Selective Inhibitor Of Nuclear Export Compound Research Articles

Abstract 2086: Effect of XPO1 inhibition on colorectal cancer tumorigenesis

Abstract Colorectal Cancer (CRC) is the second leading cause of cancer-related death in the U.S. A subset of individuals faces a notably higher likelihood of developing CRC within their lifetime. Hence, there is a compelling need for innovative chemopreventive treatments aimed at minimizing CRC tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. The overexpression of XPO1 has been identified across several cancers, including CRC. XPO1 interacts with a diverse spectrum of more than 1000 proteins. The overexpression of XPO1 leads to the excessive removal of tumor suppressors from the nucleus, rendering these proteins functionally inert. To counteract the overexpression of XPO1 in tumors, a novel class of drugs known as Selective Inhibitors of Nuclear Export (SINE) has been developed. Among these, Eltanexor (KPT-8602) has emerged as a promising therapeutic agent, demonstrating fewer side effects compared to its precursors, and is currently under evaluation in Phase 1/2 clinical trials. The treatment effects of SINE compounds on CRC remain largely unknown. We have assessed Eltanexor's ability to limit CRC tumorigenesis using a genetic CRC model in Apcmin/+ mice. Our findings indicate that Eltanexor substantially reduces tumor burden by approximately 3-fold and induces a significant reduction of tumors exceeding 1mm in size. Furthermore, a xenograft experiment using the human CRC cell line, HCT116, reveals nearly a 3-fold reduction in tumor size upon Eltanexor treatment. In both CRC models, Eltanexor exhibits noteworthy tolerability. Additionally, XPO1 protein levels are consistently diminished in both models, aligning with our in vitro observations. To further ascertain Eltanexor's specificity for CRC tumors versus normal tissue, we conducted drug sensitivity assays using organoids derived from Apcmin/+ tumors and wild-type mice small intestine tissue. We found that Apcmin/+ tumoroids have heightened sensitivity to Eltanexor-treatments, when compared to wild-type organoids. In an endeavor to elucidate Eltanexor's potent in vivo tumorigenesis inhibitory effects, we conducted multiple assays which revealed a reduction in COX-2 RNA and protein expression levels in Eltanexor-treated cells and reduced COX-2 protein in the tumors of Eltanexor-treated Apcmin/+ mice. In CRC, COX-2 is implicated in CRC development and is currently the target of multiple CRC chemopreventive agents. Our observations reveal that Eltanexor-treated CRC cells exhibit reduced COX-2 gene expression through both inhibiting COX-2’s promoter and its mRNA post-transcriptional stability. Collectively, our findings underscore XPO1 as a potent target for inhibiting CRC tumorigenesis. Future studies will aim to further assess how Eltanexor is impacting COX-2’s promoter, while additionally testing Eltanexor’s tumorigenesis-inhibiting potential in a colitis-induced CRC mouse model. Citation Format: Andrew E. Evans, Dan A. Dixon. Effect of XPO1 inhibition on colorectal cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2086.

The Clinical Menin Inhibitor Ziftomenib and the Nuclear Export Inhibitor Selinexor Synergistically Inhibit the Growth of MLL-r AML

