Chromosome Region Maintenance Research Articles

Identification of a Novel Post-transcriptional Transactivator from the Equine Infectious Anemia Virus.

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions. IMPORTANCE Nuclear export of viral transcripts is a crucial step for viral gene expression and viral replication in lentiviruses, and this export is regulated by a post-transcriptional transactivator, Rev, that is shared by all lentiviruses. Here, we report that the equine infectious anemia virus (EIAV) encodes a novel viral protein, Grev, and demonstrated that Grev, like Rev, mediates the expression of the viral protein Mat by binding to the first exon of mat mRNAs via the chromosome region maintenance 1 (CRM1) pathway. Grev is encoded by a single-spliced transcript containing two exons, whereas Rev is encoded by a multiple-spliced transcript containing four exons. Moreover, Rev is able to mediate EIAV Gag/Pol expression by binding to rev-responsive element (RRE) located within the Env-coding region, while Grev cannot. Therefore, the present study demonstrates that EIAV encodes two post-transcriptional regulators, Grev and Rev, suggesting that post-transcriptional regulation patterns in lentivirus are diverse and complex.

Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1-mediated nuclear export.

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor.

The Exportin, XPO2, Is a Novel Therapeutic Target in Pediatric, Adolescent and Young Adult AML

The active transport of proteins across the nuclear membrane is regulated by nuclear transport receptors termed importins and exportins, depending on their direction of transport. Nuclear transport receptors contribute to cancer development, through abnormal localization of protein cargos. Increased expression of the exportin, exportin-1/chromosome region maintenance 1 (XPO1/CRM1), has been associated with high risk AML and shorter survival times and XPO1 inhibitors show signs of efficacy in AML. While previous data demonstrate a functional role for XPO1 in AML, less is known about the exportin, XPO2, (alias CSE1L). Despite XPO2 RNAi depletion screens demonstrating a higher genetic dependence in leukemia versus solid tumors/non-hematologic malignancies (p = 0.0394), the functional impact of inhibiting XPO2 has never been studied in leukemia. We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes in AML, along with KPNB1 and XPO1 (DepMap 22Q2 Public+Score, Chronos, www.depmap.org). An RNAi depletion screen confirmed these findings, with XPO2 identified within the top 0.2% of essential genes in AML (Achilles+DRIVE+Marcotte, DEMETER2, www.depmap.org). Gene dependency scores indicated a greater reliance on XPO2, compared to XPO1 (p <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (p = 0.0014) and overall survival (p = 0.00091) in pediatric and adolescent/young adult (AYA) AML patients from the TARGET dataset. Analyzing the BeatAML dataset, increased XPO2 mRNA expression was also associated with decreased overall survival in AML patients <40 years, but not in patients >40 years (p <0.0001). We analyzed protein expression databases and identified XPO2 as the highest expressed member of the exportin family in AML cells lines and AML primary patient samples. By immunoblot analysis, increased expression of XPO2 was detected in 7/7 primary AML samples and 8 AML cell lines (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX and U937), compared to normal hematopoietic cells. XPO2 mRNA was also increased in 5/5 primary AML samples and 8/8 AML cell lines, compared to normal hematopoietic cells, as measured by qRT-PCR. To confirm the functional importance of XPO2 in AML, we knocked down XPO2 with shRNA in AML cell lines. Genetic silencing of XPO2 decreased growth and viability of OCI-AML2, TEX and NB4 cells. Gambogic acid (GA), a natural product from Garcinia hanburyi, has been identified as a covalent inhibitor of XPO2-mediated nuclear transportation. In OCI-AML2, TEX and NB4 cell lines, gambogic acid decreased growth and viability of AML cell lines at nanomolar concentrations (TEX cells IC50 = 369nM, OCI-AML2 cells IC50 = 433nM, NB4 cells IC50 = 161nM). To uncover the mechanism of how XPO2 mediates cell death in AML we interrogated a publicly available BioID database (PXD007976) and performed gene set enrichment analysis on XPO2 targets using gprofiler. Processes related to ribosome biogenesis were significantly enriched in XPO2 cargo, whereas XPO1 cargo were enriched for processes related to snRNA transcription and transport (p <0.05). In summary, we identified XPO2 as an essential gene for AML. XPO2 is preferentially increased in AML cell lines and primary patient samples compared to normal hematopoietic cells. Increased XPO2 expression is associated with worse clinical outcomes in pediatric and AYA patients with AML. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemia initiation and progression. Thus, this work demonstrates a new dependency of AML on XPO2, as well as highlights a new therapeutic target for this disease.

