CRM1 Inhibitor Research Articles

KPT-330, a potent and selective CRM1 inhibitor, exhibits anti-inflammation effects and protection against sepsis

Sepsis, a systemic inflammatory response caused by infection or injury, is still one of the most important causes of death in clinical patients. The ongoing search for the pathogenesis of sepsis and novel therapeutic methods are highly urgent. In this study, we hypothesized that KPT330, a potent and specific small molecule inhibitor of CRM1, could reduce inflammation and attenuate the severity of sepsis. In LPS-induced sepsis model in vivo, administration of KPT330 increased survival rate and ameliorated LPS-induced lung injury, with suppressed levels of TNF-α, IL-6 and HMGB1 in the circulation and decreased macrophage and PMN subpopulations in peritoneal cavity. In vitro investigations showed that KPT330 dose-dependently inhibited LPS-triggered proinflammatory cytokines production including TNF-α, IL-6 and HMGB1 in macrophages. Furthermore, KPT330 treatment significantly suppressed TNF-α and IL-6 mRNA expression and inhibited HMGB1 necleocytoplasmic translocation by inhibiting CRM1 distribution. Moreover, the mechanism analysis demonstrated that KPT330 exerted anti-inflammation effects by inhibiting the production of pro-inflammatory cytokines through suppressing activation of NF-κB and p38 signaling. Thus, pharmacologic stimulation of KPT330 may present a promising therapeutic strategy for sepsis.

Inhibition of CRM1 activity sensitizes endometrial and ovarian cell lines to TRAIL-induced cell death

BackgroundCRM1 enrichment has been shown to be indicative of invasive as well as chemoresistant tumors. On the other hand, TRAIL, a powerful and specific anti-tumoral agent, has yet to be used effectively to treat gynecological tumors in patients. In the present study, we examined if CRM1, a nuclear exporter capable of mediating protein transport, could be a relevant target to restore chemosensitivity in chemoresistant cells. We thus explored the hypothesis that CRM1-driven nuclear exclusion of tumor suppressors could lead to chemoresistance and that CRM1 inhibitors could present a novel therapeutic approach, allowing sensitization to chemotherapeutic agents.MethodsOvarian cancer cell lines, as well as endometrial cancer cell lines, were treated with leptomycin B (LMB), cisplatin and TRAIL, either singly or in combination, in order to induce apoptosis. Western blot and flow cytometry analysis were used to quantify caspases activation and apoptosis induction. Immunofluorescence was used to determine nuclear localization of p53. Colony formation assays were performed to determine therapeutic effectiveness; p53 siRNA were used to establish p53 role in sensitization. Additional information from GEO database and Prognoscan allowed us to contextualise the obtained results. Finally, qRT-PCR was performed to measure apoptotic regulators expression.ResultsTRAIL and LMB combination therapy lead to cleavage of caspase-3 as well as the appearance of cleaved-PARP, and thus, apoptosis. Further experiments suggested that sensitization was achieved through the synergistic downregulation of multiple inhibitor of apoptosis, as well as the activation of apoptotic pathways. p53 was enriched in the nucleus following LMB treatments, but did not seem to be required for sensitization; additional experiments suggested that p53 opposed the apoptotic effects of LMB and TRAIL. Results obtained from public data repositories suggested that CRM1 was a driver of chemoresistance and poor prognostic; DR5, on the other hand, acted as as a marker of positive prognostic.ConclusionsTaken together, our results suggest that the use of CRM1 inhibitors, in combination to chemotherapeutic compounds, could be highly effective in the treatment of gynecological malignancies.