CONCLUSION Menin is a scaffold protein that interacts with oncogenic histone-lysine-N-methyltransferase MLL1 (KMT2A)-fusion protein (FP) complex in MLL-rearranged ( MLL-r) AML. Menin inhibitors that evict menin from this interaction have been shown to be active against MLL-r and NPM1MT leukemia by modulating the expression of the leukemogenic homeobox A9 ( HOXA9) gene and its co-factor, MEIS1. Recent evidence showed that menin inhibitors can activate a tumor-suppressive network via a non-canonical transcriptional program through UTX. On the other hand, selective inhibitor of nuclear export (SINE) against XPO1/CRM1 has been shown to have robust anti-leukemia activity via enrichment of tumor suppressor proteins in the nucleus. In the present study, we hypothesized that menin and nuclear export inhibition would synergistically suppress AML cell proliferation and possibly sensitize menin inhibitor-resistant cells. We have used the clinical-stage menin inhibitor ziftomenib and SINE compound selinexor to simultaneously target menin-KMT2A protein-protein interactions and nuclear export. To assess growth inhibition, an ATP-based cell proliferation assay was used. Combination synergy was determined using CalcuSyn Version 2.0 synergy software. Stem-like progenitor cells were isolated using the StemSpan CD34+ expansion kit (StemCell Tech). Colony formation efficiency was determined using a Methocult assay (StemCell Tech). Gene and protein expression was detected using quantitative real-time PCR and western blotting. Flow cytometric analysis was performed to detect cell death and cell cycle status. We have performed the whole transcriptomic analysis via the RNA sequencing approach. The clinical candidate drug ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11 and MOLM13) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor stem cells derived from primary patient samples but was not toxic to normal CD34+ human hematopoietic progenitor stem cells (HPSCs). Menin inhibitor treatment caused the downregulation of menin protein along with its downstream targets HOXA9, MEIS1, and FLT3 in western blot analysis. Interestingly, selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination enhanced menin down-expression, suggesting a molecular basis for the synergy between the two compounds. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. The combination showed increased CD11b, a marker of monocytic differentiation in MV4-11 treated cells. The combination also significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. Compared to controls, ziftomenib and selinexor-treated samples individually showed increased apoptotic and decreased G2/M populations, significantly enhanced in the combination. We observed differential expression of certain mRNAs and micro RNAs (miRs) in MV4-11 treated cells. The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by ziftomenib and selinexor, which are enhanced further in the combination group. On the contrary, CENPK, CKS2, KIF18A, and H2BC4 mRNAs were downregulated by a single agent or combination. Among miRs, hsa-miR-34a-5p, hsa-miR-142-3p, hsa-miR-26b-5p were upregulated, and hsa-miR-33b-3p, hsa-miR-4454, hsa-miR-210-3p and hsa-miR-130b-3p [reported to be an essential lineage-specific codriver of MLL-AF4 leukemia] were downregulated by single agent or combination. Such mRNAs and miRs panels could serve as biomarkers for treatment response. MV4-11 cell line-derived xenografts in NSG mice showed a significant difference in survival in the combination arm compared to any single agent. Metronomic dosing also showed robust improvement in survival in the combination arm. Patient-derived xenografted NSG mice model using human primary MLLr re-transplantable leukemia cells showed improved survival in the combination arm [Fig. 1B]. These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export is a viable strategy for the treatment of MLL-r AML. Ongoing experiments with functional proteomic analysis will be presented at the ASH meeting.

Impacting T-cell fitness in multiple myeloma: potential roles for selinexor and XPO1 inhibitors.

Competent T-cells with sufficient levels of fitness combat cancer formation and progression. In multiple myeloma (MM), T-cell exhaustion is caused by several factors including tumor burden, constant immune activation due to chronic disease, age, nutritional status, and certain MM treatments such as alkylating agents and proteasome inhibitors. Many currently used therapies, including bispecific T-cell engagers, anti-CD38 antibodies, proteasome inhibitors, and CART-cells, directly or indirectly depend on the anti-cancer activity of T-cells. Reduced T-cell fitness not only diminishes immune defenses, increasing patient susceptibility to opportunistic infections, but can impact effectiveness MM therapy effectiveness, bringing into focus sequencing strategies that could modulate T-cell fitness and potentially optimize overall benefit and clinical outcomes. Certain targeted agents used to treat MM, such as selective inhibitors of nuclear export (SINE) compounds, have the potential to mitigate T-cell exhaustion. Herein referred to as XPO1 inhibitors, SINE compounds inhibit the nuclear export protein exportin 1 (XPO1), which leads to nuclear retention and activation of tumor suppressor proteins and downregulation of oncoprotein expression. The XPO1 inhibitors selinexor and eltanexor reduced T-cell exhaustion in cell lines and animal models, suggesting their potential role in revitalizating these key effector cells. Additional clinical studies are needed to understand how T-cell fitness is impacted by diseases and therapeutic factors in MM, to potentially facilitate the optimal use of available treatments that depend on, and impact, T-cell function. This review summarizes the importance of T-cell fitness and the potential to optimize treatment using T-cell engaging therapies with a focus on XPO1 inhibitors.

Nuclear Export in Non-Hodgkin Lymphoma and Implications for Targeted XPO1 Inhibitors.

