Browsing by Subject "Karyopherins"
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Item Identification of Oncogenic KRAS-Associated Vulnerabilities in Non-Small Cell Lung Cancer(2016-05-26) Kim, Ji Mi; Lum, Lawrence; White, Michael A.; Fontoura, Beatriz; Cobb, Melanie H.Activating mutations in KRAS are frequently involved in the pathogenesis of non-small cell lung cancer (NSCLC), the disease responsible for the most cancer-related deaths in the US. Despite intensive efforts to develop drugs that directly interfere with KRAS activity over the past decade, no effective inhibitor has been developed. As an alternative, synthetic-lethal therapeutic opportunities are being pursued using large-scale, RNAi-based, functional genomics platforms. We first addressed two major challenges associated with RNAi-based primary synthetic-lethal screens; a prevalent miRNA-like behavior of siRNA and cell line-dependent phenotypic diversity within intra-lineage KRAS-driven cancer. In consideration of these, we performed a whole-genome synthetic-lethal siRNA screen, powered by 106 NSCLC lines and integrated with gene set enrichment analysis. This identified components of nuclear transport machinery as selectively essential for KRAS mutant NSCLC lines. We found that pharmacological inhibition of a key nuclear export receptor, XPO1 (a.k.a. CRM1), was sufficient to induce robust and selective apoptosis in KRAS mutant NSCLC cells in vitro and to cause significant impairment of KRAS mutant tumor growth in vivo. Mechanistically, XPO1-depedent nuclear export machinery was required to maintain NFκB-mediated survival signaling. We discovered that a subset of KRAS mutant NSCLC lines bypassed the addiction to XPO1-dependent nuclear export via YAP1 activation as a consequence of previously unappreciated co-occurring loss-of-function mutations in FSTL5 and mutations in Hippo pathway. The intrinsic resistance was reversed by coadministration of YAP1/TEAD inhibitor. Thus, our study suggests that XPO1 can be exploited for a promising therapeutic opportunity for KRAS mutant lung cancer and provides strategies for genomics-guided application of clinically available XPO1 inhibitors.Item Inhibition of Karyopherin-β1-Mediated Nuclear Import of Lineage-Defining Transcription Factors in Small Cell Lung Cancer(2021-05-01T05:00:00.000Z) Kelenis, Demetra Patrica; Minna, John D.; Johnson, Jane E.; Cobb, Melanie H.; McFadden, David G.Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that accounts for approximately 16% of lung cancer diagnoses. Recent genomic studies support the classification of this disease into four different subtypes based on the expression of the lineage-defining transcription factors ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), and YAP1 (SCLC-Y). Together, the SCLC-A and SCLC-N subtypes account for a majority of SCLC. ASCL1 and NEUROD1 are Class II bHLH transcription factors that command the expression of SCLC oncogenes and are known to drive distinct transcriptional programs, conferring SCLC-A and SCLC-N with different molecular and physiological features. ASCL1 has also been shown to be required for tumor formation in SCLC mouse models, and, where tested, both ASCL1 and NEUROD1 play key roles in maintaining growth of SCLC-A and SCLC-N cell lines. Together, these findings suggest that strategies to inhibit ASCL1 and NEUROD1 activity may represent an attractive SCLC therapy, and provide new insight into the underlying plasticity of the major SCLC subtypes. ASCL1 and NEUROD1 are translated in the cytoplasm and must be transported into the nucleus in order to regulate gene expression. Interestingly, it has been shown that NEUROD1 is selectively imported into the nucleus via the nuclear transport receptor Karyopherin-β1, or KPNB1 (also known as Importin-β1) in several mouse and human cell lines. Additionally, a muscle-specific bHLH transcription factor, MYOD, has also been shown to be imported into the nucleus by KPNB1, suggesting a common nuclear import mechanism for Class II bHLH transcription factors. However, whether ASCL1 is also imported into the nucleus via the same mechanism, and whether the nuclear import of NEUROD1 is mediated by KPNB1 in the setting of SCLC, had not been tested. Here, I 1. identified KPNB1 as a nuclear import receptor for ASCL1 and NEUROD1 in SCLC, 2. showed that KPNB1 inhibition disrupts SCLC- A patient derived xenograft (PDX) growth in vivo, and preferentially disrupts the growth of ASCL1/NEUROD1+ SCLC in vitro, and 3. compared the changes in gene expression following the inhibition of ASCL1 or KPNB1 activity across in vitro and in vivo models of SCLC.Item Nuclear Export Receptor CRM1 Recognizes Nuclear Export Signals with Diverse Conformations(2017-04-13) Fung, Ho Yee Joyce; Nam, Yunsun; Chook, Yuh Min; Grishin, Nick V.; Yu, Hongtao; Zhang, XuewuThe Chromosome Region of Maintenance 1 or CRM1 protein facilitates export of hundreds of proteins and RNA molecules from eukaryotic cell nuclei. CRM1 recognizes its protein cargoes by their 8-15 residues-long nuclear export signals or NESs, which bind to a hydrophobic groove in CRM1. NESs are highly variable in sequence and structure. Their sequences are described by multiple sequence patterns of four variably-spaced hydrophobic residues, and three previous structures showed CRM1-bound NESs adopting either helix-strand or mostly extended conformations while the CRM1 groove remains unchanged. The plasticity of CRM1-NES interaction and the repertoire of NES