Nuclear Export Protein Research Articles

The pro-oncogenic noncanonical activity of a RAS•GTP:RanGAP1 complex facilitates nuclear protein export.

Canonical RAS signaling, including PI3K/AKT- and RAF/MEK-dependent activities, results mainly from RAS•GTP interaction with its effectors at the plasma membrane. Here, we identified a fundamental, oncogenic, noncanonical RAS•GTP activity that increases XPO1-dependent export of nuclear protein cargo into the cytoplasm and is independent of PI3K/AKT and RAF/MEK signaling. This RAS-dependent step acts downstream from XPO1 binding to nuclear protein cargo and is mediated by a perinuclear protein complex between RAS•GTP and RanGAP1 that facilitates hydrolysis of Ran•GTP to Ran•GDP, which promotes release of nuclear protein cargo into the cytoplasm. The export of nuclear EZH2, which promotes cytoplasmic degradation of the DLC1 tumor suppressor protein, is a biologically important component of this pro-oncogenic activity. Conversely, preventing nuclear protein export contributes to the antitumor activity of KRAS inhibition, which can be further augmented by reactivating the tumor suppressor activity of DLC1 or potentially combining RAS inhibitors with other cancer treatments.

Selinexor as a Therapeutic Target: Advances in Non-small Cell and Small Cell Lung Cancer Treatment Strategies.

Selinexor treats lung cancer, particularly non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). This review summarizes the prevalence and types of lung cancer and emphasizes the challenges associated with current treatments like resistance and limited effectiveness. Selinexor is a selective inhibitor of nuclear export (SINE) that has emerged as a potential therapy that targets the nuclear export of tumor suppressor proteins. The mechanisms of selinexor, its potential in combination therapies, and challenges like side effects and drug resistance are explained in this review. Key findings highlight the effectiveness of selinexor in preclinical studies, particularly against KRAS-mutant NSCLC and in combination with chemotherapy for SCLC. The review concludes with a discussion of future directions and underscores the potential of selinexor to improve the treatment strategies for lung cancer.

The nuclear export protein XPO1 provides a peptide ligand for natural killer cells.

XPO1 (Exportin-1/CRM1) is a nuclear export protein that is frequently overexpressed in cancer and functions as a driver of oncogenesis. Currently small molecules that target XPO1 are being used in the clinic as anticancer agents. We identify XPO1 as a target for natural killer (NK) cells. Using immunopeptidomics, we have identified a peptide derived from XPO1 that can be recognized by the activating NK cell receptor KIR2DS2 in the context of human leukocyte antigen-C. The peptide can be endogenously processed and presented to activate NK cells specifically through this receptor. Although high XPO1 expression in cancer is commonly associated with a poor prognosis, we show that the outcome of specific cancers, such as hepatocellular carcinoma, can be substantially improved if there is concomitant evidence of NK cell infiltration. We thus identify XPO1 as a bona fide tumor antigen recognized by NK cells that offers an opportunity for a personalized approach to NK cell therapy for solid tumors.

Unveiling the Antiviral Potential of Minocycline: Modulation of Nuclear Export of Viral Ribonuclear Proteins during Influenza Virus Infection.

Influenza A virus (IAV) poses a global threat worldwide causing pandemics, epidemics, and seasonal outbreaks. Annual modification of vaccines is costly due to continual shifts in circulating genotypes, leading to inadequate coverage in low- and middle-income countries like India. Additionally, IAVs are evolving resistance to approved antivirals, necessitating a search for alternative treatments. In this study, the antiviral role of the FDA-approved antibiotic minocycline against IAV strains was evaluated in vitro and in vivo by quantifying viral gene expression by qRT-PCR, viral protein levels by Western blotting, and viral titers. Our findings demonstrate that minocycline at a non-toxic dose effectively inhibits IAV replication, regardless of viral strain or cell line. Its antiviral mechanism operates independently of interferon signaling by targeting the MEK/ERK signaling pathway, which is crucial for the export of viral ribonucleoproteins (vRNPs). Minocycline prevents the assembly and release of infectious viral particles by causing the accumulation of vRNPs within the nucleus. Moreover, minocycline also inhibits IAV-induced late-stage apoptosis, further suppressing viral propagation. The antiviral activity of minocycline against IAVs could offer a promising solution amidst the challenges posed by influenza and the limitations of current treatments.