Abstract 87: Nuclear localization of p70 S6 kinase in ovarian cancer

Abstract Ovarian cancer is highly metastatic with a poor prognosis. The p70 S6 kinase (p70S6K) is a downstream effector of the phosphatidylinositol-3 kinase/Akt pathway that is frequently activated in ovarian cancer. Constitutive activation of p70S6K is significantly more often in malignant ovarian tumors than in normal or benign tissues, suggesting a role for p70S6K in tumor development and progression. In addition to its well-established role in cell growth and survival, we showed for the first time that p70S6K plays a pivotal role in other aspects of tumor progression such as metastasis. Here, we further showed that this was associated with the nuclear localization of p70S6K. Treatment of cells with the CRM1 inhibitor, leptomycin B, retained p70S6K in the nucleus. Growth factor stimuli led to p70S6K phosphorylation and triggered nuclear translocation of p70S6K. The phosphorylation was sensitive to p70S6K inhibitors. We further identified a potential region that was important in its nuclear localization. Expression of nuclear localization signal-p70S6K enhanced tumor cell migration and invasion when compared to non-targeted p70S6K. Our findings identify a region responsible for the nuclear entry of p70S6K and demonstrate a functional significance of nuclear p70S6K in tumor progression (This work is supported by RGC grant 17103417). Citation Format: Jing ZHOU, Sally K. TO, Alice S. WONG. Nuclear localization of p70 S6 kinase in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 87.

Crm1 knockdown by specific small interfering RNA reduces cell proliferation and induces apoptosis in head and neck cancer cell lines.

Head and neck squamous cell carcinoma (HNSCC) is the most common and most aggressive type of head and neck cancer. Current approaches for the treatment of HNSCC are not sufficient to increase the patient survival or to reduce the high recurrence rate. Consequently, there is a need to explore the molecular characteristics of this cancer in order to discover potential therapeutic target molecules. The overexpression of chromosome region maintenance 1 (Crm1), responsible for the transport of different classes of macromolecules from the nuclear membrane to the cytoplasm, in various cancer cells has made it an attractive target molecule in cancer research. It has been reported that transcription factors, which are the target cargo proteins of Crm1, have critical roles in regulating intracellular processes via their expression levels and functions, which in turn are regulated by the cell cycle and signaling proteins. Previous findings show that head and neck cancer cells overexpress Crm1 and that these cells become highly dependent on Crm1 function. The results of this study show that after decreasing Crm1 expression levels in HNSCC cells through either treatment with specific Crm1 RNA interference (siRNA) or the selective Crm1 inhibitor leptomycin B (LMB), cell viability, proliferation, migration, and wound-healing abilities decreased, suppressing tumorigenic properties through the induction of apoptosis. Crm1 is a powerful diagnostic biomarker because of its central role in cancerogenesis, and it has a high potential for the development of targeted Crm1 molecules or synthetic agents, such as LMB, as well as for the improvement of the clinical features in head and neck cancer.

Nuclear export of ubiquitinated proteins via the UBIN-POST system

Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.

Gemcitabine and 5‐FU disrupt nuclear transport and subsequent localization of p21, p27 and p53 in cancer cells

Altered nuclear transport plays a role in the mislocalization of tumor suppressor proteins in certain cancers. In addition, the disruption of nuclear transport and the nuclear pore complex during apoptosis are important mechanisms of action in several different chemotherapies. We previously demonstrated that the chemotherapy 5‐Fluorouracil (5‐FU) alters nuclear transport by disrupting the Ran gradient and increasing nuclear pore permeability. This block in nuclear transport may be advantageous to overcome Crm1‐induced excessive nuclear export in some cancers or with certain cases of drug resistance. Other work has shown that an additional chemotherapy, gemcitabine, is more effective when in combination with Crm1 inhibition. Therefore, we were interested in determining if gemcitabine and 5‐FU work effectively alone and in combination to disrupt nuclear transport via mislocalization of the transport protein Ran. We utilized immunofluorescence to determine the localization of Ran, which is normally localized to the nucleus. We show here that while each of these drugs mislocalized Ran at both 6h and 24h time points, and that gemcitabine counteracts the effects of 5‐FU in combination treatments to induce no change in Ran localization. This may be due to gemcitabine inhibiting 5‐FU's calcium‐mediated mechanism for inducing nuclear pore permeability. Additionally, we were interested in determining how these drugs affected the localization of three cargo proteins regulated by Crm1‐mediated export: p53, p27 and p21. These proteins have important function in the nucleus to control cell cycle progression and induction of apoptosis. Therefore, inhibition of Crm1 export should lead to nuclear accumulation of these factors and a potential increase in their function as tumor suppressors. We show here that 5‐FU and gemcitabine have opposing effects on the nuclear localization of both p53 and p21, and that the drugs again have counteractive effects when in combination treatments. Both 5‐FU and gemcitabine decrease the nuclear localization and expression of p27, while the combination treatment shows an additive effect on both localization and expression. The effects of these drugs on protein localization may be related to an additional block in nuclear import, as well as an induction of nuclear pore permeability downstream of the induction of apoptosis. Further research will help to characterize the effects of 5‐FU and gemcitabine on nuclear transport and subsequent protein localization. This may help to determine how these drugs may be more effectively used in the treatment of specific cancers and in specific drug combinations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 324: Investigating the dependence of canine bladder transitional cell carcinoma on activated mutant BRAF

