<div>Abstract<p>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 <i>NGFR</i> 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 <i>NGFR</i> 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 <i>in vivo</i> 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 <i>in vitro</i>. 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.</p></div>
Abstract Introduction: Selinexor is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. XPO1 also mediates the export of key signaling molecules in multiple myeloma (MM) including c-MYC mRNA and NF-κB. Selinexor is currently being investigated in phase 2 clinical trials in MM in combination with dexamethasone, pomalidamide, lenalidomide, bortezomib and carfilzomib. In preclinical studies selinexor has been shown to synergize broadly with these anti-MM drugs making it an excellent candidate partner for combination therapies in MM. To identify additional synergistic pairings, we investigated the use of selinexor in combination with panobinostat, a pan-histone deacetylase inhibitor (HDAC) recently approved by the FDA in combination with bortezomib for 3rd line treatment of MM, as a potential treatment for MM both in-vitro and in-vivo. Methods: The effects of selinexor and panobinostat alone or in combination on cell viability were tested on MM1.S and NCI-H929 cell lines using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, MM.1S cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of selinexor and panobinostat alone or in combination and with the therapeutic dose of selinexor. Tumor growth was monitored for 17 days and% Tumor Growth Inhibition (%TGI) was determined. Xenografts were harvested and analyzed microscopically and by immunohistochemestry (IHC). Results: Selinexor-panobinostat combination was highly effective both in-vitro and in-vivo. In MTT assays, both selinexor and panobinostat demonstrated low IC50 values (nanomolar) and when combined, they synergistically/ additively inhibit proliferation. Gene expression and western blot analyses showed that the combination treatment leads to a synergistic reduction in c-MYC mRNA and protein levels. In addition, an overall reduction in expression of anti-apoptotic signaling molecules including NF-κB and p21 and an increase of expression of pro-apoptotic molecules including cleaved caspase 3 and BAX were observed. Importantly, in-vivo, while%TGI of sub-therapeutic doses of selinexor and panobinostat measured 58% and 52% respectively, the combination showed a synergistic effect, measuring 93%. Importantly this high%TGI exceeded the effect of the therapeutic dose of selinexor alone 79%. Conclusion: Selinexor-panobinostat combination synergizes to induce apoptosis in MM cells and amplifies anti-tumor effect in a MM xenograft model. These data provide rational support for study of selinexor/ panobinostat combination in clinical trials. Citation Format: Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4720.
Abstract Background: Melanoma is the most deadly skin cancer and represents 4.6% of all cancers diagnosed within the US (American Cancer Society). Risk factors for melanoma are almost entirely related to pale skin color and sunburns. Selinexor is a clinically approved oral inhibitor of nuclear exportation protein Exportin 1 (XPO1/CRM1). In preclinical studies, selinexor significantly inhibited growth of A375 and CHL-1 human melanoma cell lines in vivo at well tolerated doses (Yang et al. 2014). Interleukin 12 (IL-12) is a cytokine produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells in response to antigenic stimulation. IL-12 drives the differentiation of T-cells into T helper (Th1) cells, in addition to stimulating T-cell and NK cell activity, specifically through the activation of interferon-γ (IFN-γ). IL-12 exerts antitumor activity through IFN-γ-dependent and independent mechanisms, which include modulation of the immune system and anti-angiogenesis (Zhang et al. 2016). The objective of this study was to compare the impact and scheduling of recombinant mouse IL-12 alone and in combination with selinexor on the growth of murine B16F10 melanoma cells grown as subcutaneous tumors in C57B/6 mice. Methods: Sixty, 6-7 weeks old female C57B/6 mice were inoculated subcutaneously in the left flank with 1 x 105 B16F10 cells. Treatment was initiated when the tumors reached a mean volume of 100 mm3 (standard deviation ± 6.3 mm3). Mice were allocated to 6 groups of 10 such that mean tumor volume in each group was within the range of 11.8 to 13.4 mm3, then treated with: oral selinexor (10mg/kg, twice weekly on day 0 and 2) combined with a pre-dose of subcutaneous IL-12 (1µg/kg, once weekly on day -2); selinexor with post-dose IL-12 (1µg/kg, once weekly on day 3); selinexor alone; IL-12 alone (day -2); IL-12 alone (day 3); or vehicle. Animal weight was recorded daily, and tumors were measured 3 days a week to record percent of tumor growth inhibition (%TGI). Results: Both selinexor and IL-12 demonstrated single agent activity against B16F10 tumor in vivo. All treatment groups showed significant tumor growth inhibition as compared to vehicle. The group receiving selinexor with pre-dose IL-12 showed the largest tumor inhibition (%TGI=92%). Significant weight loss compared to vehicle occurred only in the single agent selinexor group (p=0.039). Conclusions: Synergistic anti-cancer activity of selinexor and IL-12 warrants further clinical investigation. Further mechanistic studies to understand the effects of IL-12 priming on increasing the effectiveness of selinexor will be undertaken. Citation Format: Shira Orr, Leah Henegar, Christopher J. Walker, Feng Wang, Trinayan Kashyap, Marie Maloof, Kathleen Martyn, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Selinexor synergizes with IL-12 to inhibit tumor growth in syngeneic mouse models of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1122.
