XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53-p21 and p27 pathways.

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression.

The first-in-class inhibitor of exportin-1 (XPO1) selinexor is currently under clinical investigation in combination with the BTK inhibitor ibrutinib for patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkin lymphoma. Selinexor induces apoptosis of tumour cells through nuclear retention of tumour suppressor proteins and has also recently been described to modulate natural killer (NK) cell and T cell cytotoxicity against lymphoma cells. Here, we demonstrate that XPO1 inhibition enhances NK cell effector function against primary CLL cells via downregulation of HLA-E and upregulation of TRAIL death receptors DR4 and DR5. Furthermore, selinexor potentiates NK cell activation against CLL cells in combination with several approved treatments; acalabrutinib, rituximab and obinutuzumab. We further demonstrate that lymph node associated signals (IL-4 + CD40L) inhibit NK cell activation against CLL cells via upregulation of HLA-E, and that inhibition of XPO1 can overcome this protective effect. These findings allow for the design of more efficacious combination strategies to harness NK cell effector functions against CLL.

Exportin(g) precision oncology beyond genomics with XPO1 inhibition.

We urgently need to identify new druggable targets for pediatric cancers. Coutinho et al.1 identified and validated XPO1 aberrant activity in patients with Wilms tumor (WT) and malignant rhabdoid tumor (MRT) using public databases and report an early proof-of-concept bench-to-bedside translation of XPO1 inhibition in pediatric WT and MRT.

P2RY2-AKT activation is a therapeutically actionable consequence of XPO1 inhibition in acute myeloid leukemia.

Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.

CRM1 Promotes Capsid Disassembly and Nuclear Envelope Translocation of Adenovirus Independently of Its Export Function.

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.

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.

Bench to bedside radiosensitizer development strategy for newly diagnosed glioblastoma.

Glioblastoma is the most common primary brain malignancy and carries with it a poor prognosis. New agents are urgently needed, however nearly all Phase III trials of GBM patients of the past 25 years have failed to demonstrate improvement in outcomes. In 2019, the National Cancer Institute Clinical Trials and Translational Research Advisory Committee (CTAC) Glioblastoma Working Group (GBM WG) identified 5 broad areas of research thought to be important in the development of new herapeutics for GBM. Among those was optimizing radioresponse for GBM in situ. One such strategy to increase radiation efficacy is the addition of a radiosensitizer to improve the therapeutic ratio by enhancing tumor sensitivity while ideally having minimal to no effect on normal tissue. Historically the majority of trials using radiosensitizers have been unsuccessful, but they provide important guidance in what is required to develop agents more efficiently. Improved target selection is essential for a drug to provide maximal benefit, and once that target is identified it must be validated through pre-clinical studies. Careful selection of appropriate in vitro and in vivo models to demonstrate increased radiosensitivity and suitable bioavailability are then necessary to prove that a drug warrants advancement to clinical investigation. Once investigational agents are validated pre-clinically, patient trials require consistency both in terms of planning study design as well as reporting efficacy and toxicity in order to assess the potential benefit of the drug. Through this paper we hope to outline strategies for developing effective radiosensitizers against GBM using as models the examples of XPO1 inhibitors and HDAC inhibitors developed from our own lab.

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.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

PSEN1-selective gamma-secretase inhibition in combination with kinase or XPO-1 inhibitors effectively targets T cell acute lymphoblastic leukemia.

