CRM1-mediated Export Research Articles

Abstract B067: CRM1 inhibitor selinexor induces DNA damage repair-related vulnerability in prostate cancer cells

Abstract Background: Advanced metastatic prostate cancer is the second leading cause of death in men in the United States. Androgen deprivation therapy has been used as a front-line therapy; however, despite initial success, it fails subsequently and results in the development of castration-resistant prostate cancer. Due to these unmet therapeutic needs, there is an urgency to identify novel targets to develop next-generation therapeutics. Chromosome region maintenance 1 (CRM1) is one of the seven members of mammalian export proteins that function to export the proteins using nuclear export signal (NES). Several tumors, including prostate cancer, overexpress CRM1 to efflux tumor suppressor proteins to the cytoplasm and exhibit dysregulated nuclear export. CRM1-mediated export can lead to functional inactivation of p53, cyclinD1, p16, p21, and p27. Essential DNA repair proteins such as Rad51 and BRCA1 exhibit leucine-containing hydrophobic residues rich in NES and are known to be shuttled by CRM1. Based on this, we hypothesize that CRM1 inhibition may affect the expression, kinetics, localization, and stability of DNA repair network proteins. Methods: Androgen receptor-positive and negative prostate cancer cells were treated with Selinexor. The dose of the drug and duration was dependent on the endpoint measurement. Cell death was monitored using MTT assay and western blot measurement of cleaved PARP. Total RNA and cell lysate were collected to measure the relative levels of BRCA-1, RAD51, p53, and other members of the DNA repair pathway (HR and NHEJ). DNA damage was estimated using the number of micronuclei and immunofluorescence staining for γ-H2AX and COMET assay. Results: Selinexor treatment in prostate cancer cells affects the functioning of the DNA repair network and can be combined with inhibitors of DNA repair proteins. Citation Format: Rajendra Kumar, Janet Mendonca, Yuhan Yang, Abhishek Shetty, Lillian Wilson, Kavya Boyapati, Deven Topiwala, Keerti Soundappan, Eleni Panagopoulos, Michael Carducci, Sushant Kachhap. CRM1 inhibitor selinexor induces DNA damage repair-related vulnerability in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B067.

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.

Staufen-2 functions as a cofactor for enhanced Rev-mediated nucleocytoplasmic trafficking of HIV-1 genomic RNA via the CRM1 pathway.

Nucleocytoplasmic shuttling of viral elements, supported by several host factors, is essential for the replication of the human immunodeficiency virus (HIV). HIV-1 uses a nuclear RNA export pathway mediated by viral protein Rev to transport its Rev response element (RRE)-containing partially spliced and unspliced transcripts aided by the host nuclear RNA export protein CRM1. The factor(s) interacting with the CRM1-Rev complex are potential antiretroviral target(s) and could serve as a retroviral model system to study nuclear export machinery adapted by these viruses. We earlier reported that cellular Staufen-2 interacts with Rev, facilitating viral-RNA export. Here, we identified the formation of a complex between Staufen-2, CRM1 and Rev. Molecular docking and simulations mapped the interacting residues in the RNA-binding Domain 4 of Staufen-2 as R336 and R337, which were experimentally verified to be critical for interactions among Staufen-2, CRM1 and Rev by mutational analysis. Staufen-2 mutants defective in interaction with CRM1 or Rev failed to supplement the Rev-RNA export activity and viral production, demonstrating the importance of these interactions. Rev-dependent reporter assays and proviral DNA-construct transfection-based studies in Staufen-2 knockout cells in the presence of leptomycin-B (LMB) revealed a significant reduction in CRM1-mediated Rev-dependent RNA export with decreased virus production as compared to Staufen-2 knockout background or LMB treatment alone, suggesting the relevance of these interactions in augmenting RNA export activity of Rev. Our observations provide further insights into the mechanistic intricacies of unspliced viral-RNA export to the cytoplasm and support the notion that abrogating such interactions can reduce HIV-1 proliferation.

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.

Enhanced Activity of Exportin-1/CRM1 in Neurons Contributes to Autophagy Dysfunction and Senescent Features in Old Mouse Brain.

