HAPLN1 confers multiple myeloma cell resistance to several classes of therapeutic drugs.

Multiple myeloma (MM), a malignant plasma cell infiltration of the bone marrow, is generally considered incurable: resistance to multiple therapeutic drugs inevitably arises from tumor cell-intrinsic and tumor microenvironment (TME)-mediated mechanisms. Here we report that the proteoglycan tandem repeat 1 (PTR1) domain of the TME matrix protein, hyaluronan and proteoglycan link protein 1 (HAPLN1), induces a host of cell survival genes in MM cells and variable resistance to different classes of clinical drugs, including certain proteasome inhibitors, steroids, immunomodulatory drugs, and DNA damaging agents, in several MM cell lines tested. Collectively, our study identifies HAPLN1 as an extracellular matrix factor that can simultaneously confer MM cell resistance to multiple therapeutic drugs.

Selinexor inhibits growth of patient derived chordomas in vivo as a single agent and in combination with abemaciclib through diverse mechanisms.

Chordoma is a rare cancer that grows in the base of the skull and along the mobile spine from remnants of embryonic notochord tissue. The cornerstone of current treatments is surgical excision with adjuvant radiation therapy, although complete surgical removal is not always possible. Chordomas have high rates of metastasis and recurrence, with no approved targeted agents. Selinexor and eltanexor are selective inhibitors of nuclear export (SINE) that prevent the karyopherin protein exportin-1 (XPO1) from shuttling its cargo proteins through nuclear pore complexes out of the nucleus and into the cytoplasm. As cancer cells overexpress XPO1, and many of its cargos include tumor suppressor proteins and complexes bound to oncogene mRNAs, XPO1 inhibition can suppress oncogene translation and restore tumor suppressor protein activity in different cancer types. SINE compounds have exhibited anti-cancer activity in a wide range of hematological and solid tumor malignancies. Here we demonstrate the preclinical effectiveness of SINE compounds used as single agents or in combination with either the proteasome inhibitor, bortezomib, or the CDK4/6 inhibitor, abemaciclib, against various patient- derived xenograft (PDX) mouse models of chordoma, which included clival and sacral chordomas from adult or pediatric patients with either primary or metastatic disease, with either differentiated or poorly differentiated subtypes. SINE treatment significantly impaired tumor growth in all five tested chordoma models, with the selinexor and abemaciclib combination showing the strongest activity (tumor growth inhibition of 78-92%). Immunohistochemistry analysis of excised tumors revealed that selinexor treatment resulted in marked induction of apoptosis and reduced cell proliferation, as well as nuclear accumulation of SMAD4, and reduction of Brachyury and YAP1. RNA sequencing showed selinexor treatment resulted in differences in activated and repressed signaling pathways between the PDX models, including changes in WNT signaling, E2F pathways and glucocorticoid receptor signaling. This is consistent with SINE-compound mediated XPO1 inhibition exhibiting anti-cancer activity through a broad range of different mechanisms in different molecular chordoma subsets. Our findings validate the need for further investigation into selinexor as a targeted therapeutic for chordoma, especially in combination with abemaciclib.

A Case Report of a 58-Year-Old Woman with a Diagnosis of High-Risk Myeloma Refractory to Multiple Line of Therapy and Treated with Selinexor, Bortezomib, and Dexamethasone Prior to Allogeneic Stem Cell Transplantation.

