XPO1-mediated Nuclear Export Research Articles

Familial Thrombocytopenia-Associated Germline ETV6 P214L Mutation Results in XPO1-Mediated Nuclear Export

In recent years, a number of heterozygous germline mutations in ETV6 have been associated with familial thrombocytopenia and predisposition to hematologic malignancies. While most of these missense mutations occur in the DNA-binding-domain of ETV6, the recurrent P214L mutation is unique in that it occurs in the central disordered region of the protein. The ETV6 P214L mutation has previously been shown to disrupt the nuclear localization of ETV6 and the ability of ETV6 to act as a transcriptional repressor. Unexpectedly, we have found that the P214L missense mutation creates a recognition motif for the nuclear export protein XPO1. We have demonstrated in cellular and protein reconstitution assays that XPO1 directly engages the P214L but not the wild-type ETV6 construct. Consistent with this mechanism, we are able to fully rescue the nuclear localization of ETV6 P214L in mammalian cells with small molecule XPO1 inhibitors. We have also found that once localized to the nucleus, the P214L mutant construct exhibits functional consequences indistinguishable from wild-type ETV6 in Ba/F3 transformation assays. When looking at the evolutionary conservation of the ETV6 amino acid sequence involved in the XPO1 recognition motif, we observed that the synonymous P216L mutation in the mouse ETV6 ortholog does not create a nuclear export signal. In order to study XPO1-mediated export of ETV6 P214L and its impact on hematopoiesis, we have generated a mouse model in which two point mutations (P216L and S211I) have been introduced into a single endogenous ETV6 allele, which precisely matches the sequence of this region in human P214L carriers. Using CRISPR-Cas9, we generated 3 transgenic mouse lines: Etv6 P216L, Etv6 S211I, and Etv6 S211IP216L. Immunofluorescence of fixed bone marrow shows that ETV6 is only mislocalized in megakaryocytes from ETV6 S211I P216L mice. Complete blood counts revealed a significant decrease in platelet counts in ETV6 S211I P216L heterozygotes (485 x 10 3/µL) compared to ETV6 S211I heterozygotes (1140 x 10 3/µL, p<0.0001), ETV6 P216L heterozygotes (1065 x 10 3/µL, p<0.0006), and wild-type littermates (1254 x 10 3/µL, p<0.0001). Flow cytometry phenotyping of bone marrow from the three transgenic lines and wild-type littermates shows a significant decrease in the absolute numbers of hematopoietic stem cells (HSCs) and megakaryocyte precursors (MkPs) in ETV6 S211I P216L mice. In summary, we have determined that XPO1-mediated nuclear export is the mechanistic basis for ETV6 P214L mislocalization. We developed a mouse model that recapitulates this export signal in the endogenous ETV6 allele and found that this model demonstrates a thrombocytopenic phenotype as seen in human patients. Reductions in HSCs and MkPs suggest both a decreased fitness of HSCs, as well as dysregulated megakaryopoiesis in this model.

RNA-binding protein hnRNPU regulates multiple myeloma resistance to selinexor

Multiple myeloma (MM) is an incurable haematological cancer. Selinexor is the first-in-class selective inhibitor of nuclear export (SINE) and was newly approved for the treatment of MM. Until now, very few studies have investigated selinexor resistance in MM. Heterogeneous nuclear ribonucleoprotein U (hnRNPU) is an RNA-binding protein and a component of hnRNP complexes. Here we found that hnRNPU regulates MM sensitivity to selinexor. Cell apoptosis assays were performed to compare selinexor-induced cell death in control knockdown (CTR-KD) and hnRNPU knockdown (hnR-KD) MM cells. HnRNPU knockdown-induced nuclear protein retention was examined by proteomics array. HnRNPU-conferred mRNA translation regulation was evaluated by sucrose gradient assay, RNA electrophoresis mobility shift assay, and RNA pull-down assay. We found that hnR-KD MM cells were more sensitive to selinexor-induced cell death in vitro and in mouse model. MM patients who responded to selinexor had relatively low hnRNPU expression. In brief, hnRNPU comprehensively regulated MM sensitivity to selinexor by affecting the localization of LTV1 and NMD3, and mRNA translation of MDM2 and RAN, which were involved in XPO1-mediated nuclear export of ribosome subunits and tumor suppressors. Our discoveries indicate that hnRNPU might be a possible marker to categorize MM patients for the use of Selinexor.