Brain aging is characterized by dysfunctional autophagy and cellular senescence, among other features. While autophagy can either promote or suppress cellular senescence in proliferating cells, in postmitotic cells, such as neurons, autophagy impairment promotes cellular senescence. CRM1 (exportin-1/XPO1) exports hundreds of nuclear proteins into the cytoplasm, including the transcription factors TFEB (the main inducer of autophagy and lysosomal biogenesis genes) and STAT3, another autophagy modulator. It appears that CRM1 is a modulator of aging-associated senescence and autophagy, because pharmacological inhibition of CRM1 improved autophagic degradation in flies, by increasing nuclear TFEB levels, and because enhanced CRM1 activity is mechanistically linked to senescence in fibroblasts from Hutchinson–Gilford progeria syndrome patients and old healthy individuals; furthermore, the exogenous overexpression of CRM1 induced senescence in normal fibroblasts. In this work, we tested the hypothesis that impaired autophagic flux during brain aging occurs due to CRM1 accumulation in the brain. We found that CRM1 levels and activity increased in the hippocampus and cortex during physiological aging, which resulted in a decrease of nuclear TFEB and STAT3. Consistent with an autophagic flux impairment, we observed accumulation of the autophagic receptor p62/SQSTM1 in neurons of old mice, which correlated with increased neuronal senescence. Using an in vitro model of neuronal senescence, we demonstrate that CRM1 inhibition improved autophagy flux and reduced SA-β-gal activity by restoring TFEB nuclear localization. Collectively, our data suggest that enhanced CRM1-mediated export of proteins during brain aging perturbs neuronal homeostasis, contributing to autophagy impairment, and neuronal senescence.

Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the β-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibition, which is even sustained after clearance from CRM1 antagonists. Our results indicate CRM1 as a possible therapeutic target in NUP214-related leukemia. This is especially important, since no specific or targeted treatment options for NUP214 driven leukemia are available yet.

Using a Simple Cellular Assay to Map NES Motifs in Cancer-Related Proteins, Gain Insight into CRM1-Mediated NES Export, and Search for NES-Harboring Micropeptides.

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.

Gemcitabine and 5‐FU disrupt nuclear transport and subsequent localization of p21, p27 and p53 in cancer cells

Altered nuclear transport plays a role in the mislocalization of tumor suppressor proteins in certain cancers. In addition, the disruption of nuclear transport and the nuclear pore complex during apoptosis are important mechanisms of action in several different chemotherapies. We previously demonstrated that the chemotherapy 5‐Fluorouracil (5‐FU) alters nuclear transport by disrupting the Ran gradient and increasing nuclear pore permeability. This block in nuclear transport may be advantageous to overcome Crm1‐induced excessive nuclear export in some cancers or with certain cases of drug resistance. Other work has shown that an additional chemotherapy, gemcitabine, is more effective when in combination with Crm1 inhibition. Therefore, we were interested in determining if gemcitabine and 5‐FU work effectively alone and in combination to disrupt nuclear transport via mislocalization of the transport protein Ran. We utilized immunofluorescence to determine the localization of Ran, which is normally localized to the nucleus. We show here that while each of these drugs mislocalized Ran at both 6h and 24h time points, and that gemcitabine counteracts the effects of 5‐FU in combination treatments to induce no change in Ran localization. This may be due to gemcitabine inhibiting 5‐FU's calcium‐mediated mechanism for inducing nuclear pore permeability. Additionally, we were interested in determining how these drugs affected the localization of three cargo proteins regulated by Crm1‐mediated export: p53, p27 and p21. These proteins have important function in the nucleus to control cell cycle progression and induction of apoptosis. Therefore, inhibition of Crm1 export should lead to nuclear accumulation of these factors and a potential increase in their function as tumor suppressors. We show here that 5‐FU and gemcitabine have opposing effects on the nuclear localization of both p53 and p21, and that the drugs again have counteractive effects when in combination treatments. Both 5‐FU and gemcitabine decrease the nuclear localization and expression of p27, while the combination treatment shows an additive effect on both localization and expression. The effects of these drugs on protein localization may be related to an additional block in nuclear import, as well as an induction of nuclear pore permeability downstream of the induction of apoptosis. Further research will help to characterize the effects of 5‐FU and gemcitabine on nuclear transport and subsequent protein localization. This may help to determine how these drugs may be more effectively used in the treatment of specific cancers and in specific drug combinations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reciprocal amplification of caspase-3 activity by nuclear export of a putative human RNA-modifying protein, PUS10 during TRAIL-induced apoptosis