Abstract Transitional cell carcinoma (TCC) accounts for approximately 2% of diagnosed malignancies in canines. The majority of canine TCCs are invasive, intermediate to high grade tumors sharing similarities with human muscle invasive TCCs in risk factors, histology, sites of metastasis, and therapeutic response to single agents. Whole exome sequencing of canine TCC tumors was performed to identify somatic mutations in known cancer driver and repressor genes that could potentially contribute to canine TCC pathogenesis. A valine to glutamic acid mutation in BRAF homologous to the activating V600E mutation identified in human melanoma, colorectal and thyroid cancers was identified in 70% of sequenced tumors. Sensitivity to the BRAFV600E inhibitor Vemurafenib was tested in three BRAF mutant canine TCC cell lines (Bliley, Tyler1 and Tyler2) and two BRAF wild type canine TCC cell lines (Angus1 and Kinsey). All five canine TCC cell lines exhibited IC50s greater than 10μM, with BRAF mutant cell lines being slightly more sensitive. These sensitivity ranges are similar to those of some mutant human colorectal cancer cell lines, indicating that additional mechanisms may contribute to Vemurafenib resistance. Western blot analysis was performed to measure relative abundance of pERK, a downstream target of BRAF, in canine TCCs in response to serum starvation. All TCC cell lines showed sustained pERK expression in the absence of serum, indicating constitutive activation of the MAPK pathway. The five canine TCC lines were treated with 15μM Vemurafenib for 6 and 24 hours and their lysates were analyzed for pERK protein expression. pERK abundance was decreased in only the BRAF mutant cell lines after 6 hours of treatment. However, this decrease was less pronounced after 24 hours, suggesting that resistance mechanisms are bypassing BRAF to activate ERK. Sequence analysis of an additional panel of formalin-fixed paraffin embedded canine TCCs also revealed a mutation in RanBP2 in 31% of samples. Strikingly, the RanBP2 mutation appeared to be mutually exclusive to BRAF V to E mutant tumors with only two of the analyzed samples carrying both mutations. Significant tumor heterogeneity was implicated due to low level mutant amplification in these samples. It has been reported that loss of RanBP2 is synthetic lethal in BRAF V600E mutant colorectal cancer. Since RanBP2 forms complexes with CRM1 at the nuclear pore complex for nuclear export and at the kinetochore during mitosis, canine TCC cell lines were treated with KPT-185, a CRM1 inhibitor. BRAF mutant TCC cell lines had IC50 values ranging from 45nM to 65nM and were approximately ten-fold more sensitive than wild type cell lines. Overall, this data indicates that the pathogenesis of canine TCC likely depends on driving factors in addition to constitutive BRAF signaling, but Vemurafenib resistant BRAF mutant tumors can be targeted through inhibition of the nucleopore complex. Citation Format: Kathryn Cronise, Belen Hernandez, Daniel L. Gustafson, Dawn L. Duval. Investigating the dependence of canine bladder transitional cell carcinoma on activated mutant BRAF [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 324. doi:10.1158/1538-7445.AM2017-324