BACKGROUND Approximately 10% to 15% of patients with multiple myeloma (MM) are diagnosed with high-risk disease and have poor prognosis. Clinical trial data supports the combined use of selinexor, bortezomib, and dexamethasone (XVd) for treatment of patients with MM who have received at least 1 prior therapy. Information on the efficacy of XVd and of subsequent allogeneic stem cell transplantation (SCT) in heavily pretreated patients with high-risk MM is limited. CASE REPORT We present a case of a 58-year-old woman with high-risk MM (revised International Staging System Stage III; serum ß2-microglobulin; 8.0 mg/L; and presence of del[17p]) who had received 8 prior treatment lines, and whose disease was refractory to ixazomib, bortezomib, and all immunomodulatory agents. Before initiating XVd (once weekly 1.3 mg/m² bortezomib subcutaneously, 80 mg selinexor per os, and 40 mg dexamethasone per os), the patient had severely hypoplastic bone marrow and was transfusion dependent. After 1 cycle of XVd, she achieved a partial response, and after 4 cycles, a very good partial response (VGPR). No adverse reactions to selinexor were observed. Because of the VGPR, a haploidentical transplant was planned. At posttransplant week 4, the patient had become transfusion independent. She remained relapse-free for 13 months after initiating XVd. Maintenance treatment with lenalidomide was initiated, and following receipt of donor lymphocyte infusions due to loss of donor chimerism, the patient's light chain levels improved. CONCLUSIONS This report presents the cytogenetics and management of a heavily pretreated patient with high-risk MM treated with SVd followed by SCT.

Small Molecule Inhibitors of Nuclear Export and the Amelioration of Lupus by Modulation of Plasma Cell Generation and Survival.

OBJECTIVE: To investigate the hypothesis that selective inhibitors of nuclear export (SINE compounds), recently approved for treatment of refractory plasma cell (PC) malignancy, may have potential in the treatment of lupus. METHODS: Female NZB/NZW mice were treated with the SINE compound KPT-350 or vehicle control. Tissue specimens were harvested and analyzed by flow cytometry, using standard markers. Nephritis was monitored by determining the proteinuria score and by histologic analysis of kidney specimens. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay, and total numbers of IgG-secreting and dsDNA-specific antibody-secreting cells were assessed by enzyme-linked immunospot assay. RESULTS: KPT-350 abrogated murine lupus nephritis at both early and late stages of the disease and rapidly impaired generation of autoreactive PCs in germinal centers (GCs). SINE compounds inhibited the production of NF-κB-driven homeostatic chemokines by stromal cells, altering splenic B and T cell strategic positioning and significantly reducing follicular helper T cell, GC B cell, and autoreactive PC counts. KPT-350 also decreased levels of cytokines and chemokines involved in PC survival and recruitment in the kidney of lupus-prone mice. Exportin 1, the target of SINE compounds, was detected in GCs of human tonsils, splenic B cells of lupus patients, and multiple B cell subsets in the kidneys of patients with lupus nephritis. CONCLUSION: Collectively, our results provide support for the therapeutic potential of SINE compounds, via their targeting of several molecular and cellular pathways critical in lupus pathogenesis, including autoantibody production by plasma cells.

Nuclear Export Inhibitor KPT-8602 Synergizes with PARP Inhibitors in Escalating Apoptosis in Castration Resistant Cancer Cells.

Aberrant nuclear protein transport, often observed in cancer, causes mislocalization-dependent inactivation of critical cellular proteins. Earlier we showed that overexpression of exportin 1 is linked to higher grade and Gleason score in metastatic castration resistant prostate cancer (mCRPC). We also showed that a selective inhibitor of nuclear export (SINE) selinexor and second generation eltanexor (KPT-8602) could suppress mCRPC growth, reduce androgen receptor (AR), and re-sensitize to androgen deprivation therapy. Here we evaluated the combination of KPT-8602 with PARP inhibitors (PARPi) olaparib, veliparib and rucaparib in 22rv1 mCRPC cells. KPT-8602 synergized with PARPi (CI < 1) at pharmacologically relevant concentrations. KPT-8602-PARPi showed superior induction of apoptosis compared to single agent treatment and caused up-regulation of pro-apoptotic genes BAX, TP53 and CASPASE 9. Mechanistically, KPT-8602-PARPi suppressed AR, ARv7, PSA and AR targets FOXA1 and UBE2C. Western blot analysis revealed significant down-regulation of AR, ARv7, UBE2C, SAM68, FOXA1 and upregulation of cleaved PARP and cleaved CASPASE 3. KPT-8602 with or without olaparib was shown to reduce homologous recombination-regulated DNA damage response targets including BRCA1, BRCA2, CHEK1, EXO1, BLM, RAD51, LIG1, XRCC3 and RMI2. Taken together, this study revealed the therapeutic potential of a novel combination of KPT-8602 and PARP inhibitors for the treatment of mCRPC.