BACKGROUND: T cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype that comprises 10-15% of childhood and 20-25% of adult ALL cases. Over 70% of T-ALL patients harbor activating mutations in the NOTCH1 signaling pathway and are predicted to be sensitive to gamma-secretase inhibitors. We have recently demonstrated that selective inhibition of PSEN1-containing gamma-secretase complexes can overcome the dose-limiting toxicity associated with broad gamma-secretase inhibitors. In this study, we developed combination treatment strategies with the PSEN1-selective gamma-secretase inhibitor MRK-560 and other targeted agents (kinase inhibitors ruxolitinib and imatinib; XPO-1 inhibitor KPT-8602/eltanexor) for the treatment of T-ALL. METHODS: We treated T-ALL cell lines in vitro and T-ALL patient-derived xenograft (PDX) models in vivo with MRK-560 alone or in combination with other targeted inhibitors (ruxolitinib, imatinib or KPT-8602/eltanexor). We determined effects on proliferation of the cell lines and leukemia development and survival in the PDX models. RESULTS: All NOTCH1-signaling-dependent T-ALL cell lines were sensitive to MRK-560 and its combination with ruxolitinib or imatinib in JAK1- or ABL1-dependent cell lines synergistically inhibited leukemia proliferation. We also observed strong synergy between MRK-560 and KPT-8602 (eltanexor) in all NOTCH1-dependent T-ALL cell lines. Such synergy was also observed in vivo in a variety of T-ALL PDX models with NOTCH1 or FBXW7 mutations. Combination treatment significantly reduced leukemic infiltration in vivo and resulted in a survival benefit when compared to single treatment groups. We did not observe weight loss or goblet cell hyperplasia in single drug or combination treated mice when compared to control. CONCLUSIONS: These data demonstrate that the antileukemic effect of PSEN1-selective gamma-secretase inhibition can be synergistically enhanced by the addition of other targeted inhibitors. The combination of MRK-560 with KPT-8602 is a highly effective treatment combination, which circumvents the need for the identification of additional mutations and provides a clear survival benefit in vivo. These promising preclinical data warrant further development of combination treatment strategies for T-ALL based on PSEN1-selective gamma-secretase inhibition.

Selinexor, a novel selective inhibitor of nuclear export, reduces SARS-CoV-2 infection and protects the respiratory system in vivo.

The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1ß, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option.

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.

Discovery and biological evaluation of a small-molecule inhibitor of CRM1 that suppresses the growth of triple-negative breast cancer cells.

Dysregulation of the nuclear export machinery mediated by chromosomal maintenance 1 (CRM1, also known as exportin-1), is closely associated with various human disorders, such as breast cancer. Previously, we identified sulforaphene and its synthetic analogues as covalent inhibitors of CRM1. Herein, we describe the discovery and biological evaluation of another sulforaphene synthetic analogue, LFS-31, as a potential CRM1 inhibitor. In addition, we investigated the reversible binding mechanism of LFS-31 with CRM1 through molecular simulations coupled with bio-layer interferometry (BLI) and found relatively high binding affinity (KD = 43.1 ± 35.3 nM) between the LFS-31 and CRM1 groups. We found that LFS-31 exhibited a stronger growth suppression of triple-negative breast cancer (TNBC) cells than non-TNBC cells, and had minimal effect on normal breast cells. Pharmacological treatment of TNBC cells with LFS-31 at nanomolar concentrations led to the nuclear retention of IkBα resulting in strong suppression of NF-κB transcriptional activity and attenuated cell growth and proliferation, which collectively contributed to the antitumor responses. To the best of our knowledge, this is the first study to demonstrate the use of a sulforaphene analogue as a potent CRM1 inhibitor that targets the NF-κB signaling pathway for the targeted therapy of TNBC.

Cancer Therapy with Nanoparticle-Medicated Intracellular Expression of Peptide CRM1-Inhibitor.

INTRODUCTION: Peptides can be rationally designed as non-covalent inhibitors for molecularly targeted therapy. However, it remains challenging to efficiently deliver the peptides into the targeted cells, which often severely affects their therapeutic efficiency. METHODS: Herein, we created a novel non-covalent peptide inhibitor against nuclear export factor CRM1 by a structure-guided drug design method and targetedly delivered the peptide into cancer cells by a nanoparticle-mediated gene expression system for use as a cancer therapy. RESULTS: The nuclear export signal (NES)-optimized CRM1 peptide inhibitor colocalized with CRM1 to the nuclear envelope and inhibited nuclear export in cancer cell lines in vitro. The crystal structures of the inhibitors complexed with CRM1 were solved. In contrast to the covalent inhibitors, the peptides were similarly effective against cells harboring the CRM1 C528S mutation. Moreover, a plasmid encoding the peptides was delivered by a iRGD-modified nanoparticle to efficiently target and transfect the cancer cells in vivo after intravenous administration. The peptides could be selectively expressed in the tumor, resulting in the efficient inhibition of subcutaneous melanoma xenografts without obvious systemic toxicity. DISCUSSION: This work provides an effective strategy to design peptide-based molecularly targeted therapeutics, which could lead to the development of future targeted therapy.