Exportin-1 (XPO1) is a key player in the nuclear export pathway and is overexpressed in almost all cancers. This is especially relevant for non-Hodgkin lymphoma (NHL), where high XPO1 expression is associated with poor prognosis due to its oncogenic role in exporting proteins and RNA that are involved in cancer progression and treatment resistance. Here, we discuss the proteins and RNA transcripts that have been identified as XPO1 cargo in NHL lymphoma including tumour suppressors, immune modulators, and transcription factors, and their implications for oncogenesis. We then highlight the research to date on XPO1 inhibitors such as selinexor and other selective inhibitors of nuclear export (SINEs), which are used to treat some cases of non-Hodgkin lymphoma. In vitro, in vivo, and clinical studies investigating the anti-cancer effects of SINEs from bench to bedside, both as a single agent and in combination, are also reported. Finally, we discuss the limitations of the current research landscape and future directions to better understand and improve the clinical utility of SINE compounds in NHL.

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.

Targeting Nuclear Export Proteins in Multiple Myeloma Therapy.

Nuclear export proteins such as exportin-1 (XPO1) transport tumor-suppressor proteins and other growth-regulatory proteins from the nucleus to the cytoplasm. Overexpression of XPO1 has been observed in several cancers and correlates with shorter event-free and overall survival in multiple myeloma. Selinexor was developed as an oral first-in-class selective inhibitor of nuclear export (SINE) that inhibits XPO1. Preclinical studies in tumor cell lines and mouse models have demonstrated the efficacy of selinexor both as a single agent and in various combinations with known active antimyeloma agents. Results from the pivotal phase II STORM trial led to the US FDA approval of selinexor with dexamethasone in penta-refractory myeloma. Because of the feasibility of combining selinexor with other active antimyeloma agents, the multiarm STOMP trial was initiated and is ongoing, with impressive response rates reported in some of the combination arms thus far. The registrational phase III BOSTON trial demonstrated the superiority of selinexor in combination with bortezomib and dexamethasone as compared with bortezomib and dexamethasone in patients with relapsed refractory multiple myeloma (RRMM) who have received one to three prior anti-MM regimens. The toxicity profile of selinexor is wellestablished and predictable and may be significant unless managed aggressively and preemptively. The most common side effects are thrombocytopenia, anemia, neutropenia, fatigue, nausea, anorexia, and weight loss. Hyponatremia and cataracts seem to be class effects. Other SINE compounds are now being studied in efforts to discover agents that will potentially be better tolerated. Eltanexor is an investigational SINE compound that has shown a more positive toxicity profile in preclinical studies, with reduced central nervous system penetration and gastrointestinal side effects, and is now undergoing clinical investigation. These and other trials will further clarify the role of these innovative agents in the therapeutic advancement of RRMM.

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.

Abstract 2383: Mir-7974: An oncogenic miRNA that perpetuates gastric cancer

Abstract The global incidence of gastric cancer exceeds 1 million people per year and has a 5-year survival rate of 31%. Current chemotherapies are not effective because of genetic and epigenetic heterogeneity and late stage detection. Previously, we showed that exportin 1 (XPO1) is a therapeutic target in gastric cancer, as it facilitates the nuclear export of various cancer related tumor suppressor proteins (TSPs) through direct interaction with their nuclear export sequences (NES). XPO1 is upregulated in a variety of tumors including gastric cancer, leading to increased cytoplasmic localization of TSPs supporting malignant transformation and cancer cell proliferation, Knockdown of XPO1 leads to anti-tumor effects and pharmacologic targeting of XPO1 is achievable with selective inhibitors of nuclear export (SINE) compounds. In 2019 and 2020, the first generation XPO1 inhibitor KPT-330 was FDA approved for the use in penta-refractory multiple myeloma and non-Hodgkin's Lymphoma. Besides protein, mRNA and non-coding RNAs are also transported across the nuclear membrane. Non-coding RNA transport is generally attributed to another nuclear export protein, exportin 5 (XPO5), which plays a critical role in microRNA (miRNA) biogenesis. Nevertheless, there is emerging evidence supporting the role of XPO1 in non-coding RNA transport. In order to elucidate the role of XPO1 in non-coding RNA biology, we performed small RNA sequencing on gastric cancer cell lines treated with the XPO1 inhibitor selinexor. Our results revealed statistically significant changes in miR-7974 and miR-129-1-3p. These two miRs are recognized to influence protein modifications and cell survival. Administration of a miR-7974 mimic and a miR-129-1-3p inhibitor reduced proliferation of gastric cancer cells. Next, we evaluated the miR target genes of interest. At the mRNA level, we found upregulation of RasGEF Domain Family Member 1a (RASGEF1A), downregulation of DNA damage-regulated autophagy modulator 1 (DRAM1), downregulation of TIMP2 and downregulation of Cyclin G2 protein encoding gene CCNG2. Western Blots confirmed inhibition of AKT, RAS and Wnt signaling. Our results also showed that miR-7974 overexpression correlated with cellular XPO1 levels through the enhancement of XPO1 and its cargoes RCC1 and RANBP2. Finally inhibition of XPO1 by SINE compounds reversed the observed oncogenic phenotypes caused by these microRNAs. Our results unveil a novel interaction network between XPO1 and small non-coding RNAs and show that XPO1 induced small-noncoding microRNAs influence gastric cancer proliferation, and can be reversed by SINE compounds. This implies that modulation of microRNAs might be explored as a biomarker for XPO1 inhibitor therapy and warrants further investigation. Citation Format: Rachel E. Sexton, Mohammad Najeeb Al-Hallak, Bayan Al-Share, Yiwei Li, Amro Aboukameel, Yosef Landesman, Trinayan Kashyap, Steve Kim, Ramzi M. Mohammad, Philip A. Philip, Asfar S. Azmi. Mir-7974: An oncogenic miRNA that perpetuates gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2383.