Use of Combination Therapies Including the XPO1 Inhibitor Selinexor in Is a Potential Effective Therapeutic Strategy to Treat Myelofibrosis Patients

JAK1/2 inhibitor therapy reduces the degree of splenomegaly and improves systemic symptoms of myelofibrosis (MF) patients, but does not delay disease progression and modestly prolongs overall survival. HDM2 antagonists (nutlins) have been reported to selectively eliminate MF CD34 + cells by activating the p53 pathway. Nutlins alone target malignant cells with wild type (WT) p53 and in clinical trials, HDM2 antagonists have led to reductions in spleen size, improvement of systemic symptoms and reduction in MPN driver mutation allelic burdens (Lu et al, Blood.2012; Mascarenhas et al, Blood. 2019). Although most patients with MF have WT TP53, mutations are present in a limited number of such patients and are associated with poor outcomes. We have searched for combinations of drugs to serve as a more effective and tolerable MF stem cell depleting therapy. Exportin 1 (XPO1) mediates the nuclear export of various proteins and RNAs (Azmi, et al. Nat Rev Clin Oncol. 2021) including tumour-suppressor proteins and growth regulators, such as RB1, p53, p63, p73, p21, cyclin B1 or D1 (Azmi, 2021). XPO1 is frequently overexpressed and/or mutated in human cancers and functions as an oncogenic driver. Suppression of XPO1-mediated nuclear export has been shown to be a potential therapeutic strategy to treat MF patients (Yan, et al. Clin Cancer Res. 2019). Selinexor (SEL) is an investigational, oral XPO1 inhibitor that may inhibit multiple pathways relevant in MF including JAK/STAT, ERK, and AKT (Tantravahi, ASH 2021). In the current study, we tested the effects of SEL on apoptosis, and cell cycle arrest in JAK2V617F cells with either TP53WT or TP53mut. We also evaluated the ability of SEL alone to eliminate MF CD34 + cells and in combination with other candidate drugs such as HDM201 (HDM2 antagonist), ruxolitinib (RUX), pan-BET inhibitor (JQ1) or a Bcl-2/Bcl-xL inhibitor (navitoclax). Treatment with SEL alone induced apoptosis of both TP53WT (UKE-1) and TP53mut (SET-2) MPN CD34 + cell lines in a dose-dependent fashion. The transcript levels of TP53 as well as its downstream genes, such as p21, PUMA, NOXA (intrinsic apoptotic pathway) and DR5 and Trail (extrinsic apoptotic pathway) were increased by 2-3 fold after SEL treatment of TP53WT and TP53mut cells. Importantly, SEL also increased TP63 and TP73 by ~2 fold but decreased the transcript levels of the anti-apoptotic Bcl-xL and c-Myc (by 20% and 50%), TP63 and TP73 are TP53 family members which promote p53 downstream events independently of WT p53. In addition, treatment with SEL for 2 days increased the proportion of cells in sub-G0/G1 phase from 16% (control) to 60% in UKE-1 cells and from 15% (control) to 53% in SET-2 cells. SEL and HDM201 alone induced apoptosis of primary MF but not healthy donor (HD) CD34 + cells in a dose dependent fashion. The combination of SEL with suboptimal doses of either HDM201, RUX, JQ1 or navitoclax showed additive pro-apoptotic effects on MF CD34 + cells. Colony formation assays showed that SEL, HDM201 and navitoclax alone decreased CFU-GM colony numbers generated by MF CD34 + cells in a dose-dependent fashion, yet had no effects on HD CD34 + cells, while RUX reduced both MF and ND colony formation. Combination treatment with SEL and 50nM of HDM201 or RUX led to a greater degree of reduction of MF CFU-GM colony numbers, but only SEL+HDM201 did not affect HD CFU-GM colony numbers (Figure 1). Furthermore, JAK2 genotyping showed that treatment with SEL, Rux and HDM201 alone decreased the absolute number of JAK2V167F+ colonies. Furthermore, SEL+HDM201 and SEL+RUX decreased the number of JAK2V617F+ colonies to greater extent than either drug alone, but SEL+HDM201 treatment led to the persistence of a greater numbers of JAK2WT colonies than SEL+RUX (Table 1). SEL+navitoclax did not further decrease JAK2V167F+ colonies than SEL alone (data not showed). These data suggested that SEL induces apoptosis and inhibits MPN CD34 + cell proliferation by affecting both p53-dependent and -independent pathways. SEL alone induces apoptosis and inhibits MF progenitor cells but not HD cells. The effects of SEL combined with either RUX or an HDM2 antagonist more effectively depleted mutated MF CD34+ cells than either drug alone. The combination of SEL+HDM201 spared the reservoir of WT progenitors, and reduced JAK2 mutated progenitor cells to a great degree suggesting that this combination might be associated with less hematological toxicity and greater efficacy.