Inhibition of XPO1 with KPT-330 induces autophagy-dependent apoptosis in gallbladder cancer by activating the p53/mTOR pathway

BackgroundGallbladder cancer (GBC) is a highly aggressive malignant cancer in the biliary system with poor prognosis. XPO1 (chromosome region maintenance 1 or CRM1) mediates the nuclear export of several proteins, mainly tumor suppressors. Thus, XPO1 functions as a pro-oncogenic factor. KPT-330 (Selinexor) is a United States Food and Drug Administration approved selective inhibitor of XPO1 that demonstrates good therapeutic effects in hematologic cancers. However, the function of XPO1 and the effect of KPT-330 have not been reported in GBC.MethodsWe analyzed the correlation between XPO1 expression levels by q-PCR and clinical features of GBC patients. Cell proliferation assays were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. mRNA sequencing was used to explore the underlying mechanisms. Western blot was performed to explore the relationship between apoptosis and autophagy. The in vivo antitumor effect of KPT-330 was investigated in a nude mouse model of gallbladder cancer.ResultsWe found that high expression of XPO1 was related to poor prognosis of GBC patients. We observed that XPO1 inhibitor KPT-330 inhibited the proliferation of GBC cells in vitro. Furthermore, XPO1 inhibitor KPT-330 induced apoptosis by reducing the mitochondrial membrane potential and triggering autophagy in NOZ and GBC-SD cells. Indeed, XPO1 inhibitor KPT-330 led to nuclear accumulation of p53 and activated the p53/mTOR pathway to regulate autophagy-dependent apoptosis. Importantly, KPT-330 suppressed tumor growth with no obvious toxic effects in vivo.ConclusionXPO1 may be a promising prognostic indicator for GBC, and KPT-330 appears to be a potential drug for treating GBC effectively and safely.

A Novel, Fully Spliced, Accessory Gene in Equine Lentivirus with Distinct Rev-Responsive Element.

ABSTRACTAll lentiviruses encode the accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) within the env-encoding region. Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encoded a viral protein, named Mat, with an unknown function. The transcript mat was fully spliced and comprised parts of the coding regions of MA and TM. Interestingly, the expression of Mat depended on Rev and the chromosome region maintenance 1 (CRM1) pathway. Rev could specifically bind to Mat mRNA to promote its nuclear export. We further identified that the first exon of Mat mRNA, which was located within the Gag-encoding region, acted as an unreported RRE. Altogether, we identified a novel fully spliced transcript mat with an unusual RRE, which interacted with Rev for nuclear export through the CRM1 pathway. These findings updated the EIAV genome structure, highlighted the diversification of posttranscriptional regulation patterns in EIAV, and may help to expand the understanding of gene transcription and expression of lentivirus.IMPORTANCE In lentiviruses, the nuclear export of viral transcripts is an important step in controlling viral gene expression. Generally, the unspliced and incompletely spliced transcripts are exported via the CRM1-dependent export pathway in a process mediated by the viral Rev protein by binding to the Rev-responsive element (RRE) located within the Env-coding region. However, the completely spliced transcripts are exported via an endogenous cellular pathway, which was Rev independent. Here, we identified a novel fully spliced transcript from EIAV and demonstrated that it encoded a viral protein, termed Mat. Interestingly, we determined that the expression of Mat depended on Rev and identified that the first exon of Mat mRNA could specifically bind to Rev and be exported to the cytoplasm, which suggested that the first exon of Mat mRNA was a second RRE of EIAV. These findings provided important insights into the Rev-dependent nuclear export of completely spliced transcripts in lentiviruses.

Nuclear entry and egress of parvoviruses.