Xpo-1 Inhibition Synergizes with BCR Inhibition, Blocks Tumor Growth and Tumor-Associated Macrophages Infiltration and Prolongs Survival in a Bioluminescent Animal Model of Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is an infrequent and aggressive non-Hodgkin lymphoma usually classified as diffuse large B-cell lymphoma (DLBCL), being most of the cases more closely related to activated B-cell type (ABC). PCNSL is associated with dismal prognosis, due in part to the difficulty for many chemotherapeuticals to cross the blood brain barrier.Selinexor (KPT-330) is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inactivates XPO-1 protein and induces anti-tumor effects mainly due to forced nuclear retention and activation of tumor suppressor proteins. Selinexor, that has shown excellent brain penetrance and promising results in pre-clinical models of glioblastoma, can inhibit both BCR and NF-kB signaling in malignant B-cells.In order to provide a pre-clinical rationale for the design of new therapeutic strategies in the PCNSL, we assessed the role of XPO-1 inhibition using intracerebral xenograft murine models.To compare the sensitivity of DLBCL-derived cell lines to selinexor, we determined the inhibitory concentration 50 (IC50) in terms of survival and proliferation after 96 hours in 4 ABC and 5 GCB DLBCL cell lines. Analysis showed that DLBCL cell lines have equivalent sensitivity to selinexor, regardless the cell of origin. Since SINE compounds have been shown to inhibit BCR signaling, we next tested the potential synergy between ibrutinib and selinexor. In 3 out of 4 ABC-DLBCL cell lines there was a strong synergy. In contrast, none of the 3 GCB-DLBCL cell lines analyzed were sensitive to up to 100 µM single agent ibrutinib; interestingly, however, treatment with selinexor sensitized SUDHL4 (GCB) cells to ibrutinib and the combination index values indicated strong synergism between the two drugs.We then established an orthotopic xenograft model of PCNSL by stereotactic injection of OCI-Ly10 cells stably transfected with luciferase into the brain parenchyma of nude athymic mice. Eleven days after the injection of cells all animals had developed detectable tumors confined to the CNS. Tumor size was measured and animals were randomly distributed into drug or vehicle experimental group. At this time point mice were treated with 5mg/kg of selinexor or vehicle via oral gavage three times a week; subsequently, bioluminiscence was assessed twice a week. Treatment with selinexor significantly blocked tumoral growth (two-way ANOVA: p< 0.0001; figure 1A). Specific time-point analysis showed that differences were significant as soon as 12 days after treatment (2,255e+008 +/- 8,648e+007 vs 8,670e+006 +/- 1,981e+007). Also, treatment with selinexor increased mice survival, with a median survival of 48 days in the treatment group compared to 34 days in the vehicle group (p< 0.0001; figure 1B). At final point, histopathological analysis showed diffuse infiltration in meninges and cerebral parenquima of highly proliferative CD20-positive B-cells (virtually 100% Ki-67-positive malignant B cells). Since the infiltration of tumoral associated macrophages (TAMs) has been related to worse prognosis in different lymphomas, including PCNSL, we analyzed the infiltration by F4/80+ TAMs after two weeks of treatment with selinexor or vehicle. We observed that all tumors from vehicle-treated mice were infiltrated with TAMs, whereas treatment with selinexor significantly reduced the presence of TAMs as assessed by immunohistochemistry, especially in the parenquima. Next, we evaluated the potential synergy between ibrutinib and selinexor in vivo. For that 12 mice with equivalent tumor size were assigned to each of the following four groups: selinexor only (5mg/kg twice a week), ibrutinib only (25mg/kg daily), combination or vehicle. The combination of both drugs significantly increased survival compared to both treatments as single agent, whereas there was no significant difference between ibrutinib and selinexor treatments alone (figure 1C). Tumoral growth was equally blocked by all treatments (figure 1D). Currently, we are evaluating the effect of the combination in the infiltration and immunophenotype of TAMs, including M1 vs M2 phenotype and PD1 expression. Results will be available at the time of the meeting.Selinexor proved to be an active agent in blocking tumoral growth in DLBCL infiltrating CNS, this providing pre-clinical evidence for the development of selinexor as new therapeutic option for PCNSL or DLBCL with CNS involvement. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combining selective inhibitors of nuclear export (SINEs) with chimeric antigen receptor (CAR) Tcells for CD19‑positive malignancies.