TIRR regulates mRNA export and association with P-bodies in response to DNA damage.

To ensure the integrity of our genetic code, a coordinated network of signalling and repair proteins, known as the DNA damage response (DDR), detects and repairs DNA insults, the most toxic being double-strand breaks (DSBs). Tudor interacting repair regulator (TIRR) is a key factor in DSB repair, acting through its interaction with p53 binding protein 1 (53BP1). TIRR is also an RNA binding protein, yet its role in RNA regulation during the DDR remains elusive. Here, we show that TIRR selectively binds to a subset of messenger RNAs (mRNAs) in response to DNA damage. Upon DNA damage, TIRR interacts with the nuclear export protein Exportin-1 through a nuclear export signal. Furthermore, TIRR plays a crucial role in the modulation of RNA processing bodies (PBs). TIRR itself and TIRR-bound RNA co-localize with PBs, and TIRR depletion results in nuclear RNA retention and impaired PB formation. We also suggest a potential link between TIRR-regulated RNA export and efficient DDR. This work reveals intricate involvement of TIRR in orchestrating mRNA nuclear export and storage within PBs, emphasizing its significance in the regulation of RNA-mediated DDR.

Inhibition of RAN attenuates influenza a virus replication and nucleoprotein nuclear export

ABSTRACT Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.

XPO1 serves as a prognostic marker involving AKT/MAPK/TGFBR1 pathway in OSCC.

Exportin 1 (XPO1) is a nuclear export protein that facilitates the transportation of various substances. XPO1 promotes tumor development as a poor prognostic factor in a variety of tumors and is a therapeutic target for screening inhibitors. However, the role of XPO1 in oral squamous cell carcinoma (OSCC) has yet to be determined. The expression patterns of XPO1 mRNA in OSCC were investigated using bioinformatics tools, and the expression levels of XPO1 protein in OSCC specimens were confirmed by immunohistochemical assays. Survival analysis was conducted to evaluate the impact of XPO1 on prognosis. GO and KEGG enrichment analyses were utilized to uncover the signaling pathways mediated by XPO1. Additionally, we examined the association between XPO1 and AKT/MAPK/TGFBR1 and immune infiltration. XPO1 mRNA and protein expression levels were significantly enhanced in OSCC and associated with OSCC severity. Enhanced XPO1 expression was indicative of poor survival. Functional analysis showed that XPO1 mediated pathways associated with cell cycle and DNA replication and reduced immune infiltration in OSCC. Additionally, XPO1 mRNA and protein expression levels had significant positive relationships with AKT/MAPK/TGFBR1. XPO1, as a marker of poor prognosis in OSCC, can promote OSCC through AKT/MAPK/TGFBR1.

Effect of Selinexor on Proliferation and Apoptosis of Acute Myeloid Leukemia Kasumi-1 Cells

To investigate the effects of selinexor, a inhibitor of nuclear export protein 1 (XPO1) on the proliferation inhibition and apoptosis of Kasumi-1 cells in acute myeloid leukemia (AML). MTS method was used to detect the inhibitory effect of different concentrations of selinexor on the proliferation of Kasumi-1 cells at different time points. The apoptosis rate and cell cycle changes after treatment with different concentration of selinexor were detected by flow cytometry. Selinexor inhibited the growth of Kasumi-1 cells at different time points in a concentration-dependent manner (r 24 h=0.7592, r 48 h=0.9456, and r 72 h=0.9425). Selinexor inhibited Kasumi-1 cells growth in a time-dependent manner (r =0.9057 in 2.5 μmol/L group, r =0.9897 in 5 μmol/L group and r =0.9994 in 10 μmol/L group). Selinexor could induce apoptosis of Kasumi-1 cells in a dose-dependent manner (r =0.9732), and the apoptosis of Kasumi-1 cells was more obvious with the increase of drug concentration. The proportion of G0/G1 phase was significantly increased and the proportion of S phase was significantly decreased after the treatment of Kasumi-1 cells by selinexor. With the increase of drug concentration, the proportion of Kasumi-1 cells cycle arrest in G0/G1 phase was increased and the cell synthesis was decreased. Selinexor can promote the death of tumor cells by inhibiting Kasumi-1 cells proliferation, inducing apoptosis and blocking cell cycle.