Pus10 is a pseudouridine synthase present in Archaea and Eukarya, but not in Bacteria and yeast. It has been suggested that the human PUS10 (DOBI) gene is needed during TRAIL-induced apoptosis. We analyzed the role of PUS10 in TRAIL-induced apoptosis by immunofluorescence, immunoblotting and several indicators of apoptosis. We examined several TRAIL-sensitive cell lines and we also examined some resistant cell lines after treatment with cycloheximide. PUS10 is mainly present in the nucleus. Early during apoptosis, PUS10 translocates to mitochondria via CRM1-mediated export with the concurrent release of cytochrome c and SMAC. Caspase-3 is required for PUS10 translocation, which reciprocally amplifies the activity of caspase-3 through the intrinsic/mitochondrial pathway. This suggests that in addition to cytoplasmic factors, nuclear factors also have a direct role in the major apoptosis pathways. However, p53 is not involved in TRAIL-induced PUS10 movement. The caspase-3-mediated movement of PUS10 and the release of mitochondrial contents enhancing caspase-3 activity creates a feedback amplification loop for caspase-3 action. Therefore, any defect in the movement or interactions of PUS10 would reduce the TRAIL sensitivity of tumor cells.

Nuclear DDX3 expression predicts poor outcome in colorectal and breast cancer.

PurposeDEAD box protein 3 (DDX3) is an RNA helicase with oncogenic properties that shuttles between the cytoplasm and nucleus. The majority of DDX3 is found in the cytoplasm, but a subset of tumors has distinct nuclear DDX3 localization of yet unknown biological significance. This study aimed to evaluate the significance of and mechanisms behind nuclear DDX3 expression in colorectal and breast cancer.MethodsExpression of nuclear DDX3 and the nuclear exporter chromosome region maintenance 1 (CRM1) was evaluated by immunohistochemistry in 304 colorectal and 292 breast cancer patient samples. Correlations between the subcellular localization of DDX3 and CRM1 and the difference in overall survival between patients with and without nuclear DDX3 were studied. In addition, DDX3 mutants were created for in vitro evaluation of the mechanism behind nuclear retention of DDX3.ResultsDDX3 was present in the nucleus of 35% of colorectal and 48% of breast cancer patient samples and was particularly strong in the nucleolus. Nuclear DDX3 correlated with worse overall survival in both colorectal (hazard ratio [HR] 2.34, P<0.001) and breast cancer (HR 2.39, P=0.004) patients. Colorectal cancers with nuclear DDX3 expression more often had cytoplasmic expression of the nuclear exporter CRM1 (relative risk 1.67, P=0.04). In vitro analysis of DDX3 deletion mutants demonstrated that CRM1-mediated export was most dependent on the N-terminal nuclear export signal.ConclusionOverall, we conclude that nuclear DDX3 is partially CRM1-mediated and predicts worse survival in colorectal and breast cancer patients, putting it forward as a target for therapeutic intervention with DDX3 inhibitors under development in these cancer types.

Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the interaction between annulate lamellae pore complexes and nuclear transport complexes in mammalian cells.

Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler׳s murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions.

A non-canonical mechanism for Crm1-export cargo complex assembly.

The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

Inter-cellular transport of ran GTPase.

Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE). Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2). Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers.

LocNES: a computational tool for locating classical NESs in CRM1 cargo proteins.

Classical nuclear export signals (NESs) are short cognate peptides that direct proteins out of the nucleus via the CRM1-mediated export pathway. CRM1 regulates the localization of hundreds of macromolecules involved in various cellular functions and diseases. Due to the diverse and complex nature of NESs, reliable prediction of the signal remains a challenge despite several attempts made in the last decade. We present a new NES predictor, LocNES. LocNES scans query proteins for NES consensus-fitting peptides and assigns these peptides probability scores using Support Vector Machine model, whose feature set includes amino acid sequence, disorder propensity, and the rank of position-specific scoring matrix score. LocNES demonstrates both higher sensitivity and precision over existing NES prediction tools upon comparative analysis using experimentally identified NESs. LocNES is freely available at http://prodata.swmed.edu/LocNES CONTACT: yuhmin.chook@utsouthwestern.edu Supplementary data are available at Bioinformatics online.

Abstract 2066: Combination therapy of human multiple myeloma using proteosome and CRM1 inhibitors.