Chimeric antigen receptor (CAR) T cells directed against CD19 (CD19.CAR T cells) have yielded impressive clinical responses in the treatment of patients with lymphoid malignancies. However, resistance and/or relapse can limit treatment outcome. Risk of tumor escape can be reduced by combining treatment strategies. Selective inhibitors of nuclear export (SINEs) directed against nuclear exportin-1 (XPO1) have demonstrated anti-tumor efficacy in several hematological malignancies. The aim of the present study was to evaluate the combination of CAR T cells with the SINE compounds eltanexor and selinexor. As expected, eltanexor and selinexor were toxic to CD19-positive malignant cells and the sensitivity of cells towards SINEs correlated with the levels of XPO1-expression in ALL cell lines. When SINEs and CAR T cells were simultaneously combined, SINEs exerted toxicity towards CAR T cells and impaired their function affecting cytotoxicity and cytokine release ability. Flow cytometry and western blot analysis revealed that eltanexor decreased the cytoplasmic concentration of the transcription factor phosphorylated-STAT3 in CAR T cells. Due to CAR T-cell toxicity, sequential use of SINEs and CAR T cells was evaluated: Cytotoxicity of CAR T cells increased significantly when target cells were pre-treated with the SINE compound eltanexor. In addition, exhaustion of CAR T cells decreased when target cells were pre-treated with eltanexor. In summary, whereas the concomitant use of SINEs and CAR T cells does not seem advisable, sequential use of SINEs and CAR T cells might improve the anti-tumor efficacy of CAR T cells.

Abstract 3086: SINE compounds demonstrated potent anti-cancer activity in PDX mouse models of chordoma

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.

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

AbstractThe 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 for the treatment of multiple myeloma

ABSTRACTIntroduction: Despite unprecedented advances in the treatment of multiple myeloma (MM), almost all patients develop a disease that is resistant to the five most commonly used and active anti-MM agents. The prognosis for this patient population is particularly poor resulting in an unmet need for additional therapeutic options. Exportin-1 (XPO-1) is a major nuclear export protein of macromolecular cargo frequently overexpressed in MM. Selinexor is a first-in-class, oral Selective-Inhibitor-of-Nuclear-Export (SINE) compound that impedes XPO-1. Based on results of the STORM-trial, selinexor in combination with dexamethasone was granted accelerated FDA approval for patients with penta-refractory MM in July 2019.Areas covered: This article summarizes our up-to-date knowledge on the pathophysiologic role of XPO-1 in MM. Furthermore, it reviews the most recent clinical data on selinexor in combination with dexamethasone and other anti-MM agents; and discusses its safety profile, management strategies; and potential future developments.Expert opinion: Selinexor represents a next-generation-novel agent with an innovative mechanism of action that marks a significant advance in the treatment of heavily pretreated MM patients. Ongoing studies investigate its therapeutic potential also in earlier lines of therapy. Additional data is needed to confirm that selinexor and other SINE compounds are a valuable addition to our current therapeutic armamentarium.

The SINE Compound KPT-350 Blocks Dystrophic Pathologies in DMD Zebrafish and Mice.