225P Nuclear exporter protein exportin 1 (XPO1) as a novel therapeutic target in pancreatic neuroendocrine tumors

225P Nuclear exporter protein exportin 1 (XPO1) as a novel therapeutic target in pancreatic neuroendocrine tumors

A CRM1-dependent nuclear export signal in Autographa californica multiple nucleopolyhedrovirus Ac93 is important for the formation of intranuclear microvesicles.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93-null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93-null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCEAutographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway.

The nuclear export protein exportin-1 in solid malignant tumours: From biology to clinical trials.

Exportin-1 (XPO1), a crucial protein regulating nuclear-cytoplasmic transport, is frequently overexpressed in various cancers, driving tumor progression and drug resistance. This makes XPO1 an attractive therapeutic target. Over the past few decades, the number of available nuclear export-selective inhibitors has been increasing. Only KPT-330 (selinexor) has been successfully used for treating haematological malignancies, and KPT-8602 (eltanexor) has been used for treating haematologic tumours in clinical trials. However, the use of nuclear export-selective inhibitors for the inhibition of XPO1 expression has yet to be thoroughly investigated in clinical studies and therapeutic outcomes for solid tumours. We collected numerous literatures to explain the efficacy of XPO1 Inhibitors in preclinical and clinical studies of a wide range of solid tumours. In this review, we focus on the nuclear export function of XPO1 and results from clinical trials of its inhibitors in solid malignant tumours. We summarized the mechanism of action and therapeutic potential of XPO1 inhibitors, as well as adverse effects and response biomarkers. XPO1 inhibition has emerged as a promising therapeutic strategy in the fight against cancer, offering a novel approach to targeting tumorigenic processes and overcoming drug resistance. SINE compounds have demonstrated efficacy in a wide range of solid tumours, and ongoing research is focused on optimizing their use, identifying response biomarkers, and developing effective combination therapies. Exportin-1 (XPO1) plays a critical role in mediating nucleocytoplasmic transport and cell cycle. XPO1 dysfunction promotes tumourigenesis and drug resistance within solid tumours. The therapeutic potential and ongoing researches on XPO1 inhibitors in the treatment of solid tumours. Additional researches are essential to address safety concerns and identify biomarkers for predicting patient response to XPO1 inhibitors.

XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53–p21 and p27 pathways

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

Abstract LB172: A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a deadly malignancy with an evolving therapeutic landscape. Although nucleic acid-based evaluations have unveiled some druggable targets, limited options are available for the majority of patients with this disease. We therefore evaluated biliary exosomes by mass spectrometry from 16 patients with CCA, which revealed a 17-fold enhancement of the nuclear export protein XPO7 in CCA-derived biliary exosomes. IHC analysis of XPO7 expression in CCA tumors from 170 patients unexpectedly demonstrated intense cytoplasmic staining in 30% of the samples, which correlated with abbreviated survival. Within the cytosol, we demonstrate that XPO7 exists in a molecular complex with the serine/threonine kinase SLK. shRNA-mediated knockdown of either XPO7 or SLK abrogated tumor organoid formation in vitro, and reduced orthotopic tumor formation and substantially altered tumor morphology using athymic mice. A kinome screen of FDA-approved drugs revealed tivozanib inhibits SLK (IC50= 81.8 nM), which we confirmed using crystallography. Tivozanib treatment reduced tumor organoid formation in vitro and induced tumor regression in vivo using murine xenografts with resulting morphology similar to XPO7 and SLK knockdown tumors. Importantly, site-directed mutagenesis of SLK was used to confirm the observed phenotypic alterations secondary to SLK inhibition. Signal transduction studies with SLK knockdown in CCA cell lines demonstrated downregulation of PI3K-AKT-mTOR signaling with further effects on DNA damage resulting in G2 cell cycle arrest. Moreover, tivozanib demonstrated anti-tumor activity and increased apoptosis in established CCA patient-derived xenografts with cytoplasmic XPO7 expression. Using ex vivo tumor slice cultures, tivozanib significantly increased tumor cell apoptosis in XPO7/SLK-expressing tumors (N=3) but not in tumors without this expression pattern (N=6). Further evaluation of these tivozanib-treated tumor slice cultures demonstrated a reduction in the expression of phosphorylated mTOR (S2448), AKT (T308), S6 (S235/236) in the high XPO7/SLK-expressing tumors but not in tumors without this expression pattern. Lastly, tivozanib monotherapy demonstrated in vivo efficacy with arrest of tumor progression (RECIST stable disease) in an ongoing clinical trial (NCT04645160). Citation Format: Priyanka P. Desai, Tahsin M. Khan, Reed I. Ayabe, Emily Smith, Surajit Sinha, Ashley Rainey, Yuri Lin, Tracey Pu, Kenneth Lubrice, Leila Sarvestani, Sarfaraz Akmal, Shahyan Rehman, Kirsten Remmert, Michael Yaffe, Craig Thomas, John Brognard, Jonathan M. Hernandez. A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB172.

A LATS2 and ALKBH5 positive feedback loop supports their oncogenic roles

N(6)-methyladenosine (m6A) critically regulates RNA dynamics in various biological processes. The m6A demethylase ALKBH5 promotes tumorigenesis of glioblastoma, while the intricate web that orchestrates its regulation remains enigmatic. Here, we discover that cell density affects ALKBH5 subcellular localization and m6A dynamics. Mechanistically, ALKBH5 is phosphorylated by the large tumor suppressor kinase 2 (LATS2), preventing its nuclear export and enhancing protein stability. Furthermore, phosphorylated ALKBH5 reciprocally erases m6A from LATS2 mRNA, thereby stabilizing this transcript. Unexpectedly, LATS2 depletion suppresses glioblastoma stem cell self-renewal independent of yes-associated protein activation. Additionally, deficiency in either LATS2 or ALKBH5 phosphorylation impedes tumor progression in mouse xenograft models. Moreover, high levels of LATS2 expression and ALKBH5 phosphorylation are associated with tumor malignancy in patients with gliomas. Collectively, our study unveils an oncogenic positive feedback loop between LATS2 and ALKBH5, revealing a non-canonical branch of the Hippo pathway for RNA processing and suggesting potential anti-cancer interventions.