conformations were unclear. Many NES sequences also seem incompatible with the asymmetric and seemingly structurally invariant NES-bound CRM1 groove. I developed a general strategy to crystallize CRM1 bound to NES peptides in order to study how diverse sequences bind CRM1. In the first study, I solved crystal structures of CRM1 bound to NESs with unusual sequences, which bound the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome. In the second study, I solved eight additional structures of diverse NESs, which show peptide conformations ranging from mostly loop-like to all-helical NESs, occupying the CRM1 groove to different extents. Comparison of >13 structures show a total of 5-6 different NES conformations where the only conserved structural element is one turn of helix, which has dihedral angles that proceed from helical to β-strand. All NESs also participate in hydrogen bonds with the human CRM1 Lys568 side chain, which functions as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs. We now have comprehensive structural knowledge for NESs of most known patterns. The structural information obtained is now the foundation for a new peptide docking/modeling approach to improve the accuracy of NES prediction.Item Nuclear Export Signal Recognition by CRM1 Carrying the Oncogenic E571K Mutation and Structure-Based NES Prediction(2020-08-01T05:00:00.000Z) Baumhardt, Jordan Matthew; Erzberger, Jan; Chook, Yuh Min; Grishin, Nick; Lin, Milo; Fontoura, BeatrizNuclear-cytoplasmic trafficking is an essential cellular process in eukaryotes that maintains and regulates the spatial distribution of key cellular processes in the nucleus and the cytoplasm. CRM1 is the major nuclear export mediator, which facilitates nuclear export of hundreds of protein and RNA molecules. CRM1 is essential to the survival of eukaryotic cells and is overexpressed or mutated in a variety of cancers. This involvement of CRM1 in cancer cell survival has led to the development of novel small molecule inhibitors such as XpovioTM or Selinexor, which has been approved for treatment of advanced and relapsed multiple myeloma and is also in many clinical trials for a variety of cancers. In addition, the point mutant CRM1(E571K) is found in a variety of tumors and is highly prevalent in some B-cell lymphomas. A deep understanding of how CRM1 recognizes the diverse NESs in hundreds to thousands of cargos is needed to understand the role of CRM1 in cancer pathogenesis and the mechanism of action of its inhibitors. My work has focused on expanding our foundational knowledge of CRM1-NES interactions in the presence and absence of the oncogenic E571K mutation. Previous studies measured the affinities of CRM1 interactions with 22 different nuclear export signals (NESs) from protein cargos using a low-throughput differential photobleaching assay. However, hundreds of very diverse CRM1 cargos and their NESs have yet to be characterized, limiting our understanding of the biological roles of CRM1 in disease. I developed a high-throughput, quantitative fluorescence polarization assay to measure the affinity of >100 NES peptides for WT CRM1 and for the oncogenic CRM1(E571K) to understand how the mutation affects NES binding. The large body of CRM1-NES affinity data was also used to develop a new structure-based NES prediction method. The CRM1 oncogenic mutation E571K is highly prevalent in some subtypes of B-cell lymphomas and can drive tumors in mouse B-cell models, but the mechanism of tumorigenesis is unclear. Using structural and biophysical approaches, I studied the recognition of 27 diverse NESs by CRM1(E571K) and showed that while most cargos are unaffected by the mutation, a small subset of highly charged NESs have greater than 10-fold affinity changes for the cancer mutant. To study whether these affinity changes cause nuclear export defects in cells, I used CRISPR/Cas to generate generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K. HEK 293 cells with CRM1WT/E571K or CRM1E571K/E571K had decreased proliferation and cells with homozygous CRM1E571K/E571K had obvious cell cycle defects. The eIF4E-Transporter which binds 10-fold weaker to CRM1(E571K) is mislocalized in HEK 293 cells with CRM1(E571K) and in chronic lymphocytic leukemia patient cells burdened with the CRM1 mutation. Additionally, I solved crystal structures of CRM1 bound to covalent KPT inhibitors, which showed that the mutation site, position 571 of CRM1, is located far from the bound drugs and thus unlikely to substantially affect their ability to inhibit CRM1. In order to understand the effects of the E571K on CRM1 activity and further illuminate possible cancer mechanisms, it is crucial to identify many more NESs across the entire human proteome. Therefore, NES prediction is of great interest, but existing sequence-based approaches give high false positive rates. My work also shows that may NES-like peptides are not accessible in the full-length cargos to to CRM1, in ways that many existing NES predictors cannot identify. To improve NES prediction, our collaborators in the Grishin Lab utilized CRM1-NES crystal structures as templates to develop a structure-based NES predictor and incorporated full-length protein context considerations to accurately identify novel NESs that are likely to be functional. I contributed to this work by expanding the CRM1-NES structural dataset and I helped to interpret modeling outliers to iteratively improve the CRM1-bound NES models. I also assisted the Grishin