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease that is caused by the loss of functional dystrophin protein in cardiac and skeletal muscles. DMD patient muscles become weakened, leading to eventual myofiber breakdown and replacement with fibrotic and adipose tissues. Inflammation drives the pathogenic processes through releasing inflammatory cytokines and other factors that promote skeletal muscle degeneration and contributing to the loss of motor function. Selective inhibitors of nuclear export (SINEs) are a class of compounds that function by inhibiting the nuclear export protein exportin 1 (XPO1). The XPO1 protein is an important regulator of key inflammatory and neurological factors that drive inflammation and neurotoxicity in various neurological and neuromuscular diseases. Here, we demonstrate that SINE compound KPT-350 can ameliorate dystrophic-associated pathologies in the muscles of DMD models of zebrafish and mice. Thus, SINE compounds are a promising novel strategy for blocking dystrophic symptoms and could be used in combinatorial treatments for DMD.

Abstract 3476: Evaluable antitumor activity in metastatic pancreatic adenocarcinoma with specific inhibitor of nuclear export based treatment

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease in urgent need of newer therapeutic modalities. Earlier we have shown that in PDAC, the over-expression of the nuclear exporter protein Exportin-1 (XPO1) leads to functional inactivation of tumor suppressor proteins (TSPs; FOXO3a, p27, Par-4 etc.) through mis-localization. We demonstrated that inhibition of XPO1 by CRISPR/Cas9 validated Selective Inhibitor of Nuclear Export (SINE) Selinexor and analogs restores the anti-tumor function of multiple TSPs leading to PDAC cell death and tumor inhibition in orthotopic models. Methods: Here we evaluate the synergy between SINE compounds and standard of care gemcitabine-nab-paclitaxel in PDAC models in vitro, in vivo and in a Phase Ib/2 trial (NCT02178436). Results: Selinexor and second generation SINE compound eltanexor synergized with gemcitabine (GEM) and nab-paclitaxel leading to suppression of PDAC growth, induction of apoptosis, and superior spheroid disintegration of PDAC derived cancer stem cells (CSCs). The observed synergy was due in part to enhanced nuclear localization of TSPs and suppression of both CSCs and epithelial-to-mesenchymal transition (EMT) markers. Label-Free quantitative (LFQ) proteome profiling with nuclear and cytoplasmic enrichment showed superior enhancement in nuclear protein fraction in combination treatment. The protein class with highest percent of nuclear retention were DNA binders. Selinexor and eltanexor as single agent (used at MTD) inhibited the growth of PDAC-CSC and two patient derived (Pdx) sub-cutaneous xenografts (p&amp;lt;0.01). In combination experiment, selinexor-GEM-nab-paclitaxel used at sub-MTD dose could significantly suppress the growth of PDx tumors. Molecularly, we observed down-regulation of CRM1 and target TSPs ex vivo. In a Phase 1b/2 study examining patients with metastatic pancreatic cancer, 9 patients were exposed to selinexor (60 mg oral) with GEM (1000 mg/m2 IV) and nab-paclitaxel (125 mg/m2 IV) once weekly (Mondays) for 3 weeks. Evaluable responses were observed in patients on this trial. 2 patients showed partial response (PR) and 2 had stable disease. Outstanding objective response was observed in 1 patient who demonstrated remission for 16 months and remained alive for 22 months. Remarkable and sustained reduction in CA19-9 levels were observed in the responding patient. Conclusions: These results rationally fortify selinexor-gemcitabine-nab-paclitaxel as new and effective therapy for metastatic PDAC and strengthen our ongoing Phase II study. Citation Format: Asfar S. Azmi, Yosef Landesman, Michael Kauffman, Sharon Shacham, Gabriel Mpilla, Amro Aboukameel, Steve Kim, Mandana Kamgar, Anteneh Tesfaye, Ramzi M. Mohammad, Philip A. Philip. Evaluable antitumor activity in metastatic pancreatic adenocarcinoma with specific inhibitor of nuclear export based treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3476.

Phase 1b study of selinexor, a first-in-class selective inhibitor of nuclear export (SINE) compound, in combination with doxorubicin in patients (pts) with locally advanced or metastatic soft tissue sarcoma (STS).