Abstract Purpose. Despite improved treatments using proteosome inhibitor- and immunomodulator-based (thalidomide) therapies human multiple myeloma (MM) still remains an incurable disease. New therapeutic targets or unique drug-combinations are needed to further improve treatment outcomes. Our purpose was to investigate the use of small-molecule inhibitors of the nuclear export (SINE) in combination with proteosome inhibitors (PI), bortezomib (BTZ) and carfilzomib (CFZ) as a potential treatment for MM both in vitro and ex vivo. Methods. Previously, we reported that blocking nuclear export of topo IIα with SINE sensitized MM cells to topo II poisons. In this study, we used SINE inhibitors developed by Karyopharm Therapeutics, KPT-185, -249 and a new clinical compound KPT-330. In addition we investigated the use of KOS-2464 (BMS), an effective and relatively non-toxic analog of the known CRM1 inhibitor LMB. To test the efficacy of these inhibitors, we treated human MM cell lines, RPMI-8226, U266B1 and NCI-H929 with SINE +/- PI. MM cell lines or MM patient bone marrow aspirates were placed at high-density conditions and treated for 20 hours with KPT-drugs (300 nM) or KOS-2464 (10 nM), either concurrently or sequentially with 10-40 nM BTZ or CFZ. Cells were then assayed for cell viability by an automated CellTiter-Blue (Promega) analysis. Additional experiments were performed to investigate time-course of drug action and dose-dependence. In addition, treated cell lines or CD138+/light chain+ patient MM cells were assayed for induction of apoptosis by cleaved caspase 3 staining and flow cytometry. To determine if the SINE blocked CRM1-mediated export, treated cells were separated into nuclear and cytoplasmic fractions and assayed for export of p53 and topo IIα. Immunofluorescent microscopy was used to confirm fractionation results. In addition, CRM1 expression levels were measured in all treated cell lines by both Western blot and quantitative PCR. Results. SINE used in combination with PI were highly effective against both MM cell lines and patient MM cells. We found that SINE blocked nuclear export of both p53 and topo IIα as shown by nuclear/cytoplasmic fractionation and also by immunocytochemical staining. Using the CellTiter-Blue assay, we found that all SINE tested had low IC50 values (nanomolar) as single agents and functioned synergistically with BTZ and CFZ (CI &amp;lt;0.5). SINE/PI combination therapy also induced apoptosis in MM cell cultures and patient MM cells (ex vivo), as shown by caspase 3 cleavage and flow cytometric analyses. Conclusions. We found that specific, small-molecule CRM1 inhibitors, sensitized MM cells to BTZ and CFZ as shown by apoptosis and cell viability assays. Sensitization was found to be synergistic. In addition, CRM1 inhibiting activity was verified by nuclear/cytoplasmic fractionation and immunocytochemical staining in MM cells. This combination therapy may be an effective treatment for multiple myeloma. Citation Format: Joel G. Turner, Jana L. Dawson, Christopher L. Cubitt, Michael Kauffman, Sharon Shacham, Daniel M. Sullivan. Combination therapy of human multiple myeloma using proteosome and CRM1 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2066. doi:10.1158/1538-7445.AM2013-2066

CRM1 Inhibition Sensitizes Drug Resistant Human Myeloma Cells to Topoisomerase II and Proteasome Inhibitors both In Vitro and Ex Vivo

Multiple myeloma (MM) remains an incurable disease despite improved treatments, including lenalidomide/pomalidomide and bortezomib/carfilzomib based therapies and high-dose chemotherapy with autologous stem cell rescue. New drug targets are needed to further improve treatment outcomes. Nuclear export of macromolecules is misregulated in many cancers, including in hematological malignancies such as MM. CRM1 (chromosome maintenance protein-1) is a ubiquitous protein that exports large proteins (>40 kDa) from the nucleus to the cytoplasm. We found that small-molecule Selective Inhibitors of Nuclear Export (SINE) prevent CRM1-mediated export of p53 and topoisomerase IIα (topo IIα). SINE's CRM1-inhibiting activity was verified by nuclear-cytoplasmic fractionation and immunocytochemical staining of the CRM1 cargoes p53 and topo IIα in MM cells. We found that SINE molecules reduced cell viability and induced apoptosis when used as both single agents in the sub-micromolar range and when combined with doxorubicin, bortezomib, or carfilzomib but not lenalidomide, melphalan, or dexamethasone. In addition, CRM1 inhibition sensitized MM cell lines and patient myeloma cells to doxorubicin, bortezomib, and carfilzomib but did not affect peripheral blood mononuclear or non-myeloma bone marrow mononuclear cells as shown by cell viability and apoptosis assay. Drug resistance induced by co-culture of myeloma cells with bone marrow stroma cells was circumvented by the addition of SINE molecules. These results support the continued development of SINE for patients with MM.

Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65

The phosphoprotein 65 (pp65) of human cytomegalovirus is a prominent target of the antiviral CD8 T lymphocyte response. This study focused on investigating the properties of pp65 that render it a privileged antigen. It was found that pp65 was metabolically stable. The tegument protein was introduced into MHC class I presentation following its delivery via non-replicating dense bodies. No ubiquitination was found on particle-associated pp65. Proof was obtained that pp65 was a nucleocytoplasmic shuttle protein, using heterokaryon analyses. Based on this finding, inhibition experiments showed that presentation of particle-derived pp65 by HLA-A2 was sensitive to the impairment of the CRM1-mediated nuclear export pathway. The data support the idea that particle-derived pp65 can serve as a nuclear reservoir for proteasomal processing and MHC class I presentation, following its CRM1-dependent nuclear export. The presentation of pp65-derived peptides was also impaired by CRM1-inhibition following de novo synthesis of the tegument protein. However, pp65 protein levels were also reduced when blocking CRM1-mediated export after transient expression. This indicated that pp65 expression rather than direct interference with its own nuclear export was responsible for its reduced presentation in this case. The functionality of CRM1-mediated nuclear export is thus important for the presentation of pp65-derived peptides in the context of MHC class I on organ cells, both after exogenous uptake and after de novo synthesis of the tegument protein, but different mechanisms may account for either case.

CAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

Abstract 1771: KOS-2464, a novel CRM1 inhibitor and a potential drug for multiple myeloma and leukemia

Abstract Purpose: Human multiple myeloma (MM) remains an incurable disease despite improved treatments that include bortezomib- and lenalidomide-based therapies and high-dose chemotherapy with autologous stem cell rescue. New therapeutic targets are needed to further improve treatment outcomes. Our purpose was to investigate the use of a small-molecule inhibitor (KOS-2464) of the nuclear export receptor CRM1 and whether this inhibitor sensitized drug-resistant MM to topoisomerase IIα (topo IIα) inhibitors. Experimental Procedures: In previous studies, we have shown that MM cells possess intrinsic drug resistance to topo II inhibitors due to a CRM1-mediated export of topo IIα in high cell density microenvironments, such as found in the diseased bone marrow. Blocking nuclear export of topo IIα with a CRM1 inhibitor may sensitize MM cells to topo II poisons. In this study, we investigated a novel, small molecule (585 MW), analog of the known CRM1 inhibitor LMB. To test the efficacy of this inhibitor, we treated human MM cell lines, RPMI-8226 and NCI-H929 and AML cell lines Kg1a and HL-60, with KOS-2464 +/− the topo IIα inhibitors doxorubicin (dox) or daunomycin. MM cells were placed at high-density drug resistant conditions and were treated for 20 hours with KOS-2464 (at concentrations of 1 nM, 10 nM and 100 nM), followed by treatment with 2 µM dox or daunomycin for four hours. Cells were then assayed for cell viability by an automated CellTiter-Blue (Promega) analysis. In addition, treated cells were assayed for induction of apoptosis by cleaved caspase 3 staining and flow cytometry. To determine if the CRM1 inhibitor blocked CRM1-mediated export, treated cells were separated into nuclear and cytoplasmic fractions and assayed for export of p53 and topo IIα. Immunofluorescent microscopy was used to confirm fractionation results. Results: KOS-2464 was used in combination with the topo IIα drugs dox and daunomycin to overcome intrinsic drug resistance in MM and AML cells. We found that KOS-2464 blocked nuclear export of both p53 and topo IIα as shown by nuclear/cytoplasmic fractionation and also by immunocytochemical staining. Using the CellTiter-Blue assay, we found that KOS-2464 had low IC50 values (2 nM) as single agent and functioned synergistically with dox (combination index 0.268). KOS-2464/dox combination therapy also induced apoptosis in MM cell cultures, as shown by caspase 3 cleavage and flow cytometric analyses. Conclusions: We found that a specific, small-molecule CRM1 inhibitor KOS-2464, sensitized MM and AML cells to dox and daunomycin as shown by apoptosis and cell viability assays. Sensitization was found to be synergistic. In addition, CRM1 inhibiting activity was verified by nuclear/cytoplasmic fractionation and immunocytochemical staining in MM cells. This compound may be a potential candidate for future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1771. doi:1538-7445.AM2012-1771