Abstract 1031: Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest

Abstract NPM1 mutated acute myeloid leukemia (AML) is a distinct entity in the WHO classification of hematopoietic cancers. It displays a specific phenotype characterized by favorable prognosis and upregulation of HOX cluster genes. NPM1 is a multifunctional nucleolar chaperone. Mutations in NPM1 result in cytoplasmic protein localization (NPM1c+) through the acquisition of a nuclear export signal (NES) at the C-terminus. The most frequent NPM1 mutation is a heterozygous 4bp insertion in exon 12 (mutA). The role of NPM1 in leukemogenesis is still a matter of debate and there is no direct proof that cytoplasmic localization of mutant NPM1 is necessary for maintenance of leukemia. We recently developed a CRISPR strategy for highly efficient gene editing in hematopoietic cells. We hypothesized that NPM1 mutA could be specifically targeted due to its 4bp insertion, avoiding the WT allele. Using an sgRNA spanning the insertion site (NPM1c sgRNA), we aimed to introduce indels adjacent to the mutation and disrupt the C-terminal NES. To test our hypothesis, we used the NPM1 mutated OCI-AML3 cell line. After transfection of OCI-AML3 with NPM1c sgRNA, while NPM1mutA allele showed 70-90% indel frequencies, the NPM1wt allele was intact. To determine whether the novel edited alleles generated NPM1 that was re-localized to the nucleus, we cloned the alleles into a GFP-NPM1 fusion construct and observed nuclear localization after transfection. Consistent with this finding, immunofluorescence revealed that edited OCI-AML3 cells had lost nearly all cytoplasmic NPM1. Return of NPM1 protein to the nucleus was followed by terminal differentiation and cell cycle arrest in G1 phase (controls 45±3%, NPM1c sgRNA 68±1.5%). Cell growth (3.7-4 fold decrease in cell counts in NPM1c sgRNA samples), colony forming ability (16-20 fold reduction in colonies in NPM1c sgRNA samples) and engraftment in xenograft models (significant loss of indels at NPM1mutA allele in engrafted cells) were also significantly impaired after NPM1mutA targeting. Furthermore, RNA sequencing on NPM1mutA-targeted and control OCI-AML3 cells revealed almost complete loss of expression of the HOXA and HOXB cluster genes as well as MEIS1 in treated cells (4.5 average fold reduction of HOXA9-A13; 5.6 average fold reduction of HOXB7-B13; 5 fold reduction of MEIS1). Allele-specific editing is a powerful tool to probe mechanistic aspects of oncogene dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c+ is necessary for OCI-AML3 cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization such as CRM1 inhibitors are attractive candidates for clinical success in NPM1-mutated AML. Citation Format: Lorenzo Brunetti, Michael C. Gundry, Anna G. Guzman, Ilaria Gionfriddo, Federica Mezzasoma, Francesca Milano, Maria Paola Martelli, Brunangelo Falini, Margaret A. Goodell. Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest [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 1031. doi:10.1158/1538-7445.AM2017-1031

CRM1 inhibitory and antiproliferative activities of novel 4′-alkyl substituted klavuzon derivatives

Klavuzons are 6-(naphthalen-1-yl) substituted 5,6-dihydro-2H-pyran-2-one derivatives showing promising antiproliferative activities in variety of cancer cell lines. In this work, racemic syntheses of nine novel 4′-alkyl substituted klavuzon derivatives were completed in eight steps and anticancer properties of these compounds were evaluated. It is found that size of the substituent has dramatic effect over the potency and selectivity of the cytotoxic activity in cancerous and healthy pancreatic cell lines. The size of the substituent can also effect the CRM1 inhibitory properties of klavuzon derivatives. Strong cytotoxic activity and CRM1 inhibition can be observed only when a small substituent present at 4′-position of naphthalen-1-yl group. However, these substituents makes the molecule more cytotoxic in healthy pancreatic cells rather than cancerous pancreatic cells. Among the tested compounds 1,2,3,4-tetrahydrophenanthren-9-yl substituted lactone was the most cytotoxic compound and its antiproliferative activity was also tested in 3D spheroids generated from HuH-7 cell lines.