3123 Background: Selinexor is a first-in-class SINE compound with single-agent activity in STS. We undertook this study to determine the safety, tolerability and efficacy of selinexor in combination with doxorubicin in pts with incurable STS. Methods: This phase 1b study was conducted using a bayesian model (modified toxicity probability index). Patients with locally advanced or metastatic STS received selinexor at either 60 or 80mg weekly PO plus doxorubicin (75mg/m2 IV q21 days, max 6 cycles). Pts with stable disease (SD) or better (per RECIST 1.1 criteria) after 6 cycles of combination treatment received selinexor monotherapy until disease progression or unacceptable toxicity. Disease assessments were made with standard imaging after every 2 cycles. Results: 24 pts (19F/5M, ECOG 0/1: 12/12, median age 58.5 years [range 34-74]) were enrolled. Disease subtypes included leiomyosarcoma (n = 6), malignant peripheral nerve sheath tumor (n = 3) and other sarcomas (n = 15). Three pts at 60mg selinexor and 21 pts at 80mg selinexor were treated. The most common G3 drug related adverse events were hematological, neutropenia n = 13 (54%), anemia n = 6 (25%). There were 4 dose-limiting toxicities (2 febrile neutropenia, 1 vomiting and 1 unresolved fatigue) all at the 80mg dose level, but does not satisfy criteria for maximum tolerated dose. Two patients had clinically significant and relevant drop in ejection fraction, presenting with cardiac symptoms. Of the 24 evaluable pts 4 (17%) had a partial response, 16 (67%) had SD as best response and SD &gt; 16 weeks was seen in 13 pts (54%). PK analysis of selinexor did not demonstrate changes compared to single agent profile. The estimated median PFS and OS are 5.5 (95% CI:4.1-7.0) and 9.4 (6.6-13.8) months. Conclusions: Our initial data demonstrate that the combination of selinexor at 80mg with doxorubicin is tolerable and is associated with clinical benefit. Longer term follow up of available patients will be needed to understand toxicity profile. Clinical trial information: NCT03042819.

Clinical Response and Tolerability of Selinexor in Acute Myeloid Leukemia and Other Hematologic Malignancies - a Systematic Review

IntroductionSelinexor, a SINE (selective inhibitor of nuclear export) compound, inhibits exportin 1 (XPO1) involved in transport of tumor suppressor proteins leading to apoptosis of tumor cells. XPO1 is overexpressed in variety of cancer including ovarian cancer, pancreatic cancer, glioma, osteosarcoma, leukemia, lymphoma, and multiple myeloma. The aim of this study is to summarize clinical response and adverse events of selinexor in hematological neoplasms.MethodsA comprehensive literature search on PubMed, Embase, AdisInsight and Clinicaltrials.gov was completed on July 12, 2018. Studies focusing on efficacy and/or adverse events of selinexor in patients with hematological neoplasms were included for the review.ResultsOut of 321 studies found on initial search, we finalized 15 studies (8 phase I and 7 phase I/II) after screening by two reviewers. AMLSelinexor in combination with high-dose cytarabine and mitoxantrone has shown overall response rate (ORR) of 70% among 20 patients with acute myeloid leukemia (AML), Wang et al., 2018. Out of 12 newly diagnosed AML (ND AML) patients, 11 (92%) patients showed response with complete response (CR) in 7, CR with incomplete recovery (CRi) in 3 and partial response (PR) in 1 patient. Among 8 relapsed/refractory AML (R/R AML) patients, only 3 patients showed CR while 5 had treatment failure (TF), ORR in this subset was 38%. In 81 evaluable R/R AML patients receiving selinexor as monotherapy only 14% of the patients showed response while 31% patients had disease progression (PD) along with grade ≥3 hematological adverse events (AEs) of thrombocytopenia, anemia and neutropenia in 19%, 15% and 13% patients, respectively (Garzon et al., 2017). MMRelapsed refractory multiple myeloma patients receiving selinexor combined with pomalidomide and dexamethasone have achieved ORR of 60% with CR in 1 and PR in 5 patients (Chen et al., 2016 n=10) with grade ≥3 neutropenia in 8 patients. In another regimen with doxorubicin and dexamethasone the clinical benefit rate (CBR) was 26% with 15% overall response (Rachid et al., 2017 n=27). Grade ≥3 neutropenia, thrombocytopenia and hyponatremia occurred in 33%, 33% and 30% of patients, respectively. NHLKuruvilla et al. observed ORR in 31% patients with relapsed refractory non-Hodgkin lymphoma with single-agent selinexor. Grade ≥3 thrombocytopenia, neutropenia and anemia occurred in 47%, 32% and 27% patients, respectively. The efficacy of selinexor in phase I and I/II clinical trials is given in table 1 while toxicity is mentioned in table 2. With selinexor, the most common hematological and nonhematological AEs noted were thrombocytopenia and hyponatremia, respectively. ConclusionSelinexor based combination regimens have shown better clinical response against AML as compared to monotherapy. The efficacy results in multiple myeloma and other hematological malignancies are also encouraging. The adverse events like cytopenias were common as in other chemotherapy regimens. DisclosuresNo relevant conflicts of interest to declare.