lab in using this structure-based predictor to map somatic mutations found in cancer patients that may disrupt nuclear export of the NES-containing proteins. Overall, I have added >100 new CRM1-NES binding affinity measurements and 16 CRM1 (WT or E571K) crystal structures to the field. These results have shown how the oncogenic E571K mutation binds very differently to small subset of highly charged cargos, and provided a robust dataset to develop a new structure-based NES prediction tool. Future work will build on this foundation to clearly illustrate how nuclear export defects lead to oncogenesis, and potentially give key insights into improving use of CRM1-targeted therapeutics.Item Recognition Mechanisms of Nuclear Localization and Export Signals(2009-06-19) Süel, Katherine Elizabeth; Chook, Yuh MinThe transport of proteins between the nucleus and cytoplasm of cells is mediated by the Karyopherin beta family of proteins. Karyopherin betas recognize their substrates through either a nuclear localization or export signal depending on the direction of transport. Even though there are ten yeast import Karyopherin betas, for the past thirteen years there was only one well characterized nuclear localization signal, the classical nuclear localization signal. However, a second signal, the proline-tyrosine nuclear localization signal recognized by Karyopherin beta2, was recently identified through X-ray crystallography and biochemical studies of Karyopherin beta2 bound to one of its substrates. These studies identified rules for the recognition of the proline-tyrosine nuclear localization signal by Karyopherin beta2. The signal must have overall basic charge, structural disorder and a weak consensus sequence of an amino-terminal basic or hydrophobic-enriched region followed by a carboxyl-terminal arginine residue separated from a proline and tyrosine residue by two to five residues. The proline-tyrosine nuclear localization signal is also recognized by the Saccharomyces cerevisiae homolog of Karyopherin beta2, Karyopherin 104, demonstrating the generality of this import mechanism across eukaryotes. Thermodynamic analyses of the two known substrates of Karyopherin 104, Hrp1p and Nab2p, revealed physical properties governing its binding. The proline-tyrosine nuclear localization signal is an extended signal with significant sequence diversity. The signal is comprised of three binding epitopes, each of which can have varying energetic strengths in different substrates. The multivalent nature of the signal increases the diversity of the signal as well as the difficulty of identifying new substrates. A bioinformatics search identified putative proline-tyrosine nuclear localization signals which were validated through biochemical studies. Additionally, one of the proteins identified, Tfg2p, was verified as a bona fide Karyopherin 104 substrate. Analysis of Tfg2p's cellular localization revealed that its nuclear localization was not solely determined by the presence of a nuclear localization signal, but was also dependent on its retention in the nucleus. Furthermore, crystallographic studies of substrate Snurportin1 bound to the export karyopherin CRM1 revealed that its nuclear export signal has two binding epitopes implying that the multivalent nature of targeting signals may not be limited to the proline-tyrosine nuclear localization signal.Item Structural and Biochemical Studies of Multiple Importin-Histone Interactions(2016-04-13) Soniat, Michael Maurice, II; Rizo-Rey, José; Chook, Yuh Min; Jiang, Youxing; Goodman, Joel M.; Li, BingMultiple Importins can bind the N-terminal tails of histones H3 and H4, and import them into the nucleus to be assembled into the nucleosomes. However, it is not known what sequence elements in the histone tails are recognized by each of the Importins. Through structural and quantitative biochemical analysis, I identified binding determinants in the N-terminal tails of histones H3 and H4 for each of seven different human Importins (Impα, Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9). Crystal structure of the H3 tail bound to Kapβ2 identified H3 tail residues 11-27 as the important binding element, which resembles a PY-NLS that is missing the canonical proline-tyrosine motif. This same N-terminal basic segment of H3 is also important for binding Impβ, Imp4, Imp5, Imp7, Imp9, and Impα. In addition, a C-terminal IK-NLS-like motif at residues 35-40 of H3 is also used to bind Imp5, Imp7, Imp9 and Impα. Interactions of the H4 tail with the same Importins show a similar trend of relative affinities as the H3 tail, though at least 10-fold weaker. Similar to the H3 tail, the H4 tail also uses one or two basic regions to bind the Importins. I also studied the effects of histone tail acetylation on Importin-histone interactions and showed that acetylation of Lys14 of the H3 tail impairs binding to all six Importins and Impα while acetylation of Lys18 of H3 tail and acetylation of Lys5 and Lys12 of the H4 tail had only mild effects on binding to the Importins. Lastly, I studied Importin binding to the H3/H4 dimer and showed that only one Importin molecule binds each H3/H4 dimer. Furthermore, the Importin-binding trend with the H3/H4 dimer is very different than with the N-terminal tails alone suggesting additional interactions with the histone fold domains of H3 and H4. Overall, I have mapped Importin-binding determinants for the H3 and H4 and revealed acetylation effects on Importin-histone binding.