Abstract 2089: A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications

Abstract The majority of RAS oncogenic activity is thought to be mediated by its canonical signaling, mainly via RAS-GTP interactions with its effectors RAF and PI3K at the plasma membrane, which respectively activate MEK/ERK and AKT/mTOR pathways. However, in this study, we have identified a new, fundamental, pro-oncogenic, non-canonical activity of RAS-GTP that increases the Exportin 1 (XPO1)-dependent export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. The nuclear protein export machinery is known to depend on a trimeric protein complex composed of RAN-GTP:XPO1:nuclear protein cargo. Unexpectedly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP) at the nuclear envelope, which facilitates the release of nuclear protein cargo by increasing the hydrolysis of RAN-GTP to RAN-GDP. We have determined that RAS-GTP and RAN-GAP form this complex in primary human lung tumors, patient derived xenografts, and human lung cancer cell lines. Interestingly, the RAS-GTP:RAN-GAP complex is found at the nuclear envelope and in the cytoplasm, but not at the plasma membrane. The RAS-dependent step mediated by the RAS-GTP:RAN-GAP complex acts downstream of XPO1 by increasing RAN-GTP hydrolysis, which promotes protein cargo release. Our fundamental findings have important clinical implications. For example, in human lung cancer models that harbor a KRAS-G12C mutation, the combined inhibition of MEK and PI3K (MEKi + PI3Ki) does not inhibit tumor growth to the same degree as a KRAS-G12C inhibitor. Remarkably, adding an XPO1 inhibitor to MEKi + PI3Ki inhibits tumor growth to the same degree as the KRAS-G12C inhibitor. One critical pro-oncogenic target of the RAS-GTP:RAN-GAP complex is nuclear export of the EZH2 methyltransferase, whose increase in the cytoplasm leads to a direct methylation of the cytoplasmic DLC1 tumor suppressor protein, leading to ubiquitin-dependent proteasomal degradation of DLC1 protein. Analysis of a KRAS mutant cell line from which the DLC1 gene has been disrupted by CRISPR-Cas9 technology indicates that DLC1 makes an important contribution to the growth inhibition by inhibitors that reduce nuclear protein export. These findings establish nuclear protein export as a critical, pro-oncogenic, non-canonical RAS function that is mediated by the RAS-GTP:RAN-GAP complex and provide a mechanistic explanation for poor clinical responses to combined inhibition of MEK and PI3K, which do not regulate the nuclear protein activity identified in this study. Citation Format: Brajendra K. Tripathi, Nicole H. Hirsh, David Hansen, Xiaolan Qian, Marian E. Durkin, Dunrui Wang, Alex G. Papageorge, Ross Lake, Yvonne A. Evrard, Adam Marcus, Suresh S. Ramalingam, Mary Dasso, Karen Vousden, James H. Doroshow, Kylie J. Walters, Douglas R. Lowy. A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications [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 2089.

Abstract 7269: Karyopherin α-2, a nuclear export protein, promotes cancer progression in lung adenocarcinoma

Abstract Introduction: Karyopherin α-2 (KPNA2), a nucleocytoplasmic transport protein, regulates the nuclear import of macromolecules and contributes to carcinogenesis. Actually, KPNA2 is upregulated in various types of cancer tissues and serum from cancer patients. In particular, it is associated with cancer invasiveness and poor prognosis in patient. Here, we tried to present the molecular mechanism and clinical significance of KPNA2 in lung cancer progression and metastasis. Method: First, we retrospectively analyzed the data extracted from EMR for patients with non-small cell lung cancer receiving curative surgical resection in Uijeongbu and Bucheon St. Mary’s Hospitals (n=165). Immunohistochemistry staining was performed on the tumor microarray samples using antibodies against KPNA2. The functional assay and gene silencing was conducted. Result: The clinical and pathological information of the experimental group is as follows. Based on the TMA staining, mean KPNA2 score was 36.5 [0-240] and median value was 10. Based on the TMA staining, patients with high KPNA2 expression were related to lymphatic invasion (p = 0.00782), advanced stage (p = 0.0202), and current smoker (p = 0.0074). KPNA2 expression was higher in squamous cell carcinoma than in adenocarcinoma histotype. Patients with high KPNA2 expression showed shorter survival than those with low KPNA2 (median OS (months), 72.8 vs 42.2, P &amp;lt; 0.0001; median RFS (months), 72.8 vs 32.1, P &amp;lt; 0.0011) and relevant to high recurrence (recurrence rate (%), 24.7 vs 44.7, p = 0.007). Further, high KPNA2 expression was associated with poor survival outcomes. Knockdown of KPNA2 in lung cancer cells suppressed the invasion and migration of lung cancer cells and inhibited metastasis in a mouse model of lung cancer. Inversely, KPNA2 overexpression promoted cancer cell growth, invasion, and migration. Moreover, ectopic expression of KPNA2 made lung cancer cells less sensitive to anticancer drugs, Talazoparib and Olaparib, indicating that KPNA2 enhances drug resistance in lung cancer cells. Conclusion: Collectively, this study suggest that KPNA2 promotes invasiveness and metastasis and is associated with poor patient outcomes; therefore, KPNA2 will be a good therapeutic target or a biomarker in lung adenocarcinoma. Citation Format: Inyoung Cheon, Seoree Kim, Sengran Cho, Heejin Lee, Jung-Sook Yoon, Ji Hyun Lee, Sang Hoon Chun, Hye Sung Won, Soon Auck Hong, Yoon Ho Ko, Young-Ho Ahn. Karyopherin α-2, a nuclear export protein, promotes cancer progression in lung adenocarcinoma [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 7269.