Targeting XPO1 Combined with Radiotherapy to Enhance Systemic Anti-tumor Effects in Non-Small Cell Lung Cancer

The combination of radiation and radiosensitizing chemotherapeutic agents have shown promising anti-tumor effects in NSCLC. Acting as an oncogenic driver, XPO1 is frequently overexpressed and/or mutated in lung cancer. Thus, suppression of XPO1-mediated nuclear export presents a unique therapeutic strategy. We hypothesize that XPO1 inhibition combined with radiotherapy (XRT) may remodel the tumor immune microenvironment (TIME) and reduce radioresistance, thus enhance systemic anti-tumor effects. Herein, we optimized a small molecule inhibitor, WJ01024, which can bind to XPO1 and antagonize its activity to inhibit nuclear export. In the C57BL/6 mouse subcutaneous tumor model, we evaluated the ability of different treatment regimens containing oral WJ01014 single or combined with XRT (one fractions of 15 Gy) in tumor control and tumor recurrence inhibition. The effects of each treatment regimen on the alterations of immunophenotypes, including the quantification, activation, proliferative capacity, exhaustion marker expression, and memory status, were evaluated by flow cytometry. In our study, we found that the overexpression of XPO1 was associated with poor prognosis and survival in radioresistant patients with NSCLC. The combination therapy of WJ01024 and XRT resulted in an increase of apoptosis and a decrease of proliferation compared to monotherapy, which was closely correlated with tumor regression and improved survival in the C57BL/6 mouse subcutaneous tumor model. Notably, we found that WJ01024 were shown to enhance the therapeutic effect of XRT by remodeling TIME. Compared with XRT, the addition of WJ01024 increased the infiltration and proliferation of radiation-stimulated CD8+ T cells, which especially promoted the production of interferon-γ and granzyme B. Moreover, the combination therapy also reversed the immunosuppressive effect of radiation on the percentage of Tregs and exhausted T cells in mouse xenografts. Thus, the TIME was significantly improved in combination therapy. Strikingly, mechanistic studies suggested that the activation of cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) signaling pathway is required to reshape TIME and produce synergistic anti-tumor effect with the combination of WJ01024 and XRT. Our findings suggest that WJ01024 might be a potential synergistic treatment for radiotherapy to control the proliferation of NSCLC cells, promote tumor regression and prolong survival in mouse model of NSCLC by activating cGAS/STING signaling, and this in turn potentiate the immune microenvironment.

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Differential impact of exportin-1-mediated nuclear export of RNAs on the RNA content of extracellular vesicle subpopulations

Extracellular vesicles (EVs) are membrane-enclosed subcellular structures released by all cell types. EVs have important roles in both cellular homeostasis and intercellular communication. Recent progress in the field revealed substantial heterogeneity of EVs even within the size-based EV categories. Here we addressed the question whether the exportin-1 (XPO1)-mediated nuclear export of RNAs contributed to the EV heterogeneity. Size-based populations were separated from the conditioned media of three cell lines (U937, THP-1 and 5/4E8) in steady-state condition. The effects of activation and leptomycin B treatment (to inhibit the XPO1-mediated nuclear export of RNAs) were also tested in the case of the two monocytic cell lines. Agilent Pico and Small chips were used to characterize RNAs, fragment analysis was performed, and EV-associated miRNAs were tested by Taqman assays. As expected, we found the highest small RNA/total RNA ratio and the lowest rRNA/total RNA proportion in small EVs (~ 50–150 nm). Profiles of the small RNAs within different size-based EV categories significantly differed based on the activation status of the EV releasing cells. Leptomycin B had a differential inhibition on the tested small RNAs in EVs, even within the same EV size category. A similar heterogeneity of the EV miRNA content was observed upon cellular activation and nuclear export inhibition. Here we complement the already existing knowledge on EV heterogeneity by providing evidence that the RNA cargo varies depending on the EV size-based category, the releasing cell type, the functional status of the releasing cells and the exportin-1-mediated nuclear export of RNAs.