Abstract Introduction: Chordoma is an ultra-rare cancer found in the base of the skull and the mobile spine that originates from embryonic remnants of the notochord. It is often resistant to standard chemotherapy and most systemic anti-cancer treatment, and surgical removal followed by radiation therapy remains the mainstay of current treatment. However, due to the difficult location of chordoma, complete surgical removal is not always possible. In addition, chordoma has a high rate of metastasis and recurrence. Therefore, novel and effective treatment options for chordoma are needed. Selective inhibitor of nuclear export (SINE) compounds selinexor and eltanexor are a class of novel oral drugs that target XPO1 (exportin-1/ CRM1) and exhibit anti-cancer activity across a wide range of solid and hematological malignancies. In July 2019, selinexor was approved by the US FDA to treat patients with multiple myeloma. To investigate the preclinical efficacy and tolerability of SINE compounds in chordoma, two PDX (patient derived xenograft) mouse models of chordoma were tested. Methods: Two SINE drugs, selinexor at 5 mg/kg x 4 times a week and eltanexor at 10 mg/kg x 5 times a week, alone or in combination with bortezomib were used to treat chordoma CF466 and SF8894 PDX mouse models. CF466 PDX was derived from a patient with metastatic sacral chordoma whereas SF8894 PDX was derived from a patient with recurrent clival chordoma. Tumor volume and mouse body weight were monitored during the study. Tumors were collected at the end of 6 weeks of the study for histological, and immunohistochemistry (IHC)-based biomarker analyses. Results: Both selinexor and eltanexor demonstrated potent anti-cancer activity compared to controls. Tumor growth inhibition (TGI) of selinexor was 58% and 78% in the CF466 and SF8894 models, respectively, and TGI of eltanexor was 55% in the SF8894 model. No significant difference in body weight was observed among different treatment groups. Bortezomib did not exhibit anti-tumor activity in these models nor did it have additive/synergistic effects when combined with selinexor or eltanexor. CF466 tumors from mice treated with selinexor showed increased apoptosis, decreased cell proliferation and lowered cell density when compared with the controls. In addition, IHC analysis showed that selinexor treatment increased nuclear retention of eIF4E and tumor suppressor proteins APC, SMAD4 and FOXO3A, and decreased the expression of proteins that may play a role in chordoma tumor biology, such as SHH, GLI1 and SOX9. Conclusions: SINE compounds effectively inhibit tumor growth in PDX mouse models of chordoma. The anti-cancer effects are likely achieved through regulation of multiple signaling pathways. Further investigation of SINE compounds as treatment options for chordoma is warranted. Citation Format: Hua Chang, Leah Henegar, Trinayan Kashyap, Thaddeus J. Unger, Sharon Shacham, Josh Sommer, Patty Cogswell, Stacy Fechner, Michael J. Wick, Joan Levy, Yosef Landesman. SINE compounds demonstrated potent anti-cancer activity in PDX mouse models of chordoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3086.
Abstract Background: Pediatric high-grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG) account for the majority of pediatric brain tumor deaths and respond poorly to chemotherapy. Selinexor, a nuclear export inhibitor, is effective against HGG and DIPG in in vitro and in vivo models, but resistance to treatment develops. We previously identified the NF-κB pathway as a likely mediator of selinexor’s activity in these tumors. NF-κB transcriptional activity is regulated by an inhibitor, IKB-α, whose levels are in turn regulated by ubiquitination and proteasomal degradation. IKB-α is a client of exportin-1 (XPO1); its nuclear levels are increased by selinexor treatment, leading to inhibition of NF-κB. We subsequently identified proteasome inhibitors as potentially synergistic with selinexor in HGG and DIPG through a screen of all FDA-approved chemotherapy agents. Proteasome inhibition has also been shown to synergize with selinexor treatment in multiple myeloma and osteosarcoma. Methods: We treated HGG cell lines (BT245 and GBM1) and DIPG cell lines (DIPG4, DIPG7 and SF7761) for five days with selinexor in combination with each of three proteasome inhibitors, bortezomib, carfilzomib and marizomib, and assayed cell viability at the conclusion of treatment. In each experiment, cells were treated with selinexor, a proteasome inhibitor, and a combination of the two drugs at several constant ratios. IC50 values were computed for each drug acting alone, and the combination index (CI) of the two drugs acting together was computed using the Chou-Talalay method. We also treated SF7761 cells with a combination of radiation (8Gy), selinexor, and a proteasome inhibitor. Results: The proteasome inhibitors had widely varying IC50 values in the cell lines treated, ranging from 1nM to 5µM. The CI for the combination of selinexor and each proteasome inhibitor was consistently less than 1 (indicating a synergistic relationship) in the cell lines tested. We found that radiation and proteasome inhibition had an antagonistic relationship (CI>1), radiation and selinexor a synergistic relationship (CI<1), and the combination of all three was mixed, with some combinations being synergistic and others antagonistic. Conclusions: Selinexor and proteasome inhibitors show promise as a combination therapy for HGG and DIPG. We are conducting in vivo experiments to further explore this combination for subsequent clinical trial use. Citation Format: John DeSisto, Patrick Flannery, Trinayan Kashyap, Rakeb Lemma, Shelby Mestnik, Andrew Kung, Rajeev Vibhakar, Yosef Landesman, Adam Green. Synergistic effects of the XPO1 inhibitor selinexor with proteasome inhibitors in pediatric high-grade glioma and diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2017-1946
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.