In vitro toxicity and efficacy of verdinexor, an exportin 1 inhibitor, on opportunistic viruses affecting immunocompromised individuals.

Infection of immunocompromised individuals with normally benign opportunistic viruses is a major health burden globally. Infections with viruses such as Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi’s sarcoma virus (KSHV), adenoviruses (AdV), BK virus (BKPyV), John Cunningham virus (JCPyV), and human papillomavirus (HPV) are significant concerns for the immunocompromised, including when these viruses exist as a co-infection with human immunodeficiency virus (HIV). These viral infections are more complicated in patients with a weakened immune system, and often manifest as malignancies resulting in significant morbidity and mortality. Vaccination is not an attractive option for these immune compromised individuals due to defects in their adaptive immune response. Verdinexor is part of a novel class of small molecules known as SINE (Selective Inhibitor of Nuclear Export) compounds. These small molecules demonstrate specificity for the nuclear export protein XPO1, to which they bind and block function, resulting in sequestration of XPO1-dependent proteins in the nucleus of the cell. In antiviral screening, verdinexor demonstrated varying levels of efficacy against all of the aforementioned viruses including previously with HIV. Studies by other labs have discussed likely mechanisms of action for verdinexor (ie. XPO1-dependence) against each virus. GLP toxicology studies suggest that anti-viral activity can be achieved at a tolerable dose range, based on the safety profile of a previous phase 1 clinical trial of verdinexor in healthy human volunteers. Taken together, these results indicate verdinexor has the potential to be a broad spectrum antiviral for immunocompromised subjects for which vaccination is a poor option.

Phase II study of the oral selective inhibitor of nuclear export (SINE) KPT-335 (verdinexor) in dogs with lymphoma

BackgroundChemotherapeutic options for the treatment of canine lymphoma have not changed in several decades necessitating the identification of new therapeutics to improve patient outcome. KPT-335 (verdinexor) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) that exhibited anti-tumor activity against non-Hodgkin lymphoma in a prior phase I study. The objective of this phase II study was to expand upon the initial findings and assess the activity and safety in a larger population of dogs with lymphoma.ResultsFifty-eight dogs with naïve or progressive B-cell and T-cell lymphoma were enrolled in this clinical trial. KPT-335 was administered orally in one of three dosing groups, based on the previously established biologically active dose of 1.5 mg/kg three times weekly. Treatment with single-agent, orally administered KPT-335 resulted in an objective response rate (ORR) of 37%, of which dogs with T-cell lymphoma had an ORR of 71%. KPT-335 was well tolerated in all dose groups with grade 1–2 anorexia being the most common adverse event. Anorexia was responsive to symptomatic and supportive medications, including prednisone.ConclusionsThese data demonstrate that KPT-335 has biologic activity in canine lymphoma, and support continued evaluation of SINE compounds such as KPT-335 in combination with standard chemotherapeutics in canine lymphoma.

Anti-tumor efficacy of Selinexor (KPT-330) in gastric cancer is dependent on nuclear accumulation of p53 tumor suppressor

Exportin-1 (XPO1) controls the nucleo-cytoplasmic trafficking of several key growth regulatory and tumor suppressor proteins. Nuclear export blockade through XPO1 inhibition is a target for therapeutic inhibition in many cancers. Studies have suggested XPO1 upregulation as an indicator of poor prognosis in gastric cancer. In the current study, we investigated the anti-tumor efficacy of selective inhibitors of nuclear export (SINE) compounds KPT-185, KTP-276 and clinical stage selinexor (KPT-330) in gastric cancer. XPO1 was found to be overexpressed in gastric cancer as compared to adjacent normal tissues and was correlated with poor survival outcomes. Among the 3 SINE compounds, in vitro targeting of XPO1 with selinexor resulted in greatest potency with significant anti-proliferative effects at nano molar concentrations. XPO1 inhibition by selinexor resulted in nuclear accumulation of p53, causing cell cycle arrest and apoptosis. Also, inhibition of XPO1 lead to the cytoplasmic retention of p21 and suppression of survivin. Orally administered selienxor caused significant inhibition of tumor growth in xenograft models of gastric cancer. Furthermore, combination of selinexor with irinotecan exhibited greater anti-tumor effect compared to individual treatment. Taken together, our study underscores the therapeutic utility of XPO1 targeting in gastric cancer and suggests the potential benefits of XPO1 inhibition in-combination with chemotherapy.