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.

Venetoclax response is enhanced by selective inhibitor of nuclear export compounds in hematologic malignancies.

The selective inhibitor of nuclear export (SINE) compounds selinexor (KPT-330) and eltanexor (KPT-8602) are from a novel class of small molecules that target exportin-1 (XPO1 [CRM1]), an essential nucleo-cytoplasmic transport protein responsible for the nuclear export of major tumor suppressor proteins and growth regulators such as p53, p21, and p27. XPO1 also affects the translation of messenger RNAs for critical oncogenes, including MYC, BCL2, MCL1, and BCL6, by blocking the export of the translation initiation factor eIF4E. Early trials with venetoclax (ABT-199), a potent, selective inhibitor of BCL2, have revealed responses across a variety of hematologic malignancies. However, many tumors are not responsive to venetoclax. We used models of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) to determine in vitro and in vivo responses to treatment with venetoclax and SINE compounds combined. Cotreatment with venetoclax and SINE compounds demonstrated loss of viability in multiple cell lines. Further in vitro analyses showed that this enhanced cell death was the result of an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays, coinciding with activation of p53 and loss of MCL1. Treatment with SINE compounds and venetoclax combined led to a reduction in tumor growth in both AML and DLBCL xenografts. Immunohistochemical analysis of tissue sections revealed that the reduction in tumor cells was partly the result of an induction of apoptosis. The enhanced effects of this combination were validated in primary AML and DLBCL patient cells. Our studies reveal synergy with SINE compounds and venetoclax in aggressive hematologic malignancies and provide a rationale for pursuing this approach in a clinical trial.

Exportin 1 Inhibition Induces Nerve Growth Factor Receptor Expression to Inhibit the NF-κB Pathway in Preclinical Models of Pediatric High-Grade Glioma.

High-grade glioma (HGG) is the leading cause of cancer-related death among children. Selinexor, an orally bioavailable, reversible inhibitor of the nuclear export protein, exportin 1, is in clinical trials for a range of cancers, including HGG. It inhibits the NF-κB pathway and strongly induces the expression of nerve growth factor receptor (NGFR) in preclinical cancer models. We hypothesized that selinexor inhibits NF-κB via upregulation of NGFR. In HGG cells, sensitivity to selinexor correlated with increased induction of cell surface NGFR expression. Knocking down NGFR in HGG cells increased proliferation, anchorage-independent growth, stemness markers, and levels of transcriptionally available nuclear NF-κB not bound to IκB-α, while decreasing apoptosis and sensitivity to selinexor. Increasing IκB-α levels in NGFR knockdown cells restored sensitivity to selinexor. Overexpression of NGFR using cDNA reduced levels of free nuclear NF-κB, decreased stemness markers, and increased markers of cellular differentiation. In all HGG lines tested, selinexor decreased phosphorylation of NF-κB at serine 536 (a site associated with increased transcription of proliferative and inflammatory genes). Because resistance to selinexor monotherapy occurred in our in vivo model, we screened selinexor with a panel of FDA-approved anticancer agents. Bortezomib, a proteasome inhibitor that inhibits the NF-κB pathway through a different mechanism than selinexor, showed synergy with selinexor against HGG in vitro Our results help elucidate selinexor's mechanism of action and identify NGFR as a potential biomarker of its effect in HGG and in addition suggest a combination therapy strategy for these challenging tumors.

Down-regulation of AR splice variants through XPO1 suppression contributes to the inhibition of prostate cancer progression.

Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC.

Selinexor reduces the expression of DNA damage repair proteins and sensitizes cancer cells to DNA damaging agents.