1489MO A phase Ib/II study of selinexor as single agent and in combination with imatinib in patients with advanced gastrointestinal stromal tumor (GIST): SeliGIST/GEIS-41 trial

Currently approved agents in metastatic GIST focus on targeted suppression of KIT or PDGFRA oncogenic activation. However, their clinical benefit after imatinib (IM) progression is still modest, suggesting the co-operation of KIT/PDGFRA-independent mechanisms in GIST cell survival. Selinexor is an oral, selective inhibitor of XPO1-mediated nuclear export, and preclinical studies evidenced antitumoral activity in GIST as single agent and in combination with IM in both IM-sensitive and IM-resistant models.

Abstract 1061: Exportin 1 (XPO1) inhibition alone or in combination as a novel therapeutic strategy in osteosarcoma

Abstract Background: Osteosarcoma is an aggressive bone cancer in which therapeutic advancements have been limited over the last 30 years, in part due to genomic heterogeneity. The combination of high-throughput drug screening platforms that efficiently pinpoint drug sensitivities with patient-derived cross-species models is an innovative approach to address the critical need to identify novel treatment strategies for osteosarcoma patients. Methods: We performed high-throughput drug screens on patient-derived osteosarcoma cell lines D418 (canine) and 17-3x (human), followed by validation of the top compounds, to identify drug sensitivities and novel therapeutic combinations. Results: High-throughput drug screens using 2100 bioactive compounds show that osteosarcoma cell lines D418 and 17-3x exhibited sensitivity to standard-of-care chemotherapy drugs, inhibitors of XPO1 nuclear export, and proteasome inhibitors. The XPO1 inhibitor, verdinexor (VER), and the proteasome inhibitor, bortezomib (BORT), induced dose-dependent cytotoxicity in multiple osteosarcoma cell lines (D418, IC50VER: 3187 nM, IC50BORT: 2.8 nM; 17-3x IC50VER: 679 nM, IC50BORT: 10.9 nM). In addition, dual XPO1 and proteasome inhibition synergistically reduced cell proliferation in D418 (synergy score=12.89) and 17-3x (synergy score=17.87) cell lines (p <0.05). Selinexor (SEL), an FDA approved XPO1 inhibitor used in combination with bortezomib to treat multiple myeloma, also demonstrated dose-dependent single-agent activity in patient-derived osteosarcoma cell lines (D418, IC50SEL: 370 nM; 17-3x IC50SEL: 101 nM). With drug screening of 119 oncology compounds in combination with selinexor in 17-3x cells, XPO1 inhibition again shows synergistic activity with proteasome inhibition in osteosarcoma. Conclusions: Inhibition of XPO1-mediated nuclear export is a promising therapeutic strategy in osteosarcoma. These effects may be further potentiated when used in combination with other agents, such as proteasome inhibitors. Additional drug screening and validation assays are underway to identify novel synergistic agents for use in combination with XPO1 inhibitors in osteosarcoma. Citation Format: Laurie Graves, Gabrielle Rupprecht, Erdem Altunel, Etienne M. Flamant, Sneha Rao, Dharshan Sivara, Alexander L. Lazarides, Sarah M. Hoskinson, Maya U. Sheth, Serene Cheng, So Young Kim, Kathryn E. Ware, Anika Agarwal, Mark M. Cullen, Casey Syal, Laura E. Selmic, Jeffrey I. Everitt, Shannon J. McCall, Cindy Eward, Trinayan Kashyap, Marie Maloof, Christopher J. Walker, Yosef Landesman, Lars Wagner, William C. Eward, David S. Hsu, Jason A. Somarelli. Exportin 1 (XPO1) inhibition alone or in combination as a novel therapeutic strategy in osteosarcoma [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 1061.

Clinical Outcomes in Patients (Pts) with Dose Reduction of Selinexor in Combination with Bortezomib, and Dexamethasone (XVd) in Previously Treated Multiple Myeloma from the Boston Study

Clinical Outcomes in Patients (Pts) with Dose Reduction of Selinexor in Combination with Bortezomib, and Dexamethasone (XVd) in Previously Treated Multiple Myeloma from the Boston Study

Reversible disruption of XPO1-mediated nuclear export inhibits respiratory syncytial virus (RSV) replication