Parvoviruses are small non-enveloped single-stranded DNA viruses, which depend on host cell nuclear transcriptional and replication machinery. After endosomal exposure of nuclear localization sequence and a phospholipase A2 domain on the capsid surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to the small capsid diameter of 18-26 nm, intact capsids can potentially pass into the nucleus through nuclear pore complexes (NPCs). This might be facilitated by active nuclear import, but capsids may also follow an alternative entry pathway that includes activation of mitotic factors and local transient disruption of the nuclear envelope. The nuclear entry is followed by currently undefined events of viral genome uncoating. After genome release, viral replication compartments are initiated and infection proceeds. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly during virus-induced S/G2 cell cycle arrest. Nuclear egress of capsids occurs upon nuclear envelope degradation during apoptosis and cell lysis. An alternative pathway for nuclear export has been described using active transport through the NPC mediated by the chromosome region maintenance 1 protein, CRM1, which is enhanced by phosphorylation of the N-terminal domain of VP2. However, other alternative but not yet uncharacterized nuclear export pathways cannot be excluded.

Abstract 5848: Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein

Abstract Mutant KRAS is found in &amp;gt;30% of lung adenocarcinomas, and tumors with mutant KRAS usually have poor outcomes with current therapies. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have uncovered for reactivating the DLC1 tumor suppressor protein, which is frequently downregulated in a wide range of human cancers. Although several mechanisms account for DLC1 downregulation, here we have focused on a subset of mutant KRAS lung tumor lines that express wild type DLC1 mRNA but lack detectable DLC1 protein. We have recently reported that cytoplasmic EZH2 protein directly methylates Lysine-678 of DLC1 protein leading to its ubiquitination by CUL-4A and consequently, proteasomal degradation. In this study, we have found a specific inhibitor of CRM1 protein (Chromosomal Region Maintenance 1, also known as Exportin 1 [XPO1]) substantially reduces cytoplasmic EZH2 level in several mutant KRAS lung cancer cell lines demonstrating that cytoplasmic EZH2 depends on its nuclear export by CRM1. CRM1, which is the major mammalian nuclear export regulator, is frequently overexpressed and/or mutated in human cancer, including lung adenocarcinomas, and these phenotypes are associated with poor prognosis. Endogenous CRM1 and EZH2 protein colocalized and formed a complex in several lung cancer lines and patient-derived tumor xenografts implying the cancer relevance of this interaction. In addition to blocking the cytoplasmic export of EZH2, pharmacologic inhibition of CRM1 increased the steady-state level of DLC1 protein in several mutant KRAS lung cancer lines and patient-derived xenografts. The tumor suppressor activity of the post-translationally stabilized DLC1 protein was further increased by kinase inhibitors of AKT and SRC, which reverse the direct phosphorylation and attenuation of DLC1 tumor suppressor functions by these kinases. The three-drug combination of inhibitors of CRM1 (Selinexor), AKT (MK-2206), and SRC (Saracatinib), which is well-tolerated in mice, induced potent inhibition of tumor xenograft growth and was associated with induction of markers of apoptosis and senescence. Although each of these inhibitors has multiple targets, CRISPR/Cas9 knockout of DLC1 abolished most of the tumor growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant target for the antitumor activity of the drug combination. Interestingly, KRAS siRNA knockdown phenocopied CRM1 inhibition, as it reduced cytoplasmic EZH2, DLC1 methylation, and DLC1 degradation. Our findings suggest CRM1 inhibitors, when given in conjunction with AKT and/or SRC kinase inhibitors, may have clinical efficacy against lung tumors with mutant KRAS. Citation Format: Brajendra K. Tripathi, Nicole Hirsh, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, James H. Doroshow, Douglas R. Lowy. Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5848.

Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells alleviates myocardial injury by targeting thioredoxin-interacting protein-mediated hypoxia-inducible factor-1α pathway.