Abstract 4530: Nuclear export inhibitor cooperates with PARP inhibitor to suppress the growth of metastatic castration resistant prostate cancer in vivo

Abstract Background: Aberrant nuclear protein transport, often observed in cancer, causes mislocalization-dependent inactivation of critical cellular proteins. Earlier we showed that overexpression of nuclear export protein exportin 1 (XPO1) is linked to higher grade and Gleason score in metastatic castration resistant prostate cancer (mCRPC). The network topology computational approach (NTCP) determined a higher synthetic lethal score between XPO1 and poly (ADP-ribose) polymerase (PARP1). We showed that selective inhibitor of nuclear export (SINE) could synergized with PARP inhibitors in mCRPC cell lines. Here we evaluated the efficacy of SINE and PARP inhibitor (PARPi) combination in cell line derived as well as patient derived xenograft (CDX and PDX) models and deciphered the mechanism of synergy. Methods: For CDX model, the 22rv1 mCRPC cells were grown as subcutaneous xenografts in ICR-SCID male mice. For PDX model, tissue was collected from Champions Oncology (CTG-3581) and grown subcutaneously in CEIA/NOG male mice. SINE dosed orally at 10-15 mg/kg twice a week and PARPi dosed orally at 50 mg/kg daily. For in vitro mechanistic study, 22rv1 cells were subjected to RNAseq and proteomic analysis after treatment. Data analysis was performed using iPathwayGuide (advaitabio.com). Results: The CDX and PDX showed pronounced anti-cancer efficacy by this combination compared to single agents without any significant weight loss. Survival analysis in CDX model demonstrated enhanced benefits to the mice of combination group. Immunohistochemistry (IHC) revealed apoptotic cell death in the combination group which is evident from cleaved caspase 3 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). To decipher the mechanism of synergy we performed transcriptomic (RNAseq) and proteomic analysis in vitro. We observed downregulation of DNA replication related gene minichromosome maintenance complex component 6 (MCM6) and cell division cycle 6 (CDC6) in the combination treatment. Gene set enrichment analysis (GSEA) also showed low enrichment scores for DNA replication. Proteomic analysis revealed a down regulation of DNA replication modulators such as HMGB2 and DNAJC9 which work on DNA coiling and histone respectively. Conclusions: Taken together, this study revealed the therapeutic potential of SINE-PARPi combination via targeting DNA damage response pathway in mCRPC. Further evaluation of molecular synergy in the xenograft models are underway through RNA interference (RNAi) technique and Digital Spatial Profiling (DSP). Citation Format: Md Hafiz Uddin, Amro Aboukameel, Husain Y. Khan, Sahar F. Bannoura, Frank Cackowski, Rafic Beydoun, Gregory Dyson, Seongho Kim, Julie Boerner, Vinod Shidham, Ramzi M. Mohammad, Boris C. Pasche, Asfar S. Azmi, Elisabeth I. Heath. Nuclear export inhibitor cooperates with PARP inhibitor to suppress the growth of metastatic castration resistant prostate cancer in vivo [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 4530.

