoplasmic transport of IL-33, but not the secretion of IL-33. Second, a neo-form murine amino-terminal p40 fragment gasdermin D (Gsdmd), whose generation was independent of inflammatory caspase-1 and caspase-11, dominated cytosolic secretion of IL-33 by forming pores in the cell membrane. Either the blockade of stress granule assembly or the abolishment of p40 production through amino acid mutation of residues 309-313 (ELRQQ) could efficiently prevent the release of IL-33 in murine epithelial cells. Our findings indicated that targeting stress granule disassembly and Gsdmd fragmentation could reduce IL-33-dependent allergic airway inflammation.

Brain Cell Type-Specific Nuclear Proteomics Is Imperative to Resolve Neurodegenerative Disease Mechanisms.

Neurodegenerative diseases (NDs) involve complex cellular mechanisms that are incompletely understood. Emerging findings have revealed that disruption of nuclear processes play key roles in ND pathogenesis. The nucleus is a nexus for gene regulation and cellular processes that together, may underlie pathomechanisms of NDs. Furthermore, many genetic risk factors for NDs encode proteins that are either present in the nucleus or are involved in nuclear processes (for example, RNA binding proteins, epigenetic regulators, or nuclear-cytoplasmic transport proteins). While recent advances in nuclear transcriptomics have been significant, studies of the nuclear proteome in brain have been relatively limited. We propose that a comprehensive analysis of nuclear proteomic alterations of various brain cell types in NDs may provide novel biological and therapeutic insights. This may be feasible because emerging technical advances allow isolation and investigation of intact nuclei from post-mortem frozen human brain tissue with cell type-specific and single-cell resolution. Accordingly, nuclei of various brain cell types harbor unique protein markers which can be used to isolate cell-type specific nuclei followed by down-stream proteomics by mass spectrometry. Here we review the literature providing a rationale for investigating proteomic changes occurring in nuclei in NDs and then highlight the potential for brain cell type-specific nuclear proteomics to enhance our understanding of distinct cellular mechanisms that drive ND pathogenesis.

Ribosomal quality control in repeat‐associated non‐AUG translation of GC rich repeats

Nucleotide repeat expansions cause multiple neurodegenerative disorders including C9orf72‐associated amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD) and Fragile X‐associated tremor/ataxia syndrome (FXTAS). C9 ALS/FTD results from a GGGGCC (G4C2) hexanucleotide repeat expansions within an intron of C9orf72while FXTAS is caused by CGG repeat expansions in the 5’UTR of FMR1. These repeat‐containing RNAs elicit toxicity at least in part by triggering repeat‐associated non‐AUG (RAN) translation, a non‐canonical initiation process that generates toxic proteins from GC rich repeats. As repetitive RNA elements form strong RNA secondary structures might impact translational elongation, we explored the impact of ribosome‐associated quality control (RQC) pathways on RAN translation. RQC rescues stalled ribosomes and prevents translation of aberrant transcripts, with specific roles for both the mammalian nuclear export mediator factor (NEMF, homologous of Rqc2 in yeast) and the E3 ubiquitin ligase Listerin. Here we show that depletion of NEMF markedly increases the production of RAN products from both G4C2 and CGG repeats. This effect appears to be mediated post transcriptionally and does not involve the nuclear‐cytoplasmic transport functions of the protein. Ongoing studies are characterizing how loss of Listerin impacts RAN translation and repeat RNA stability while exploring whether NEMF meditated CAT‐tailing of RAN translation products impacts their turnover and toxicity.

Progerin and Its Role in Accelerated and Natural Aging

Well-known theories of aging suggest that a certain metabolic defect negatively affects vital activity of the cell, be it oxidative stress, the accumulation of lesions in DNA, the exhaustion of telomeres, or distorted epigenetic processes. The theory of aging considered in the review postulates that an accumulation of progerin on the inner side of the nuclear envelope underlies the above defects. Progerin is a defective precursor of the lamin A nuclear matrix protein in which the C-terminal cysteine, which is removed normally, is retained and modified with a hydrophobic oligoisoprene chain. Progerin molecules attach with their hydrophobic processes to the inner membrane of the nuclear envelope, pushing away the adjacent fibrils of the nuclear matrix and the chromatin periphery. This changes the morphology and shape of the nucleus and alters the properties of the nuclear envelope and pore complexes embedded in it. As progerin accumulates in the nucleus, structural distortions increase in the nucleus, further distorting the nuclear-cytoplasmic transport of macromolecules and leading to the above defects in cell metabolism. This leads to increasing cell death and aging of the body over time. This mechanism of aging has been identified in patients with Hutchinson-Gilford progeria syndrome (HGPS). Mass progerin production in HGPS is caused by the point mutation c.1824C→T in exon 11 of the LMNA gene, which codes for lamins A and C. The mutation stimulates non-standard splicing of the primary transcript during the formation of the lamin A precursor mRNA, thus causing progerin production. Children with progeria who have received the mutation from one of their parents age rapidly and die before 15 years of age. Approaches to progeria treatment are aimed at preventing the formation of progerin or destroying the progerin that has already accumulated. In the latter case, a promising strategy is to use rapamycin or its analogs and other substances and techniques that activate autophagy to purify the cell from progerin. Although in much smaller amounts, progerin is found in progeria-free people and may therefore play a role in natural aging. A maximum age that a person can reach is possible to estimate by taking account of the role that progerin plays in telomere shortening. Encouraging preliminary results achieved in purifying cells from progerin provide a means to develop an optimal procedure for periodic purification of the human body from progerin in order to reduce the rate of aging.