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-1α via inhibition of CRM1 in human osteosarcoma and hepatoma cells associated with an increased radiosensitivity.

BACKGROUND: The 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. METHODS: Human 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. RESULTS: Selinexor 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. CONCLUSION: Targeting the HIF pathway by Selinexor might be an attractive tool to overcome hypoxia-induced radioresistance.

Selinexor in combination with topotecan in patients with advanced or metastatic solid tumors: Results of an open-label, single-center, multi-arm phase Ib study.

Background Selinexor, a first-in-class, oral selective inhibitor of nuclear export (SINE) compound inhibits Exportin-1(XPO1), had demonstrated synergistic activity with many chemotherapies and conferred in vivo antitumor efficacy in hematologic as well as solid tumors. Methods This open-label, single-center, multi-arm phase 1b study used a standard 3 + 3 design and a "basket type" expansion. Selinexor with intravenous topotecan was given in one of the 13 parallel arms. Patients with advanced or metastatic relapsed/refractory solid tumors following prior systemic therapy, or in whom the addition of selinexor to standard chemotherapy deemed appropriate, were eligible. Results Fourteen patients with the median age of 61 years (range, 22-68years) were treated, and the most common cancer types were gynecological cancers; ovarian (n = 5), endometrial (n = 2), and 1 each with fallopian tube and vaginal cancers. Of the 14 patients treated, 12 (86 %) had at least one treatment-related adverse event (TRAE). The most common TRAEs were anemia (71 %), thrombocytopenia (57 %), hyponatremia (57 %), vomiting (57 %), fatigue (50 %), nausea (50 %), and neutropenia (36 %). Two patients had dose limiting toxicities. One patient dosed at selinexor 80 mg had grade 3 nausea and vomiting and one patient dosed at selinexor 60 mg experienced grade 4 neutropenia and thrombocytopenia. Of the 13 efficacy evaluable patients, one (8 %) with endometrial cancer achieved unconfirmed partial response (uPR) and the time-to-treatment failure (TTF) was 48 weeks, whereas 6 of the 13 (46 %) patients had stable disease (SD) contributing to the clinical benefit rate of 46 %. The median TTF for all patients was 9 weeks (range, 2-48weeks). Conclusions Once weekly selinexor in combination with topotecan was viable and showed some preliminary tumor efficacy. The recommend phase 2 dose of selinexor was 60 mg once weekly in combination with IV topotecan.Trial registration: NCT02419495. Registered 14 April 2015, https://clinicaltrials.gov/ct2/show/NCT02419495.

Chemoproteomic Profiling of Covalent XPO1 Inhibitors to Assess Target Engagement and Selectivity.

Selinexor, a covalent XPO1 inhibitor, is approved in the USA in combination with dexamethasone for penta-refractory multiple myeloma. Additional XPO1 covalent inhibitors are currently in clinical trials for multiple diseases including hematologic malignancies, solid tumor malignancies, glioblastoma multiforme (GBM), and amyotrophic lateral sclerosis (ALS). It is important to measure the target engagement and selectivity of covalent inhibitors to understand the degree of engagement needed for efficacy, while avoiding both mechanism-based and off-target toxicity. Herein, we report clickable probes based on the XPO1 inhibitors selinexor and eltanexor for the labeling of XPO1 in live cells to assess target engagement and selectivity. We used mass spectrometry-based chemoproteomic workflows to profile the proteome-wide selectivity of selinexor and eltanexor and show that they are highly selective for XPO1. Thermal profiling analysis of selinexor further offers an orthogonal approach to measure XPO1 engagement in live cells. We believe these probes and assays will serve as useful tools to further interrogate the biology of XPO1 and its inhibition in cellular and in vivo systems.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer.

BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.