Abstract 4574: Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma

Abstract Introduction: A novel KRAS inhibitor, MRTX1133, specifically targeting the G12D isoform, prevalent in pancreatic ductal adenocarcinoma (PDAC), holds promise for a substantial PDAC patient population. Nevertheless, given the clinical experience with sotorasib (a KRASG12C inhibitor) in lung cancer, it's anticipated that resistance to the KRASG12D inhibitor will eventually emerge in most PDAC patients. This underscores the need to identify strategies that can enhance the efficacy of KRAS inhibitors in PDAC. Emerging evidence suggests that the nuclear export protein exportin 1 (XPO1) is a druggable vulnerability in KRAS mutant cancers. XPO1 plays a pivotal role in shuttling various critical tumor suppressors, genome surveillance proteins, and transcription factors out of the nucleus and is frequently overexpressed in various cancers, including PDAC. In this study, we employed both in vitro and in vivo models to assess the potential of KPT8602, an investigational and next-generation selective XPO1 inhibitor, to synergize with the KRASG12D inhibitor MRTX1133 and sensitize MRTX1133-resistant PDAC cells. Methods: We examined the cytotoxic and molecular effects of KRASG12D inhibitor MRTX1133 in combination with nuclear export blocker KPT8602 in KRASG12D mutant 2D and 3D cellular/spheroid models. The preclinical antitumor efficacy of the combination was evaluated in a KRASG12D mutant PDAC cell derived xenograft (CDX) model. Results: KPT8602 synergized with MRTX1133 in vitro yielding suppressed proliferation of KRASG12D mutant PDAC cells. Similar synergistic effects were also seen in 3D cultures of KRASG12D mutant PDAC cancer cell lines and patient derived primary tumor cells where the combination suppressed spheroid formation and reduced spheroid viability. Also, the combination inhibited the clonogenic potential of KRASG12D mutant PDAC cells. In addition, we developed a PDAC cell line resistant to MRTX1133 and observed that such MRTX1133-resistant cell line was acutely sensitive to treatment with KPT8602. Mechanistically, KPT8602 and MRTX1133 combination led to reduction in protein expression of KRAS downstream effectors and cell cycle markers. Furthermore, a combined administration of KPT8602 and MRTX1133 at sub-optimal doses resulted in remarkable tumor growth inhibition in a KRASG12D mutant HPAC CDX mice model. Moreover, KPT8602 as a maintenance therapy also prevented tumor relapse. Conclusion: This is the first study demonstrating that nuclear export blocker KPT8602 can synergize with KRASG12D inhibitor MRTX1133 and has the potential to target MRTX1133-resistant PDAC cells. This study provides a rationale for combining MRTX1133 with KPT8602 for the treatment of PDAC patients with KRASG12D mutant tumors. Citation Format: Husain Yar Khan, Amro Aboukameel, Md Hafiz Uddin, Sahar F. Bannoura, Khaled Keffri, Medha Jasti, Rachael Virga, Sarah Motorwala, Khalil Choucair, Tarik Hadid, Mohammad Najeeb Al-Hallak, Eliza Beal, Miguel Tobon, Rafic Beydoun, Greg Dyson, Steve Kim, Misako Nagasaka, Philip A. Philip, Basel El-Rayes, Herbert Chen, Anthony Shields, Ramzi Mohammad, Boris Pasche, Asfar Azmi. Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma [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 4574.

Efficacy of Selinexor in Relapsed/Refractory Multiple Myeloma (RRMM) Patients with del17p and Other High-Risk Abnormalities (A Retrospective Single-Center Study).