Leptomycin B alters the subcellular distribution of CRM1 (Exportin 1)

CRM1 (chromosome maintenance region 1, Exportin 1) binds to nuclear export signals and is required for nucleocytoplasmic transport of a large variety of proteins and RNP complexes. Leptomycin B (LMB), the first specific inhibitor of CRM1 identified, binds covalently to cysteine 528 in the nuclear export signal binding region of CRM1 leading to the inhibition of protein nuclear export. Although the biochemical mechanisms of action of CRM1 inhibitors such as LMB are well studied, the subcellular effects of inhibition on CRM1 are unknown. We have found that LMB causes CRM1 to redistribute from the nucleus to the cytoplasm in A549 cells. A significant decrease in nuclear CRM1 coupled with an increase in cytoplasmic CRM1 was sustained for up to 4 h, while there was no change in total CRM1 protein in fractionated cells. Cells expressing an LMB insensitive HA-tagged CRM1-C528S protein were unaffected by LMB treatment, whereas HA-tagged wildtype CRM1 redistributed from the nucleus to the cytoplasm with LMB treatment, similar to endogenous CRM1. GFP-tagged CRM1 protein microinjected into the cytoplasm of A549 cells distributed throughout the cell in untreated cells remained primarily cytoplasmic in LMB-treated cells. Upon nuclear microinjection, GFP-CRM1 translocated to and accumulated in the cytoplasm of LMB-treated cells. Thus, LMB binds to CRM1 and causes its redistribution to the cytoplasm by inhibiting its nuclear import. Decreasing the nuclear availability of CRM1 likely contributes to the accumulation of CRM1 cargo proteins in the nucleus, suggesting a new mechanism of action for LMB.

Nuclear Export of Ubiquitinated Proteins Determines the Sensitivity of Colorectal Cancer to Proteasome Inhibitor.

Although proteasome inhibitors such as bortezomib had significant therapeutic effects in multiple myeloma and mantel cell lymphoma, they exhibited minimal clinical activity as a monotherapy for solid tumors, including colorectal cancer. We found in this study that proteasome inhibition induced a remarkable nuclear exportation of ubiquitinated proteins. Inhibition of CRM1, the nuclear export carrier protein, hampered protein export and synergistically enhanced the cytotoxic action of bortezomib on colon cancer cells containing wild-type p53, which underwent G2-M cell-cycle block and apoptosis. Further analysis indicated that tumor suppressor p53 was one of the proteins exported from nuclei upon proteasome inhibition, and in the presence of CRM1 inhibitor KPT330, nuclear p53, and expression of its target genes were increased markedly. Moreover, knockdown of p53 significantly reduced the synergistic cytotoxic action of bortezomib and KPT330 on p53+/+ HCT116 cells. In mice, KPT330 markedly augmented the antitumor action of bortezomib against HCT116 xenografts as well as patient-derived xenografts that harbored functional p53. These results indicate that nuclear p53 is a major mediator in the synergistic antitumor effect of bortezomib and KPT330, and provides a rationale for the use of proteasome inhibitor together with nuclear export blocker in the treatment of colorectal cancer. It is conceivable that targeting nuclear exportation may serve as a novel strategy to overcome resistance and raise chemotherapeutic efficacy, especially for the drugs that activate the p53 system. Mol Cancer Ther; 16(4); 717-28. ©2016 AACR.