BACKGROUNDExtracellular vesicles (EVs) derived from hypoxia-preconditioned (HP) mesenchymal stem cells (MSCs) have better cardioprotective effects against myocardial infarction (MI) in the early stage than EVs isolated from normoxic (NC)-MSCs. However, the cardioprotective mechanisms of HP-EVs are not fully understood.AIMTo explore the cardioprotective mechanism of EVs derived from HP MSCs.METHODSWe evaluated the cardioprotective effects of HP-EVs or NC-EVs from mouse adipose-derived MSCs (ADSCs) following hypoxia in vitro or MI in vivo, in order to improve the survival of cardiomyocytes (CMs) and restore cardiac function. The degree of CM apoptosis in each group was assessed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/PI assays. MicroRNA (miRNA) sequencing was used to investigate the functional RNA diversity between HP-EVs and NC-EVs from mouse ADSCs. The molecular mechanism of EVs in mediating thioredoxin-interacting protein (TXNIP) was verified by the dual-luciferase reporter assay. Co-immunoprecipitation, western blotting, and immunofluorescence were performed to determine if TXNIP is involved in hypoxia-inducible factor-1 alpha (HIF-1α) ubiquitination and degradation via the chromosomal region maintenance-1 (CRM-1)-dependent nuclear transport pathway.RESULTSHP-EVs derived from MSCs reduced both infarct size (necrosis area) and apoptotic degree to a greater extent than NC-EVs from CMs subjected to hypoxia in vitro and mice with MI in vivo. Sequencing of EV-associated miRNAs showed the upregulation of 10 miRNAs predicted to bind TXNIP, an oxidative stress-associated protein. We showed miRNA224-5p, the most upregulated miRNA in HP-EVs, directly combined the 3’ untranslated region of TXNIP and demonstrated its critical protective role against hypoxia-mediated CM injury. Our results demonstrated that MI triggered TXNIP-mediated HIF-1α ubiquitination and degradation in the CRM-1-mediated nuclear transport pathway in CMs, which led to aggravated injury and hypoxia tolerance in CMs in the early stage of MI. CONCLUSIONThe anti-apoptotic effects of HP-EVs in alleviating MI and the hypoxic conditions of CMs until reperfusion therapy may partly result from EV miR-224-5p targeting TXNIP.

RETRACTED ARTICLE: The abnormal expression of chromosomal region maintenance 1 (CRM1)-survivin axis in ovarian cancer and its related mechanisms regulating proliferation and apoptosis of ovarian cancer cells

Statement of Retraction Jing Zhang, Xinyan Xu, Yongfeng Chen, Xiaoju Guan, Hong Zhu, Yuhong Qi- 2022, The abnormal expression of chromosomal region maintenance 1 (CRM1)-survivin axis in ovarian cancer and its related mechanisms regulating proliferation and apoptosis of ovarian cancer cells, Bioengineered 13(1), doi: 10.1080/21655979.2021.2012416 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Selected Articles from This Issue

Dysregulation of the protein arginine methyltransferase 5 (PRMT5) is associated with lymphomas, lung cancers, and breast cancers. In this First Disclosure, Brehmer and colleagues outline JNJ-64619178, an inhibitor of PRMT5 with potent anti-proliferative activity. JNJ-64619178 occupies the SAM and substrate pockets of PRMT5, trapping it in a catalytically inactive state. PMRT5 inhibition by JNJ-64619178 increased alternatively spliced events and resulted in the killing of primary AML samples ex vivo. Taken together, the results support the continued clinical study of JNJ-64619178 (NCT03573310).The approval of Trastuzumab deruxtecan demonstrates the potency of the DNA topoisomerase I inhibitor DXd in antibody-drug conjugates (ADCs). DXd-ADCs exhibit a short systemic half-life and ability to optimize the drug-antibody ratio. In this First Disclosure, Okajima and colleagues outline the design of another DXd-containing ADC targeting Trophoblast cell surface antigen 2 (TROP2). Datopotamab deruxtecan is designed to have a shorter half-life than sacituzumab govitecan, a TROP2-directed ADC approved by the FDA for TNBC. Dato-DXd showed potent anti-tumor activity and was absent of severe gastrointestinal toxicity. The results support the first-in-human phase 1 study for patients with advanced solid tumors (NCT03401385).Chromosome region maintenance 1 is overexpressed in ovarian cancer and its inhibition has been shown to achieve a 30% disease control rate in recurrent disease. Here, Handley and colleagues seek to enhance the effects of selinexor through synergistic drug combinations. Their screen found the PARP inhibitor olaparib as the most likely candidate. Selinexor decreased the expression of DNA damage repair proteins, generating an increased susceptibility to PARP inhibitors. The combination reduced tumor nodules and weight in vivo in a mouse model. Their results outline an actionable combination for advanced ovarian cancer patients.The advent of KRAS inhibitors, as well as the disagreement between retrospective studies in immunotherapy and KEYNOTE-189, demonstrate the need to characterize the KRAS mutational landscape of non-small cell lung cancer (NSCLC). Therefore, Judd and colleagues sequenced 592 genes from 17,095 NSCLC specimens to assess co-occuring genomic alterations, tumor mutational burden (TMB), and PD-L1 expression. They demonstrate the rate of co-mutation depends on the KRAS mutation type. Mutated KRAS was significantly increased in PD-L1 expression compared to wild type, with KRAS G12C the most likely to be PD-L1 positive. These studies inform future therapeutic intervention for KRAS-mutated NSCLC.