INTRODUCTION: The goal of this study was to examine the effects of selinexor, an inhibitor of exportin-1 mediated nuclear export, on DNA damage repair and to evaluate the cytotoxic effects of selinexor in combination with DNA damaging agents (DDAs) in cancer cells. RESULTS: Selinexor reduced the expression of DNA damage repair (DDR) proteins. This did not induce significant DNA damage in tested cell lines. Inhibition of DDR protein expression resulted in enhanced cancer cell death when cells were pretreated with DDAs. In contrast, enhanced cell death was not detected in cells that were pretreated with selinexor then with DDAs. In vivo, single-agent selinexor, docetaxel, or cisplatin treatment resulted in 66.7%, 51.5%, and 26.6% tumor growth inhibition (TGI), respectively, in an MDA-MB-231 xenograft model. Consequently, combination treatment with docetaxel or cisplatin followed by selinexor in vivo resulted in 93.9% and 103.4% TGI, respectively. Immunohistochemical staining and immunoblot analysis of tumor sections confirmed reduced expression of DDR proteins. CONCLUSION: Selinexor treatment inhibited DDR mechanisms in cancer cell lines and therefore potentiated DNA damage-based therapy. The sequential combination of DDAs followed by selinexor increased cancer cell death. This combination is superior to each individual therapy and has a mechanistic rationale as a novel anticancer strategy. METHODS: Cancer cells treated with selinexor ± DDAs were analyzed using reverse phase protein arrays, immunoblots, quantitative PCR and immunofluorescence. Mice bearing MDA-MB-231 tumors were treated with subtherapeutic doses of selinexor, cisplatin, docetaxel and selinexor in combination with either cisplatin or docetaxel. Tumor growth was evaluated for 25 days.

Targeting the XPO1-dependent nuclear export of E2F7 reverses anthracycline resistance in head and neck squamous cell carcinomas.

Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)-dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer.

Selinexor synergizes with dexamethasone to repress mTORC1 signaling and induce multiple myeloma cell death.

Multiple myeloma (MM) is a plasma cell neoplasm that results in over 11,000 deaths in the United States annually. The backbone therapy for the treatment of MM patients almost always includes combinations with corticosteroids such as dexamethasone (DEX). We found that DEX in combination with selinexor, an inhibitor of exportin-1 (XPO1) activity, synergistically inhibits the mTOR pathway and subsequently promotes cell death in MM cells. Specifically, we show that selinexor induces the expression of the glucocorticoid receptor (GR) and when combined with dexamethasone increases GR transcriptional activity. Moreover, we found that key downstream targets of the mTOR pathway are deregulated by the combination and identified a mechanism in which GR enhances the expression of REDD1 in GR positive cells while suppressing mTOR activity and cell viability. While the single agent activity of selinexor in MM cells appears to be GR-independent, synergy with DEX depends on GR expression. These data suggest that patients with tumor cells that are GR positive will benefit substantially from the combination. The current findings are consistent with the beneficial therapeutic outcome in patients with MM when treated with the combination of selinexor and DEX. In addition, they provide a rationale for testing GR and REDD1 as predictive and prognostic markers of response, respectively, for patients treated with this beneficial combination.

Selective Inhibitors of Nuclear Export (SINE) compounds block proliferation and migration of triple negative breast cancer cells by restoring expression of ARRDC3.

Arrestin-related domain-containing protein-3 (ARRDC3) is one of 6 mammalian arrestins, which suppresses metastasis by inducing degradation of phosphorylated ß2-adrenergic receptor (ß2 AR) and integrin ß4 (ITG ß4). Our previous studies demonstrated that expression of ARRDC3 is epigentically silenced in Triple Negative Breast Cancer (TNBC) cells, and the forced expression of ARRDC3 significantly reduced the invasive potential of TNBC cells. In the current study, we found that Selective Inhibitors of Nuclear Export (SINE) compounds (KPT-185 and selinexor (KPT-330)) restore ARRDC3 expression in TNBC cell lines (MDA-MB-231 and MDA-MB-468) at both the mRNA and protein level in a dose and time course dependent manner. SINE compounds inhibit the proliferation, pro-invasive migration and anchorage independent growth of the TNBC cells by restoring ARRDC3 expression. We found that ARRDC3 expression is lower in TNBC cell lines than those of luminal breast cancer cell lines, and inversely correlated with IC50s of selinexor. Analysis of tissue microarray confirmed that ARRDC3 expression in patient samples is significantly lower in the majority of TNBC tumors relative to normal tissue. In vivo, selinexor inhibited the tumor growth of MDA-MB-231 xenografts by nearly 100% compared with vehicle treated animals. Furthermore, immunohistochemical analysis of TNBC tumors from selinexor treated mice revealed increased ARRDC3 expression versus vehicle treated animals. Our results suggest that restoration of ARRDC3 expression is an important antineoplastic mechanism of SINE compounds in TNBC, and therefore selinexor could be an effective treatment option for breast tumors with down-regulated ARRDC3.