Investigating The Spatiotemporal Distribution Patterns of PCID2 between The Nucleus And The Centrosome

PCID2 is a subunit of the TREX‐2 mRNA export complex that is also involved in the process of CRM1‐mediated nuclear protein export. Nuclear export is driven by RanGTP, which triggers a structural modification of CRM1 resulting in increased affinity for the leucine‐rich NES. Subsequent GTP hydrolysis following translocation across the NPC causes the CRM1‐cargo complex to dissociate. Karyopherins can also serve functional roles at non‐nuclear locations in the cell. For example, both CRM1 and Ran are found at the centrosome in addition to the nucleus. Specific factors facilitate mitosis by restricting the centrosome to duplicate once during the cell cycle through alternating nuclear and centrosomal localization. Specifically, CRM1 has an integral role in localizing nucleophosmin to the centrosomes to facilitate this “licensing” process. Leptomycin B inhibition of CRM1 causes dissociation of nucleophosmin from the centrosomes. In doing so it leads to centrosome reduplication within the same cell cycle. In addition to its nuclear function, PCID2 is also localized to the centrosome in a subset of cells. The function of PCID2 at the centrosome is not yet elucidated, but we demonstrate here that PCID2 is in fact present in CRM1 complexes. This evidence gives us reason to believe that PCID2 may serve a role at the centrosome that is supplementary to that of CRM1 and Ran. We hypothesize that PCID2 is localized to the centrosome as a part of the CRM1‐RanGTP complex and may translocate from the nucleus to the centrosome. To investigate the relationship between PCID2 in the nucleus and at the centrosome, we performed immunofluorescence of PCID2 in HeLa cells treated with Leptomycin B, a CRM1‐dependent nuclear export inhibitor. Preliminary results shows that LMB treatment causes a decrease in nuclear intensity of CRM1 and a potential decrease in PCID2, while causing an increase in the incidence of cells exhibiting punctate staining of PCID2. Because we know that LMB treatment leads to unregulated duplication of centrosomes, these findings suggest a correlation between increased PCID2 punctate staining and multiple centrosome duplications. This is consistent with our previous work showing that PCID2 is most commonly localized to the centrosome in G2 when duplication is occurring. These findings could also imply that PCID2 has a role in the localization of centrosomal components or mitotic proteins to the centrosome from the nucleus. Future experiments will investigate whether increased PCID2 punctate staining is indicative of a role in excess centrosome duplication or whether LMB is instead altering the CRM1/PCID2 localization pattern, causing centrosomal accumulation of PCID2.

Abstract B38: Clinical translation of nuclear export inhibitor in metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease in urgent need of newer therapeutic modalities. PDAC tumors are very heterogeneous and carry alterations in many critical pathways rendering the design of therapy against a single pathway unrealistic. The disease requires a broad form of therapy that can target the activation of multiple tumor suppressor proteins (TSPs) simultaneously. We have shown that over-expression of the nuclear exporter protein CRM1 in PDAC leads to functional inactivation of TSPs (such as FOXO3a, p27, Par-4 and p53) through their mislocalization to the cytoplasmic compartment. We clearly demonstrated that targeted inhibition of CRM1 by CRISPR/Cas9 validated Specific Inhibitor of Nuclear Export (SINE) KPT-330/Selinexor can restore the function of multiple TSPs leading to PDAC cell death and tumor inhibition in orthotopic models. More significantly, our findings show that SINE synergize with gemcitabine (GEM) and nab-paclitaxel leading to enhanced PDAC growth inhibition, apoptosis, and spheroid disintegration of PDAC derived cancer stem cells (CSCs) that undergo epithelial-to-mesenchymal transition (EMT). The observed synergy was due in part to enhanced nuclear localization of TSPs and suppression of CSC markers alongside reversal of EMT markers. RNA from quadruplet combination treatment samples were subjected to Agilent HT12 microarrays and differentially expressed genes (p<0.01) were subjected to pathway analysis. Our computational analysis demonstrated reversal of immune suppressive networks and suppression of CSC sustaining networks. Most importantly we observed activation of fibroblast specific cell death pathways highlighting an as of yet unexplored role of CRM1 in sustaining fibroblast growth that is recognized to support high desmoplastic reaction (DR) in PDAC. Our multi-model pre-clinical work has led to the approval of a Phase Ib/II clinical study involving GEM-nab-paclitaxel-Selinexor for metastatic PDAC (NCT02178436). Citation Format: Asfar S. Azmi, Irfana Muqbil, Amro Aboukameel, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad, Philip A. Philip.{Authors}. Clinical translation of nuclear export inhibitor in metastatic pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B38.