Chromosome Region Maintenance 1 (XPO1/CRM1) as an Anticancer Target and Discovery of Its Inhibitor.

Chromosome region maintenance 1 (CRM1) is a major nuclear export receptor protein and contributes to cell homeostasis by mediating the transport of cargo from the nucleus to the cytoplasm. CRM1 is a therapeutic target comprised of several tumor types, including osteosarcoma, multiple myeloma, gliomas, and pancreatic cancer. In the past decade, dozens of CRM1 inhibitors have been discovered and developed, including KPT-330, which received FDA approval for multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL) in 2019 and 2020, respectively. This review summarizes the biological functions of CRM1, the current understanding of the role CRM1 plays in cancer, the discovery of CRM1 small-molecule inhibitors, preclinical and clinical studies on KPT-330, and other recently developed inhibitors. A new CRM1 inhibition mechanism and structural dynamics are discussed. Through this review, we hope to guide the future design and optimization of CRM1 inhibitors.

RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport.

Abnormal subcellular localization of proteins is an important cause of tumorigenesis and drug resistance. Chromosome region maintenance 1 (CRM1), the nuclear export regulator of most proteins, has been confirmed to be over-expressed in various malignancies and is regarded as an efficient target. But the potential role of the CRM1 cofactor RanBP3 (Ran Binding Protein 3) is left unrevealed in chronic myeloid leukemia (CML). Here, we first detected the level of RanBP3 in CML and found an elevated RanBP3 expression in CML compared with control. Then we used shRNA lentivirus to down-regulated RanBP3 in imatinib sensitive K562 cells and resistant K562/G01 cells and found RanBP3 silencing inhibited cell proliferation by up-regulating p21, induced caspase3-related cell apoptosis, and enhanced the drug sensitivity of IM in vitro. Notably, we observed that RanBP3 silencing restored imatinib sensitivity of K562 cells in NOD/SCID mice. Mechanistically, the nuclear aggregation of SMAD2/3 revealed that tumor suppressor axis (TGF-β)-SMAD2/3-p21 was the anti-proliferation program related to RanBP3 knockdown, and the decrease of cytoplasmic ERK1/2 caused by RanBP3 interference leaded to the down-regulation of anti-apoptosis protein p(Ser112)-BAD, which was the mechanism of increased cell apoptosis and enhanced chemosensitivity to imatinib in CML. In summary, this study revealed the expression and potential role of RanBP3 in CML, suggesting that targeting RanBP3 alone or combined with TKIs could improve the clinical response of CML.

XPO1/CRM1 is a promising prognostic indicator for neuroblastoma and represented a therapeutic target by selective inhibitor verdinexor

BackgroundHigh-risk neuroblastoma patients have a 5-year survival rate of less than 50%. It’s an urgent need to identify new therapeutic targets and the appropriate drugs. Exportin-1 (XPO1), also known as chromosomal region maintenance 1, plays important roles in the progression of tumorigenesis. However, the prognostic and therapeutic values of XPO1 in neuroblastoma have not been reported.MethodsCorrelations between XPO1 expression level and clinical characteristics were analyzed using the Neuroblastoma Research Consortium (NRC) dataset and tissue microarray analysis. Cell proliferation assays, colony formation assays, apoptosis assays, cell cycle analysis were performed to analyze the anti-tumor effects of verdinexor (KPT-335) in vitro. Western blot and mRNA sequencing were performed to explore underlying mechanism. In vivo anti-tumor effects of verdinexor were studied in a neuroblastoma xenograft model.ResultsHigher XPO1 levels were associated with advanced stage and poor prognosis in neuroblastoma patients. The specific inhibitor of XPO1 verdinexor suppressed the neuroblastoma cell growth both in vitro and in vivo. Specifically, inhibition of XPO1 suppressed the neuroblastoma cell proliferation and induced cell apoptosis by nuclear accumulation of FOXO1 and RB1 in the neuroblastoma due to the inhibition of the PI3K/AKT pathway, and induced G0/G1 phase cell cycle arrest by activation of P53 function.ConclusionsXPO1 is a promising prognostic indicator for neuroblastoma and a novel target for antitumor treatment with selective inhibitor verdinexor.