Selinexor-induced thrombocytopenia results from inhibition of thrombopoietin signaling in early megakaryopoiesis.

Selinexor is the first oral selective inhibitor of nuclear export compound tested for cancer treatment. Selinexor has demonstrated a safety therapy profile with broad antitumor activity against solid and hematological malignancies in phases 2 and 3 clinical trials (#NCT03071276, #NCT02343042, #NCT02227251, #NCT03110562, and #NCT02606461). Although selinexor shows promising efficacy, its primary adverse effect is high-grade thrombocytopenia. Therefore, we aimed to identify the mechanism of selinexor-induced thrombocytopenia to relieve it and improve its clinical management. We determined that selinexor causes thrombocytopenia by blocking thrombopoietin (TPO) signaling and therefore differentiation of stem cells into megakaryocytes. We then used both in vitro and in vivo models and patient samples to show that selinexor-induced thrombocytopenia is indeed reversible when TPO agonists are administered in the absence of selinexor (drug holiday). In sum, these data reveal (1) the mechanism of selinexor-induced thrombocytopenia, (2) an effective way to reverse the dose-limiting thrombocytopenia, and (3) a novel role for XPO1 in megakaryopoiesis. The improved selinexor dosing regimen described herein is crucial to help reduce thrombocytopenia in selinexor patients, allowing them to continue their course of chemotherapy and have the best chance of survival. This trial was registered at www.clinicaltrials.gov as #NCT01607905.

A phase 1 clinical trial of single-agent selinexor in acute myeloid leukemia.

Selinexor is a novel, first-in-class, selective inhibitor of nuclear export compound, which blocks exportin 1 (XPO1) function, leads to nuclear accumulation of tumor suppressor proteins, and induces cancer cell death. A phase 1 dose-escalation study was initiated to examine the safety and efficacy of selinexor in patients with advanced hematological malignancies. Ninety-five patients with relapsed or refractory acute myeloid leukemia (AML) were enrolled between January 2013 and June 2014 to receive 4, 8, or 10 doses of selinexor in a 21- or 28-day cycle. The most frequently reported adverse events (AEs) in patients with AML were grade 1 or 2 constitutional and gastrointestinal toxicities, which were generally manageable with supportive care. The only nonhematological grade 3/4 AE, occurring in >5% of the patient population, was fatigue (14%). There were no reported dose-limiting toxicities or evidence of cumulative toxicity. The recommended phase 2 dose was established at 60 mg (∼35 mg/m2) given twice weekly in a 4-week cycle based on the totality of safety and efficacy data. Overall, 14% of the 81 evaluable patients achieved an objective response (OR) and 31% percent showed ≥50% decrease in bone marrow blasts from baseline. Patients achieving an OR had a significant improvement in median progression-free survival (PFS) (5.1 vs 1.3 months; P = .008; hazard ratio [HR], 3.1) and overall survival (9.7 vs 2.7 months; P = .01; HR, 3.1) compared with nonresponders. These findings suggest that selinexor is safe as a monotherapy in patients with relapsed or refractory AML and have informed subsequent phase 2 clinical development. This trial was registered at www.clinicaltrials.gov as #NCT01607892.

Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T-cell Effector Function in Mice: Implications for Combination with Immunotherapy.