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract disease in infants, young children, the elderly and immunocompromised individuals. Therapy for RSV infections is limited to high risk infants and there are no safe and efficacious vaccines. Matrix (M) protein is a major RSV structural protein with a key role in virus assembly. Interestingly, M is localised to the nucleus early in infection and its export into the cytoplasm by the nuclear exporter, exportin-1 (XPO1) is essential for RSV assembly. We have shown previously that chemical inhibition of XPO1 function results in reduced RSV replication. In this study, we have investigated the anti-RSV efficacy of Selective Inhibitor of Nuclear Export (SINE) compounds, KPT-335 and KPT-185. Our data shows that therapeutic administration of the SINE compounds results in reduced RSV titre in human respiratory epithelial cell culture. Within 24 h of treatment, RSV replication and XPO1 expression was reduced, M protein was partially retained in the nucleus, and cell cycle progression was delayed. Notably, the effect of SINE compounds was reversible within 24 h after their removal. Our data show that reversible inhibition of XPO1 can disrupt RSV replication by affecting downstream pathways regulated by the nuclear exporter.

A phase Ib/II study of selinexor in combination with imatinib in patients with advanced gastrointestinal stromal tumor (GIST): SeliGIST/GEIS-41 trial.

11534 Background: KIT or PDGFRA oncogenic activation drives GIST progression throughout the disease course. Accordingly, currently approved agents in metastatic GIST focus on the therapeutic suppression of these receptors. However, the clinical benefit after imatinib (IM) progression is still modest, suggesting the co-operation of KIT/PDGFRA-independent mechanisms in GIST cell survival. Selinexor is an oral, selective inhibitor of XPO1-mediated nuclear export, and preclinical studies evidenced antitumoral activity in GIST as single agent and in combination with IM in both IM-sensitive and IM-resistant models. Methods: The phase Ib portion studied IM 400 mg daily plus weekly selinexor in patients (pts) with IM-resistant, advanced GIST. Prior intolerance to IM was not allowed. A standard 3+3 dosing schema was utilized to determine the recommended phase II dose (RP2D) of this combination. Investigator-assessed response was evaluated every 8 weeks using RECIST 1.1. Results: At data cutoff of Sep 25, 2020, 12 pts were enrolled and received treatment with IM 400 mg and selinexor once weekly at dose levels (DL) 1 (60 mg), DL2 (80 mg) and DL3 (100 mg). Median age 57 (range 46-77), 42% female, median prior therapies 4 (range 2-7). Although only 1/6 pts developed a dose limiting toxicity (DLT) at DL3, the RP2D was defined at DL2 (IM 400 mg daily and selinexor 80 mg once weekly) based on activity data in the DL2 and the need for dose reductions in 5/6 pts at DL3 after the DLT window. All pts were evaluable for toxicity and response. One DLT occurred at DL3 (G3 nausea). Non-DLT G3/4 toxicities were anemia (1/12 pts), neutropenia (1/12 pts), vomiting (1/12 pts) and fatigue (2/12 pts). Common G1/2 toxicities were nausea (11/12 pts), vomiting (10/12 pts), neutropenia (5/12 pts) and anemia, fatigue, diarrhea, and periorbital edema (4/12 pts each). No unexpected toxicities were observed. Overall response rate in the 12 pts evaluable for response was 67% (95% CI 0.349-0.901), with 2 pts achieving PR (17%) and 6 pts SD (50%) as the best response. Clinical benefit rate (CBR = CR, PR, SD) ≥ 16 weeks was 42% (95% CI 0.157-0.723). Median progression free survival was 3.5 months (95% CI 1.7-7.3). Four pts remain on trial at data cutoff. Conclusions: IM and selinexor combination is well-tolerated and has clinical activity in heavily pretreated GIST pts. The trial is currently exploring selinexor as single agent in the IM-resistant GIST population. Clinical trial information: NCT04138381.

Selinexor versus doxorubicin in dedifferentiated liposarcoma PDXs: evidence of greater activity and apoptotic response dependent on p53 nuclear accumulation and survivin down\u2010regulation