Subcellular Localization of Epstein-Barr Virus BLLF2 and Its Underlying Mechanisms.

Epstein–Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2’s two functional nuclear localization signals (NLSs), NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (44RRPRPPVAKRRRFPR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses.

Overcoming Tyrosine Kinase Inhibitor Resistance to SEPT9-ABL1 Chimeric Fusion Protein Using CRM1 Inhibitors

ABL1 fusion genes, which have been reported in various hematological malignancies, are thought to contribute to leukemogenesis through aberrant ABL1 signaling and activating multiple downstream signals. Tyrosine kinase inhibitors (TKIs), such as imatinib and dasatinib, have been developed to inhibit the function of the most- characterized ABL1 fusion product, BCR-ABL1. Because TKIs target the ABL1 kinase domain, ABL1 fusion products are usually sensitive to TKIs both in vitro and in vivo .We previously identified the chimeric fusion gene SEPT9 - ABL1 in a case of T-prolymphocytic leukemia that was unexpectedly resistant to imatinib and dasatinib without mutations in the domains derived from ABL1. We proved that SEPT9-ABL1 exhibited TKI resistance in vitro and in vivo, as seen in theclinical course (Leuk Res 2014). The purpose of this study was to reveal the mechanism underlying TKI resistance and to overcome it in a specific way.SEPT9f-ABL1, which has the most typical characteristics among SEPT9 isoforms, was retrovirally transduced into interleukin-3 (IL-3)-dependent murine hematopoietic cell lines 32D and BaF3 cells to generate 32D/SEPT9-ABL1 and BaF3/SEPT9-ABL1 cells. 32D/BCR-ABL1 and BaF3/BCR-ABL1 cells were also generated as references. The 50% inhibitory concentration (IC50) of imatinib was higher with SEPT9-ABL1 than with BCR-ABL1 (in 32D cells and BaF3 cells, respectively), confirming that SEPT9-ABL1 contributed to TKI resistance.Because TP53 has been suggested to involved in TKI resistance, we first analyzed the TP53 expression and phosphorylation in these cells. Western blot revealed that TP53 was downregulated and less phosphorylated in the cells expressing SEPT9-ABL1 than in those with BCR-ABL1. These findings were consistent with the analysis using primary leukemic cells. Imatinib treatment reduced the TP53 expression in the cells expressing SEPT9-ABL1. When the cell pellets were fractioned, the TP53 expression in the cytoplasm was comparable between cells expressing SEPT9-ABL1 and those with BCR-ABL1 under imatinib treatment. However, the TP53 expression remained higher in the nucleus of the cells with BCR-ABL1 than in those with SEPT9-ABL1, suggesting that the decreased nuclear TP53 expression prevented the cells expressing SEPT9-ABL1 from undergoing apoptosis induced by imatinib.Because the distribution of TP53 in the nucleus and the cytoplasm is controlled by chromosomal region maintenance 1 (CRM1), a nuclear export receptor, we next evaluated the effects of CRM1 inhibition. When the cells were cultured with the combination of imatinib (1 µM) and KPT-185 (1 µM), TP53 was markedly accumulated in the cells expressing SEPT9-ABL1; however, no such accumulation was noted when cells were cultured with imatinib alone, suggesting that KPT-185 treatment enhanced the effect of imatinib in the cells expressing SEPT9-ABL1 through the inhibition of TP53 export from the nucleus. This combination dramatically increased the apoptotic cells with SEPT9-ABL1. To evaluate CRM1 inhibition in vivo, we utilized a subcutaneous tumor model with BaF3/SEPT9-ABL1 cells. Treatment with imatinib 20 mg/kg plus another CRM1 inhibitor (KPT-330, 20 mg/kg) led to the significant regression of tumors composed of BaF3/SEPT9-ABL1 cells compared to imatinib alone (p=0.029).In conclusion, decreased nuclear expression of TP53 is a possible mechanism underlying TKI resistance in cells harboring SEPT9-ABL1. CRM1 inhibition is an effective method of overcoming TKI resistance in cells with SEPT9-ABL1 through induction of the nuclear accumulation of TP53 and apoptosis. [Display omitted] DisclosuresAndo:NOVARTIS: Other: Donation to institute; Meiji Seika Pharma: Other: Donation to institute; CHUGAI PHARMACEUTICAL: Other: Donation to institute; TOYAMA CHEMICAL: Other: Donation to institute; MOCHIDA PHARMACEUTICAL: Other: Donation to institute; Sumitomo Dainippon Pharma: Other: Donation to institute; Eisai: Other: Donation to institute; Bristol-Myers Squibb: Other: Donation to institute; MSD: Other: Donation to institute; Kyowa Hakko Kirin: Other: Donation to institute; ALEXION: Other: Donation to institute; Takeda: Other: Donation to institute; Japan Blood Products Organization: Other: Donation to institute; Nippon Shinyaku: Other: Donation to institute; NIHON PHARMACEUTICAL: Other: Donation to institute; TAIHO: Other: Donation to institute; Asahi KASEI: Other: Donation to institute.