A Phase 2 Study to Evaluate the Efficacy and Safety of Selinexor in Patients with Myelofibrosis Refractory or Intolerant to JAK Inhibitors

▪Background:Selinexor is an oral, small molecule, selective inhibitor of nuclear export (SINE) compound that specifically blocks the karyopherin protein exportin 1 (XPO1, CRM1). In an shRNA library screen, we discovered that the survival of JAK2V167F mutant HEL cells is dependent on XPO1-mediated nuclear-cytoplasmic transport. Selinexor selectively suppressed primary myelofibrosis (MF) cells as compared with normal progenitor cells and induced hematologic responses in an MPN mouse model.Methods:An open label, prospective, investigator-initiated single center study is ongoing in adults with primary or secondary MF with resistance or intolerance to JAK inhibitor (JAKi) therapy with platelets > 30 K/μL and neutrophils > 500/μL. Selinexor was given orally once a week. Spleen volume was assessed by MRI at week 12 and week 24. The study was amended to include additional MRIs every 12 weeks in the year 1 and 24 weeks in the year 2. Primary end point is spleen response, defined as ≥ 35% spleen volume reduction (SVR) by MRI or CT, where applicable) at week 24. Bone marrow was evaluated at baseline and at week 24. The projected sample size of 24 will provide 83% power to reject a response rate of 15% and allow for up to a 25% dropout rate. We provide an interim report after completing 50% enrollment.Results:Between May 2019 and February 2021, 12 patients (pts) were enrolled. JAK2, CALR and MPL mutations were present in 7 (58.3%), 4 (33.3%) and 1 (8.3%) pts respectively. Eight pts (66.6%) had at least one high molecular risk mutation at baseline (Table 1). Median duration of prior JAKi therapy was 22 months (0.5 to 96 months) and 11 out of 12 were refractory to ruxolitinib at study enrollment. Median baseline spleen volume was 1454 cm 3(range 835 to 5792). Selinexor starting dose was 80 mg weekly in the first 6 pts and 60 mg for subsequent pts. At data cutoff, median duration of selinexor therapy was 36 weeks (range 11-114 weeks). One pt was not response evaluable and died due to liver abscess at week 12 (unrelated). One pt discontinued selinexor at week 18 due to grade 3 fatigue and was not evaluable for the primary end point. Of the 11 pts who had week 12 MRI or CT, 6 showed ≥ 10% SVR, 3 showed ≥ 25% SVR and 1 pt had early progression (Figure 1). At week 24, 5/9 (56%) pts had ≥ 25% SVR and 2/9 (22%) had ≥ 35% SVR (Figure 1). In 9 pts who had ≥24 weeks of selinexor, SVR ≥ 25% and ≥ 35% occurred at any point during study treatment in 4 (44%) and 3 (33%) pts, respectively. Two pts were red cell transfusion dependent at baseline; 1 became transfusion independent after 36 weeks of treatment, has not required transfusion for 49 weeks and remains on study treatment to date (114 weeks). Six pts (50%) discontinued selinexor. Reasons for treatment discontinuation are death in 1 pt, progressive disease in 1 pt, alternative treatment in 2 pts, and toxicity in 2 pts. Ten pts required dose reduction due to fatigue (1pt), anemia (1 pt), thrombocytopenia (2 pts), abdominal pain (1pt) and weight loss (5 pts). The most common treatment related adverse event was weight loss (grade 2 in 4 pts and grade 3 in 1 pt). This was manageable with treatment interruption and dose reduction, except in one pt who discontinued selinexor. As yet no changes in reticulin fibrosis MF grade were observed among 9 patients who received at least 24 weeks of treatment.Conclusions:Once weekly, oral selinexor showed single agent activity with sustained spleen responses in pts with JAKi refractory MF. Long-term administration of selinexor was well tolerated over time in MF pts. Correlatives studies including circulating inflammatory cytokine levels and mutant allele burden, as well as clonality studies by X-chromosome inactivation studies in woman, are underway and will be presented. [Display omitted] DisclosuresTantravahi: BMS: Research Funding; Novartis: Research Funding; CTI BioPharma: Research Funding; Abbvie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Consultancy, Honoraria, Research Funding. Patel: Stemline: Research Funding; Genentech: Research Funding; Roche: Research Funding. Chamoun: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Shah: Karyopharm: Current Employment. George: Celgene: Consultancy; Bristol Meyers Squibb: Consultancy; Incyte Corporation: Consultancy; Blueprint Medicines: Consultancy. Deininger: Fusion Pharma, Medscape, DisperSol: Consultancy; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Novartis: Consultancy, Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding.

SARS-CoV-2 infection triggers widespread host mRNA decay leading to an mRNA export block.

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.

Rapid Decay of Host Basal mRNAs During SARS-CoV-2 Infection Perturbs Host Antiviral mRNA Biogenesis and Export

A key feature of the mammalian antiviral response is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response to limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFNs in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. Additionally, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. Thus, inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response.Funding Information: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number F32AI145112 (J.M.B), funds from HHMI (Roy Parker), and support provided by the Office of the Vice President for Research and the Dept. of Microbiology, Immunology and Pathology at Colorado State University (Rushika Perera).Declaration of Interests: Roy Parker is a founder and consultant of Faze Medicines.

Electrostatic modulation of hnRNPA1 low-complexity domain liquid-liquid phase separation and aggregation.

Membrane-less organelles and RNP granules are enriched in RNA and RNA-binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear-cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C-terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid-liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane-less organelles, aggregation within phase-separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS-mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS-mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.

DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.