BackgroundDedifferentiated liposarcoma (DDLPS), a tumor that lacks effective treatment strategies and is associated with poor outcomes, expresses amplified MDM2 in the presence of wild-type p53. MDM2 ubiquitination of p53 facilitates its XPO1-mediated nuclear export, thus limiting p53 tumor suppressor functions. Consequently, nuclear export is a rational target in DDLPS. We directly compared the antitumor activity of the first-in class XPO1 inhibitor selinexor and doxorubicin, the standard front-line therapy in sarcomas, in DDLPS patient-derived xenografts (PDXs) and primary cell lines.MethodsDrug activity was assessed in three PDXs (and two corresponding cell lines) established from the dedifferentiated component of primary untreated retroperitoneal DDLPS with myogenic (N = 2) and rhabdomyoblastic (N = 1) differentiation from patients who underwent surgery. These models were marked by amplification of MDM2, CDK4 and HMGA2 genes.ResultsSelinexor was moderately active in the three PDXs but achieved greater tumor response compared to doxorubicin (maximum tumor volume inhibition: 46–80 % vs. 37–60 %). The PDX harboring rhabdomyoblastic dedifferentiation showed the highest sensitivity to both agents. PDX response to selinexor and doxorubicin was not associated with the extent of MDM2 and CDK4 gene amplification. Interestingly, the most chemosensitive PDX model showed the lowest extent of HMGA2 amplification. Selinexor was also more efficient than doxorubicinin in inducing an apoptotic response in PDXs and cell lines. Consistently, an increased nuclear accumulation of p53 was seen in all selinexor-treated models. In addition, a time-dependent decrease of survivin expression, with an almost complete abrogation of the cytoplasmic anti-apoptotic pool of this protein, was observed as a consequence of the decreased acetylation/activation of STAT3 and the increased ubiquitination of nuclear survivin.ConclusionsSelinexor showed a moderate antitumor activity in three DDLPS PDXs, which was, however, consistently higher than doxorubicin across all different models regardless the extent of MDM2 amplification and the histological differentiation. The depletion of survivin protein seems to significantly contribute to the induction of apoptosis through which selinexor exerts its antitumor activity.

Exportin-1 (XPO1, CRM1) inhibitors for the management of viral infection

A therapeutic gap exists between new emerging infectious diseases and effective treatments. Targeting host factors involved in the replication strategies of evolutionarily diverse viruses may bridge this gap. Exportin-1 (XPO1, also termed chromosome region maintenance 1 [CRM1]) is a cellular nuclear export receptor for cellular and viral protein and ribonucleoprotein cargos harboring a nuclear export signal. XPO1 inhibition suppresses replication of numerous evolutionarily diverse viruses and one XPO1 inhibitor has entered clinical trials for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the etiologic agent of coronavirus disease-2019 (COVID-19). This review examines current and potential strategies and challenges in targeting XPO1-mediated nuclear export for treating established, emerging and future virus infections. © 2021 Clarivate Analytics.

Novel Therapies for Relapsed or Refractory Diffuse Large B-Cell Lymphoma.

The most common type of non-Hodgkin lymphoma in adults is diffuse large B-cell (DLBCL). There is a historical unmet need for more effective therapies in the 2nd and 3rd line setting. Emerging immunochemotherapies have shown activity in small studies of heavily pre-treated patients with prolonged remissions achieved in some patients. Anti-CD19 CAR (chimeric antigen receptor) T cells are potentially curative in the 3rd line and beyond setting and are under investigation in earlier lines of therapy. Antibody-drug conjugates (ADC’s) such as polatuzumab vedotin targeting the pan-B-cell marker CD79b has proven effectiveness in multiply-relapsed DLBCL patients. Tafasitamab (MOR208) is an anti-CD19 monoclonal antibody producing prolonged remissions when combined with Lenalidomide (LEN) in patients who were not candidates for salvage chemotherapy or autologous stem cell transplant. Selinexor, an oral, small-molecule selective inhibitor of XPO1-mediated nuclear export (SINE), demonstrated prolonged activity against heavily-pretreated DLBCL without cumulative toxicity and is being investigated as part of an oral, chemotherapy-free regimen for relapsed aggressive lymphoma. This article reviews current strategies and novel therapies for relapsed/refractory DLBCL.

CRISPR/Cas9 Chemogenetic Profiling Identifies Candidate Biomarker Genes That Modulate Sensitivity to Selinexor