Structure-Guided Design of the First Noncovalent Small-Molecule Inhibitor of CRM1.

Nuclear export factor chromosome region maintenance 1 (CRM1) is an attractive anticancer and antiviral drug target that spurred several research efforts to develop its inhibitor. Noncovalent CRM1 inhibitors are desirable, but none is reported to date. Here, we present the crystal structure of yeast CRM1 in complex with S109, a substructure of CBS9106 (under clinical test). Superimposition with the LFS-829 (another covalent CRM1 inhibitor) complex inspired the design of a noncovalent CRM1 inhibitor. Among nine synthesized compounds, noncovalent CRM1 inhibitor 1 (NCI-1) showed a high affinity to human and yeast CRM1 in the absence or presence of GST-bound Ras-related nuclear protein (RanGTP). Unlike covalent inhibitors, the crystal structure showed that NCI-1 is bound in the "open" nuclear export signal (NES) groove of CRM1, simultaneously occupying two hydrophobic pockets. NCI-1 additionally inhibited the nuclear export and proliferation of cells harboring the human CRM1-C528S mutant. Our work opens up the avenue of noncovalent CRM1 inhibitor development toward a more potent, less toxic, and broad-spectrum anticancer/antiviral therapy.

PCID2 Impacts Centrosome Duplication by Regulating the Nuclear Export of BARD1 mRNA in Breast Cancer Cells

The nuclear export of proteins and other macromolecules out of the nucleus to different intracellular locations is a significant and crucial function of the cell that affects cellular processes such as DNA synthesis, RNA transcription, protein processing, the cell cycle, and apoptosis. Chromosome region maintenance (CRM1) is the major export receptor within the cell, regulating the localization of most proteins. One nuclear export cargo of CRM1 is BARD1, which dimerizes with BRCA1 to regulate DNA repair in the nucleus, while the pair also function in the cytoplasm as negative regulators of centrosome amplification during cell cycle. Despite cooperation at both of these locations BARD1 and BRCA1 are independently exported from the nucleus to the cytoplasm. Our laboratory is interested characterizing the protein PCID2, which is involved in CRM1-mediated protein export and localizes to the centrosome, where it potentially regulates centrosome duplication. We previously demonstrated that PCID2 helps mediate CRM1 dependent export of BRCA1 from the nucleus to the centrosome in Hs578T Breast Cancer cells. The present work aims to determine the role of PCID2 in BARD1 nuclear export and localization in these same cells. PCID2 siRNA knockdowns were used and confirmed via western blot, and BARD1 localization was viewed via immunofluorescence. PCID2 knockdown caused a twenty percent decrease in BARD1 protein in the nucleus and a thirty percent decrease in BARD1 protein at the centrosome. The loss of BARD1 at centrosomes was tied to a fifteen percent increase in Hs578T cells demonstrating excess centrosomes duplication. The loss of BARD1 at both locations in the absence of PCID2 suggests this protein is not involved in the CRM1 mediated export of BARD1 protein from nucleus to centrosomes, and instead changes in cellular localization of BARD1 may be due to an observed 50% decrease in total BARD1 protein with PCID2 knockdown. Interestingly, PCID2 is also a part of the TREX-2 mRNA nuclear export complex; therefore loss of PCID2 may have instead impacted the nuclear export, and translation of BARD1 mRNA. Indeed, PCID2 siRNA knockdown led to a two-fold increase in nuclear BARD1 mRNA as observed by in situ hybridization. Nuclear accumulation of BARD1 mRNA likely contributed the overall decrease in BARD1 expression. The correlation between loss of PCID2 and centrosome amplification suggests PCID2 plays impacts centrosome duplication by regulating both BARD1 mRNA export and BRCA1 protein export; with the loss of PCID2 creating an overall decrease in these negative regulators at the centrosome. Understanding PCID2’s effect on centrosome duplication helps to determine it's impact on cell cycle regulation and define it's potential role as tumor suppressor in Breast Cancer.