Selective Inhibition of Nuclear Cytoplasmic Transport As a New Treatment Paradigm in Myelofibrosis

▪Myelofibrosis (MF) is a hematopoietic stem cell neoplasm characterized by constitutive activation of JAK/STAT signaling due to mutations in JAK2, calreticulin or MPL. Many MF patients suffer from severe constitutional symptoms and have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib (RUX) reduce MF symptoms, but like all other drugs used in MF, are not curative, with persistence of mutant cells and prompt symptom rebound upon discontinuation. This defines a clinical need to identify strategies capable of inducing more profound and durable responses in MF.To identify previously unrecognized molecular vulnerabilities in MF, we infected HEL cells (homozygous for JAK2V617F) with a barcoded lentiviral shRNA library targeting ~5,000 human signal transduction genes, with 5-6 shRNAs/gene (Cellecta Human Module 1). Conditions were optimized to achieve a multitude of infection (MOI) of ~1. Barcode abundance was compared between days 0 and 9 after infection by next generation sequencing. Candidates were selected based on ≥ 15-fold reduction of abundance by ≥ 2 shRNAs targeting the same gene, similar to Khorashad et al. [Blood. 2015;125(11):1772-81]. Amongst the genes meeting these criteria, nuclear cytoplasmic transport (NCT) was significantly enriched, with RAN and RANBP2 amongst the top genes, suggesting that HEL cells may be highly dependent on NCT. For confirmation, HEL cells were stably transduced with doxycycline (DOX)-inducible shRAN. After 72 hours DOX-induced knockdown of RAN reduced viable cells by 77.3±5.5% and colony formation by 82.8±1.3% and dramatically increased apoptosis (uninduced: ~10% vs. induced: ~50%). Similar results were observed in SET-2 cells (heterozygous for JAK2V617F). We next cultured HEL and SET-2 cells with graded concentrations of the KPT-330 (selinexor, Karyopharm), an inhibitor of CRM-1, the core component of NCT, or RUX as a comparison. Selinexor was five-fold more potent than RUX against HEL cells (IC50: 98nM for KPT vs. 536 nM for RUX) and as potent as RUX in SET-2 cells (IC50:~100 nM). Importantly, RUX-resistant HEL cells (IC50:24µM) were highly sensitive to inhibition of NCT by knockdown of RAN or selinexor (IC50:160nM). Selinexor also selectively inhibited colony formation by primary MF vs. cord blood (CB) CD34+ cells (IC50:93nM for MF vs. 203nM for CB). Lastly, selinexor enhanced RUX-induced growth inhibition and apoptosis in primary MF CD34+cells cultured ex vivo for 72h (including both JAK2 mutation positive and negative MF samples, n=3 for each, and RUX resistant patient samples, n=6). Nuclear:cytoplasmic fractionation of HEL cells revealed that the expression and nuclear localization of the tumor suppressors FoxO3A and APC, but not of PP2A and nucleophosmin (NPM) were significantly increased upon knockdown of RAN, which may contribute to the increased apoptosis following NCT inhibition.To determine the in vivo effects of selinexor in MF, we induced MPN in Balb/c mice by transplanting donor marrow infected with JAK2V617F for three weeks, and then treated mice (n=13/group) with vehicle, selinexor (initial dose 20 mg/kg, 3x weekly, orally) or RUX (initial dose 50 mg/kg twice daily, orally) or the combination of RUX plus selinexor for up to 4 weeks. Combination treatment significantly reduced white blood cell counts and normalized spleen size. Compared to vehicle, selinexor alone significantly reduced GFP+cells in the spleen, and this effect was further enhanced with the combination treatment. Histopathology revealed that combination treatment restored splenic architecture, while bone marrow fibrosis was not significantly altered by selinexor or the combination. Mice in all groups, including the combined vehicle controls, experienced considerable weight loss, suggesting that toxicity may be partially due to high dose and frequent drug administration. Experiments with the next generation NCT inhibitor KPT-8602 [Etchin et al., Leukemia, 2016 Jun 24] are underway.In summary, our results suggest that MF cells are exquisitely dependent on NCT, and that NCT inhibition alone or in combination with RUX may reduce JAK2V617F allelic burden. This identifies NCT as a prime therapeutic target in MF. A phase I clinical trial of selinexor in refractory MF is in preparation. DisclosuresBaloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Deininger:BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

