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.

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.

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.

Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds.

BACKGROUND: Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330. METHODS: Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays. RESULTS: Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in >100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental and resistant cells, the extent of response was more robust in the parental cells. CONCLUSIONS: These results suggest that SINE resistance is conferred by alterations in signaling pathways downstream of XPO1 inhibition. Modulation of these pathways could potentially overcome the resistance to nuclear export inhibitors.

Biologic activity of the novel orally bioavailable selective inhibitor of nuclear export (SINE) KPT-335 against canine melanoma cell lines.

BACKGROUND: Exportin 1 (XPO1, also known as CRM1), is a chaperone protein responsible for the export of over 200 target proteins out of the nucleus. The expression and activity of XPO1 is upregulated in several human cancers and its expression is also linked to the development of chemotherapy resistance. Recent studies using both human and murine cancer cell lines have demonstrated that XPO1 is a relevant target for therapeutic intervention. The present study sought to characterize the biologic activity of an orally bioavailable selective inhibitor of nuclear export (SINE), KPT-335, against canine melanoma cell lines as a prelude to future clinical trials in dogs with melanoma. RESULTS: We evaluated the effects of KPT-335 on 4 canine malignant melanoma cell lines and found that KPT-335 inhibited proliferation, blocked colony formation, and induced apoptosis of treated cells at biologically relevant concentrations of drug. Additionally, KPT-335 downregulated XPO1 protein while inducing a concomitant increase in XPO1 messenger RNA. Lastly, KPT-335 treatment of cell lines upregulated the expression of both protein and mRNA for the tumor suppressor proteins p53 and p21, and promoted their nuclear localization. CONCLUSIONS: KPT-335 demonstrates biologic activity against canine melanoma cell lines at physiologically relevant doses, suggesting that KPT-335 may represent a viable treatment option for dogs with malignant melanoma.

The calcium/calcineurin pathway promotes hemidesmosome stability through inhibition of ß4 integrin phosphorylation.

Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the ß4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in ß4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates ß4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase ß4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates ß4 phosphorylation. We found no direct dephosphorylation of ß4 by calcineurin or association between ß4 and calcineurin, suggesting indirect regulation of ß4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate ß4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced ß4 phosphorylation, suggesting that calcineurin restricts ß4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca(2+)](i) reduces ß4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on ß4. However, MAPK activation is increased when [Ca(2+)](i) is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced ß4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased ß4 phosphorylation, an increase in [Ca(2+)](i) using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce ß4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy.

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.

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.

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.

Role of ß4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelia to the basement membrane. During squamous cell carcinoma (SCC) invasion, there is a reduction in the number of HDs, which may facilitate dissemination. Mechanisms of HD disassembly are incompletely understood. Previous work has shown that epidermal growth factor (EGF)-induced phosphorylation of the ß4 integrin on three of its serines, S(1356)S(1360)S(1364), can induce HD disassembly in normal cells. Here, we examine the role of ß4 integrin serine phosphorylation in SCC. We have found that around 60% of invasive cutaneous SCC show increased ß4 phosphorylation on S(1356) when compared with carcinoma in situ or normal tissue. To assess the mechanisms by which SCC increases ß4 phosphorylation, we performed in vitro analyses. Compared with keratinocytes, SCC cells showed increased levels of S(1356) phosphorylation in the absence of EGF, correlating with reduced HD-like structures. In addition, phospho-S(1356) signal was largely segregated from other HD components. Epidermal growth factor receptor and PKC inhibitors inhibited basal levels of S(1356) phosphorylation in SCC, suggesting that cells use intrinsic mechanisms to activate the EGF signaling pathway to induce ß4 phosphorylation. Moreover, these inhibitors stabilized HD-like structures in SCC cells and reduced their migratory ability. Mutation of S(1356)S(1360)S(1364) in SCC cells to non-phosphorylatable alanines stabilized HD-like structures and substantially reduced migration, while mutation into phosphorylation mimicking aspartate reduced HD-like structures but had no effect on migration, suggesting that serine phosphorylation function is releasing anchorage rather than promoting migration. Altogether these results suggest that ß4 serine phosphorylation may have an important role during SCC invasion by destabilizing HDs and facilitating migration.

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.

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.

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.

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.

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.

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.

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.

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.

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.