Selinexor (Seli) is a first-in-class, oral selective inhibitor of the nuclear export protein, exportin-1 (XPO1). Seli exhibits its antitumor effect through the blockage of XPO1, which increases nuclear retention of tumor suppressor proteins (TSPs), including p53, thereby limiting the translation of oncogenes, triggering cell cycle arrest and the death of malignant cells. Multiple Myeloma (MM) patients with del17p are deficient in TP53 and have a particularly poor prognosis. Given its unique mechanism of action, we investigated whether Seli has increased efficacy in RRMM patients with del17p compared to other high-risk cytogenetics (OHRC). This is an IRB-approved observational study of RRMM patients with high-risk cytogenetics (del17p, t (4;14), t (14;16) or gain 1q) or standard-risk cytogenetics treated at the Levine Cancer Institute (LCI) with a Seli-based regimen between January 2019 and December 2022. Time-to-event endpoints (PFS, OS) were evaluated using Kaplan-Meier (KM) methods. Log-rank tests compared time-to-event endpoints between cohorts [del17p vs. OHRC vs. standard risk]. We identified 40 RRMM patients with high-risk cytogenetics, including 16 patients with del17p and 24 patients with OHRC, as well as 20 with standard-risk cytogenetics. The median age was 62.5 vs. 69 vs. 65.5 years (del17p group vs. OHRC vs. standard risk). The median prior line of therapies was five (range: 3-16) with similar rates of prior autologous stem cell transplant in all arms (68.8% vs. 62.5% vs. 70.0%). The most frequently used regimens were Seli-Pomalidomide-dexamethasone(dex) or Seli-Carfilzomib-dex (Seli-Kd) in the del17p group and Seli-Kd in the OHRC and standard-risk groups. The median time to start the Seli-based regimen after initial MM diagnosis was 5.6 years for the del17p group, 4.1 years in OHRC, and 4.8 years in the standard-risk group. The median follow-up time after the start of the Seli-based regimen was 10.5 months (mos) in the del17p group, 8.4 mos in OHRC, and 10.3 mos in the standard-risk group. In the del17p group, 50% had an objective response, 41.7% in the OHRC, and 35% in the standard-risk group (p = 0.71). Depth of response was also similar across the arms (12.5% vs. 12.5% vs. 10.0% VGPR p = 0.99). The median OS was 10.9 mos in the del17p group, 10.3 mos in the OHRC, and 10.3 mos in the standard-risk group (p = 0.92). The median OS was 15.5 mos for patients who received Seli as a bridging therapy versus 9 mos for Seli use for other reasons rather than as a bridge. Overall, Seli-based regimens showed promising responses even in this heavily pretreated population. Our analysis suggests that Seli-based regimens lead to similar outcomes among RRMM patients with del17p, OHRC, and standard-risk cytogenetics. This contrasts with previously reported outcomes using combinations of novel therapies in this population, where the del17p patients often have a poorer prognosis. Interestingly, our data suggest that Seli is a particularly effective bridging modality for patients preparing for CAR-T cell therapies in our population. Further investigation into this population is warranted, including in earlier lines of therapy, in hopes of seeing a more durable response.

MRNA nuclear retention reduces AMPAR expression and promotes autistic behavior in UBE3A-overexpressing mice

UBE3A is a common genetic factor in ASD etiology, and transgenic mice overexpressing UBE3A exhibit typical autistic-like behaviors. Because AMPA receptors (AMPARs) mediate most of the excitatory synaptic transmission in the brain, and synaptic dysregulation is considered one of the primary cellular mechanisms in ASD pathology, we investigate here the involvement of AMPARs in UBE3A-dependent ASD. We show that expression of the AMPAR GluA1 subunit is decreased in UBE3A-overexpressing mice, and that AMPAR-mediated neuronal activity is reduced. GluA1 mRNA is trapped in the nucleus of UBE3A-overexpressing neurons, suppressing GluA1 protein synthesis. Also, SARNP, an mRNA nuclear export protein, is downregulated in UBE3A-overexpressing neurons, causing GluA1 mRNA nuclear retention. Restoring SARNP levels not only rescues GluA1 mRNA localization and protein expression, but also normalizes neuronal activity and autistic behaviors in mice overexpressing UBE3A. These findings indicate that SARNP plays a crucial role in the cellular and behavioral phenotypes of UBE3A-induced ASD by regulating nuclear mRNA trafficking and protein translation of a key AMPAR subunit.