GW27-e0440 Increased nuclear NT-PGC-1α via inhibition of CRM1 can enhance fatty acid oxidation in NRCM

GW27-e0440 Increased nuclear NT-PGC-1α via inhibition of CRM1 can enhance fatty acid oxidation in NRCM

Inhibiting cancer cell hallmark features through nuclear export inhibition.

Treating cancer through inhibition of nuclear export is one of the best examples of basic research translation into clinical application. Nuclear export factor chromosomal region maintenance 1 (CRM1; Xpo1 and exportin-1) controls cellular localization and function of numerous proteins that are critical for the development of many cancer hallmarks. The diverse actions of CRM1 are likely to explain the broad ranging anti-cancer potency of CRM1 inhibitors observed in pre-clinical studies and/or clinical trials (phase I–III) on both advanced-stage solid and hematological tumors. In this review, we compare and contrast the mechanisms of action of different CRM1 inhibitors, and discuss the potential benefit of unexplored non-covalent CRM1 inhibitors. This emerging field has uncovered that nuclear export inhibition is well poised as an attractive target towards low-toxicity broad-spectrum potent anti-cancer therapy.

Synergistic cytotoxic effects of selinexor and chloroquine phosphate in mantle cell lymphoma.

e19069Background: Although the cytotoxic effects of novel CRM1 inhibitor, small molecule selective inhibitors of nuclear export (SINE) on Mantle Cell Lymphoma (MCL) cells have now been established,...

CRM1 Inhibition Promotes Cytotoxicity in Ewing Sarcoma Cells by Repressing EWS-FLI1-Dependent IGF-1 Signaling.

Ewing sarcoma (EWS) is an aggressive bone malignancy that mainly affects children and young adults. The mechanisms by which EWS (EWSR1) fusion genes drive the disease are not fully understood. CRM1 (XPO1) traffics proteins from the nucleus, including tumor suppressors and growth factors, and is overexpressed in many cancers. A small-molecule inhibitor of CRM1, KPT-330, has shown therapeutic promise, but has yet to be investigated in the context of EWS. In this study, we demonstrate that CRM1 is also highly expressed in EWS. shRNA-mediated or pharmacologic inhibition of CRM1 in EWS cells dramatically decreased cell growth while inducing apoptosis, cell-cycle arrest, and protein expression alterations to several cancer-related factors. Interestingly, silencing of CRM1 markedly reduced EWS-FLI1 fusion protein expression at the posttranscriptional level and upregulated the expression of the well-established EWS-FLI1 target gene, insulin-like growth factor binding protein 3 (IGFBP3), which inhibits IGF-1. Accordingly, KPT-330 treatment attenuated IGF-1-induced activation of the IGF-1R/AKT pathway. Furthermore, knockdown of IGFBP3 increased cell growth and rescued the inhibitory effects on IGF-1 signaling triggered by CRM1 inhibition. Finally, treatment of EWS cells with a combination of KPT-330 and the IGF-1R inhibitor, linsitinib, synergistically decreased cell proliferation both in vitro and in vivo Taken together, these findings provide a strong rationale for investigating the efficacy of combinatorial inhibition of CRM1 and IGF-1R for the treatment of EWS. Cancer Res; 76(9); 2687-97. ©2016 AACR.

Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes.

The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.