Selinexor (KPT-330) is a first-in-class nuclear transport inhibitor currently in clinical trials as an anticancer agent. To determine how selinexor might affect antitumor immunity, we analyzed immune homeostasis in mice treated with selinexor and found disruptions in T-cell development, a progressive loss of CD8 T cells, and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by selinexor, suggesting that normal immune homeostasis could recover. We found that a high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week did not restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that nuclear exportins are required for T-cell development and function. We determined the minimum concentration of selinexor required to block T-cell activation and showed that T-cell-inhibitory effects of selinexor occur at levels above 100 nmol/L, corresponding to the first 24 hours post-oral dosing. In a model of implantable melanoma, selinexor treatment at 10 mg/kg with a 4-day drug holiday led to intratumoral IFNγ+, granzyme B+ cytotoxic CD8 T cells that were comparable with vehicle-treated mice. Overall, selinexor treatment leads to transient inhibition of T-cell activation, but clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T-cell functioning and development of antitumor immunity. Mol Cancer Ther; 16(3); 428-39. ©2017 AACRSee related article by Farren et al., p. 417.

The Exportin-1 Inhibitor Selinexor Exerts Superior Antitumor Activity when Combined with T-Cell Checkpoint Inhibitors.

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth in vivo We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. In vitro, selinexor increased PDCD1 and CTLA4 gene expression in leukocytes and induced CD274 gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies (P < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells (P ≤ 0.050) and CD4+ T cells with a Th1 phenotype (P ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. Mol Cancer Ther; 16(3); 417-27. ©2017 AACRSee related article by Tyler et al., p. 428.

XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma.

Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays. IκBα knockdown and NFκB activity were measured in selinexor/bortezomib-treated MM cells. We found that selinexor restored sensitivity of PI-resistant MM to bortezomib and carfilzomib. Selinexor/bortezomib treatment inhibited PI-resistant MM tumor growth and increased survival in mice. Myeloma cells from PI-refractory MM patients were sensitized by selinexor to bortezomib and carfilzomib without affecting non-myeloma cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increases in total and nuclear IκBα by selinexor/bortezomib. Proximity ligation found increased IκBα-NFκB complexes in treated MM cells. IκBα knockdown abrogated selinexor/bortezomib-induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFκB transcriptional activity. Selinexor, when used with bortezomib or carfilzomib, has the potential to overcome PI drug resistance in MM. Sensitization may be due to inactivation of the NFκB pathway by IκBα.

Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death.

The nuclear export protein, exportin-1 (XPO1/CRM1), is overexpressed in many cancers and correlates with poor prognosis. Selinexor, a first-in-class Selective Inhibitor of Nuclear Export (SINE) compound, binds covalently to XPO1 and blocks its function. Treatment of cancer cells with selinexor results in nuclear retention of major tumor suppressor proteins and cell cycle regulators, leading to growth arrest and apoptosis. Recently, we described the selection of SINE compound resistant cells and reported elevated expression of inflammation-related genes in these cells. Here, we demonstrated that NF-κB transcriptional activity is up-regulated in cells that are naturally resistant or have acquired resistance to SINE compounds. Resistance to SINE compounds was created by knockdown of the cellular NF-κB inhibitor, IκB-α. Combination treatment of selinexor with proteasome inhibitors decreased NF-κB activity, sensitized SINE compound resistant cells and showed synergistic cytotoxicity in vitro and in vivo. Furthermore, we showed that selinexor inhibited NF-κB activity by blocking phosphorylation of the IκB-α and the NF-κB p65 subunits, protecting IκB-α from proteasome degradation and trapping IκB-α in the nucleus to suppress NF-κB activity. Therefore, combination treatment of selinexor with a proteasome inhibitor may be beneficial to patients with resistance to either single-agent.

XPO1 Inhibition using Selinexor Synergizes with Chemotherapy in Acute Myeloid Leukemia by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus.

PURPOSE: Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. EXPERIMENTAL DESIGN: The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. RESULTS: Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy. CONCLUSIONS: Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC-dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142-52. ©2016 AACR.