Novel Mechanistic Observations and NES-Binding Groove Features Revealed by the CRM1 Inhibitors Plumbagin and Oridonin.

The protein chromosome region maintenance 1 (CRM1) is an important nuclear export factor and drug target in diseases such as cancer and viral infections. Several plant-derived CRM1 inhibitors including plumbagin and oridonin possess potent antitumor activities. However, their modes of CRM1 inhibition remain unclear. Here, a multimutant CRM1 was engineered to enable crystallization of these two small molecules in its NES groove. Plumbagin and oridonin share the same three conjugation sites in CRM1. In solution, these two inhibitors targeted more CRM1 sites and inhibited its activity through promoting its aggregation, in addition to directly targeting the NES groove. While the plumbagin-bound NES groove resembles the NES-bound groove state, the oridonin complex reveals for the first time a more open NES groove. The observed greater NES groove dynamics may improve cargo loading through a "capture-and-tighten" mechanism. This work thus provides new insights on the mechanism of CRM1 inhibition by two natural products and a structural basis for further development of these or other CRM1 inhibitors.

Selinexor decreases HIF-1\u03b1 via inhibition of CRM1 in human osteosarcoma and hepatoma cells associated with an increased radiosensitivity

BackgroundThe nuclear pore complexes (NPCs) are built of about 30 different nucleoporins and act as key regulators of molecular traffic between the cytoplasm and the nucleus for sizeable proteins (> 40 kDa) which must enter the nucleus. Various nuclear transport receptors are involved in import and export processes of proteins through the nuclear pores. The most prominent nuclear export receptor is chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1). One of its cargo proteins is the prolyl hydroxylase 2 (PHD2) which is involved in the initiation of the degradation of hypoxia-inducible factors (HIFs) under normoxia. HIFs are proteins that regulate the cellular adaptation under hypoxic conditions. They are involved in many aspects of cell viability and play an important role in the hypoxic microenvironment of cancer. In cancer, CRM1 is often overexpressed thus being a putative target for the development of new cancer therapies. The newly FDA-approved pharmaceutical Selinexor (KPT-330) selectively inhibits nuclear export via CRM1 and is currently tested in additional Phase-III clinical trials. In this study, we investigated the effect of CRM1 inhibition on the subcellular localization of HIF-1α and radiosensitivity.MethodsHuman hepatoma cells Hep3B and human osteosarcoma cells U2OS were treated with Selinexor. Intranuclear concentration of HIF-1α protein was measured using immunoblot analysis. Furthermore, cells were irradiated with 2–8 Gy after treatment with Selinexor compared to untreated controls.ResultsSelinexor significantly reduced the intranuclear level of HIF-1α protein in human hepatoma cells Hep3B and human osteosarcoma cells U2OS. Moreover, we demonstrated by clonogenic survival assays that Selinexor leads to dose-dependent radiosensitization in Hep3B-hepatoma and U2OS-osteosarcoma cells.ConclusionTargeting the HIF pathway by Selinexor might be an attractive tool to overcome hypoxia-induced radioresistance.

The significance of gradient expression of chromosome region maintenance protein 1 (exportin1) in large cell lymphoma

Not available.