Introduction Selinexor (KPT-330) is a first-in-class selective inhibitor of XPO1-mediated nuclear export that has recently been approved by the US FDA for the treatment of relapsed/refractory multiple myeloma and relapsed diffuse large B-cell lymphoma. Additionally, the drug is being evaluated in ongoing clinical trials (phase I to III) on late-stage haematological and solid tumours. To improve outcomes and personalize therapeutics, specific biomarkers and underlying mechanisms of response to treatment need to be uncovered. Material and method We performed genome-wide CRISPR/Cas9-mediated loss-of-function genetic screening in two multiple myeloma (KMS-28-BM and SK-MM-1) and one chronic myeloid leukemia (HAP1) cell lines. After transduction with a knockout library, cells were divided and subsequently cultured into three parallel groups (control, IC20 and IC50) for at least 20 cell doublings. This setup allows monitoring of subtle relative changes between groups. After DNA extraction, sequencing of guide RNAs and statistical analysis, we identified genes that upon disruption confer either increased susceptibility or resistance to selinexor. Results and discussion For the multiple myeloma cell lines, the TGF-β/SMAD4 signalling pathway stood out as an important mediator of resistance to selinexor. Besides SMAD4 as a major hit in both cell lines, we identified five other genes from this pathway leading to resistance in KMS-28-BM cells, suggesting that downregulation of this pathway is a potential biomarker of response to SINE treatment in multiple myeloma. In addition, screens revealed significant variability across cell lines with hit genes involved in mRNA processing, proteasomal degradation and cell cycle regulation. Interestingly, we could simultaneously uncover resistance and sensitivity genes involved in the same pathway, suggesting that these have antagonistic effects. Notably, we identified knockout of the poorly characterized gene ASB8 as a strong, common sensitizer between the three cell lines. Conclusion Until now, most chemogenetic CRISPR/Cas9 knockout screens have focused on determining resistance genes, and few have employed the technique to screen for genes that enhance cell kill. We performed such screens with selinexor and found a number of general and cell type-specific hits. Genes identified to confer increased resistance are candidate biomarkers while genes inducing increased susceptibility represent new targets for drug combination therapies. Disclosures Kwanten: Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor). Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Daelemans:Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor).

Telomerase biogenesis requires a novel Mex67 function and a cytoplasmic association with the Sm7 complex.

The templating RNA is the core of the telomerase reverse transcriptase. In Saccharomyces cerevisiae, the complex life cycle and maturation of telomerase includes a cytoplasmic stage. However, timing and reason for this cytoplasmic passage are poorly understood. Here, we use inducible RNA tagging experiments to show that immediately after transcription, newly synthesized telomerase RNAs undergo one round of nucleo-cytoplasmic shuttling. Their export depends entirely on Crm1/Xpo1, whereas re-import is mediated by Kap122 plus redundant, kinetically less efficient import pathways. Strikingly, Mex67 is essential to stabilize newly transcribed RNA before Xpo1-mediated nuclear export. The results further show that the Sm7 complex associates with and stabilizes the telomerase RNA in the cytoplasm and promotes its nuclear re-import. Remarkably, after this cytoplasmic passage, the nuclear stability of telomerase RNA no longer depends on Mex67. These results underscore the utility of inducible RNA tagging and challenge current models of telomerase maturation.

Novel carfilzomib-based combinations as potential therapeutic strategies for liposarcomas

Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib’s efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.

Weekly selinexor, bortezomib, and dexamethasone (SVd) versus twice weekly bortezomib and dexamethasone (Vd) in patients with multiple myeloma (MM) after one to three prior therapies: Initial results of the phase III BOSTON study.

8501 Background: Selinexor is an oral, selective inhibitor of XPO1-mediated nuclear export, leading to the reactivation of tumor suppressor proteins. In a phase 1b/2 study, the combination of once weekly (QW) selinexor with bortezomib and dexamethasone (SVd) was well tolerated with anti-MM activity in patients (pts) with PI-sensitive and PI-refractory disease. While twice weekly (BIW) bortezomib in combination therapy is efficacious, prolonged use is limited due to peripheral neuropathy (PN, 50-60%). The BOSTON study was designed to determine if SVd improves progression free survival (PFS), overall response rates (ORR) and reduces the rate of PN vs Vd. Methods: BOSTON is a global, phase 3, randomized study of QW SVd vs BIW Vd after 1-3 prior anti-MM regimens. The primary endpoint is PFS. Secondary endpoints include ORR, overall survival (OS) and PN (rates and EORTC QLQ-CIPN20 outcomes). Randomization is stratified by treatment with prior PI therapies, number of prior anti-MM regimens (1 vs > 1), and Revised International Staging System (R-ISS; Stage III vs I or II). Following confirmation of progressive disease, pts on Vd could cross over to either: 1) SVd for pts able to tolerate continued bortezomib or 2) selinexor and dexamethasone for pts with bortezomib intolerance. Results:402 pts were enrolled; 195 and 207 to SVd and Vd, respectively. Median age was 67 (range: 38-90). Most (59.6%) pts were > 65 years and 57.1% were male. R-ISS stage at the time of MM diagnosis was III for 18.5% of pts. Baseline characteristics were balanced across the 2 arms. SVd significantly prolonged PFS vs Vd (median 13.93 vs 9.46 months, HR = 0.70, P = 0.0066). SVd was associated with a significantly higher ORR (76.4% vs 62.3%, P = 0.0012). Median OS was not reached on SVd vs 25 months on Vd (P = 0.28). Most frequent treatment-related adverse events (grade ≥3) for SVd vs Vd were thrombocytopenia (35.9% vs 15.2%), fatigue (11.3% vs 0.5%) and nausea (7.7% vs 0%). Clinically important differences were reported on the motor, autonomic and sensory scales on CIPN20. PN rates (grade ≥2) were significantly lower with SVd vs Vd (21.0% vs 34.3%, P = 0.0013). Conclusions: BOSTON is the first phase 3 study to evaluate the clinical benefit of SVd for relapsed/refractory MM. The study met the primary endpoint: once weekly SVd significantly improved PFS and ORR compared to twice weekly Vd. Rates of PN were significantly reduced with numerically fewer deaths on SVd vs Vd. Full dataset will be presented at the meeting. Clinical trial information: NCT03110562 .

Upregulation of let-7f-2-3p by long noncoding RNA NEAT1 inhibits XPO1-mediated HAX-1 nuclear export in both in vitro and in vivo rodent models of doxorubicin-induced cardiotoxicity.

Clinical application of doxorubicin (Dox) is limited due to its undesirable side effects, especially cardiotoxicity. Several microRNAs (miRNAs) such as microRNA-140-5p and miR-23a aggravate Dox-induced cardiotoxicity. Here we demonstrate that upregulation of miRNA let-7f-2-3p by long noncoding RNA (lncRNA) NEAT1 inhibits exportin-1 (XPO1)-mediated nuclear export of hematopoietic-substrate-1 associated protein X-1 (HAX-1) in Dox-induced cardiotoxicity. Treatment of the H9c2 cells with the Dox (1μM) for 6h inhibited HAX-1 nuclear export and decreased XPO1 expression. Overexpression of XPO1 significantly attenuated the Dox-induced leakage of myocardial enzymes (creatine phosphokinase, creatine kinase-MB and lactate dehydrogenase) and cardiomyocyte apoptosis with the increased HAX-1 nuclear export. Differentially expressed miRNAs including let-7f-2-3p were selected from the Dox or vehicle-treated cardiomyocytes. TargetScan and luciferase assay showed that let-7f-2-3p targeted XPO1 3' UTR. Inhibition of let-7f-2-3p reduced Dox-induced cardiotoxicity and apoptosis by inhibiting XPO1-mediated HAX-1 nuclear export, whereas let-7f-2-3p overexpression aggravated these effects. In addition, lncRNA NEAT1 was identified as an endogenous sponge RNA to repress let-7f-2-3p expression. Overexpression of lncRNA NEAT1 abolished the increased let-7f-2-3p expression by Dox, and thereby attenuated cardiotoxicity. The loss function of let-7f-2-3p increased XPO1-mediated HAX-1 nuclear export and reduced myocardial injury in Dox (20mg/kg)-treated rats. Importantly, let-7f-2-3p inhibition in mice alleviated Dox-induced cardiotoxicity and preserved the antitumor efficacy. Together, let-7f-2-3p regulated by lncRNA NEAT1 aggravates Dox-induced cardiotoxicity through inhibiting XPO1-mediated HAX-1 nuclear export, and may serve as a potential therapeutic target against Dox-induced cardiotoxicity.

NEDD4 protects vascular endothelial cells against Angiotensin II-induced cell death via enhancement of XPO1-mediated nuclear export

NEDD4 is an E3 ubiquitin ligase containing the HECT domain, which regulates various cellular processes, but its role in vascular endothelial cells is unknown. In the present study, we found that NEDD4 bound directly to XPO1 by co-immunoprecipitation screening. In HUVECs (human umbilical vein endothelial cells), overexpression of NEDD4 reduced Ang II-induced ROS level and cell apoptosis. Ang II stimulation led to nuclear accumulation of cargoes, while overexpression of NEDD4 enhanced the XPO1-dependent nuclear export of its cargoes. KPT185, an inhibitor of XPO1, can abolished the protective effect of NEDD4 under Ang II treatment. In addition, NEDD4 could promote the interaction between XPO1 and RanBP3 via K63-linked ubiquitination of XPO1. These results suggested that NEDD4 played a protective role in vascular endothelial cell injury through regulating XPO1-mediated nuclear export.