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  • In the cytoplasm the ternary CRM

    2019-07-11

    In the cytoplasm, the ternary CRM1–cargo–RanGTP complex is disassembled by the action of Ran-binding proteins RanBP1/2 and RanGAP. A recent kinetic study showed that the Ran-binding domains (RanBDs) of the cytoplasmic proteins RanBP1 and RanBP2, but not RanGAP, accelerate dissociation of NES from CRM1 and RanGTP by over 2 orders of magnitude. Crystal structure of yeast CRM1–RanBP1–RanGTP complex showed that the NES- and RanBD-binding sites on CRM1 are distinct and that the binding of RanBD induces the movement of a long internal loop in HEAT repeat 9 (referred to as HEAT9 loop), from switch I of RanGTP to the concave side of the NES-binding cleft (the inner surface of HEAT repeats 11 and 12 of CRM1), driving rotations and translations of the α-helices constituting the NES-binding site. This results in closure of the Fenofibric acid cleft to dissociate NES. Structure-based mutagenesis of crucial hydrophobic residues of the HEAT9 loop provided strong support for this allosteric mechanism of NES release and also indicated that the HEAT9 loop functions as an allosteric autoinhibitor to stabilize CRM1 in a conformation that is unable to bind NES cargo in the absence of RanGTP. Mutational analyses also indicated that the C-terminus of CRM1 plays an important role in stabilizing CRM1 in an autoinhibited state in the absence of RanGTP,[25], [26] and so it has been proposed that the HEAT9 loop and the C-terminus of CRM1 cooperate to stabilize the NES-binding cleft in a closed state in the absence of RanGTP.[24], [26] Nevertheless, the absence of a high-resolution structure of CRM1 in isolation meant that the structural basis for autoinhibition remained obscure.
    Results and Discussion
    Materials and Methods
    Acknowledgements We thank our colleagues in Nagoya, especially Masako Koyama, Junya Kobayashi, Hidemi Hirano, and Tatsuo Hikage, for assistance and discussion. We are also indebted to the staff of Photon Factory and SPring-8 for assistance during data collection. This work was supported in part by the Sumitomo Foundation and also by JSPS/MEXT KAKENHI (18687010, 21770109, and 23770110).
    Nuclear export
    Blocking nuclear export of proteins Fig. 1 demonstrates three potential means of attenuating nuclear export of the cancer drug target topo IIα: (1) CRM1 inhibitors, (2) NES small molecule inhibitors, and (3) casein kinase 2 inhibitors, with the last preventing post-translational Fenofibric acid phosphorylation of topo IIα.
    Clinical experience with CRM 1 inhibitors
    In studies done with CRM1 inhibitors (see Table 1), single-agent CRM1 inhibitors induced apoptosis in cancer cell lines, slowed tumor growth in xenograft mice, and improved survival. However, in most cancers, single-agent CRM1 inhibitors were less likely to eliminate established tumors or reduce tumor burden. In addition, the CRM1 inhibitor\'s ability to induce apoptosis or anti-proliferative effects in cell lines improved synergistically when combined with chemotherapeutics such as ABT-737, AraC, bevacizumab, BRAF inhibitors, bortezomib, carfilzomib, cisplatin, daunomycin, decitabine, docetaxel, doxorubicin, imatinib, melphalan, oxaliplatin, paclitaxel, SN-38, nutlin-3a, or topotecan. CRM1 inhibitors have been shown to sensitize cancer cells both in vitro, in xenografts, or ex vivo to these chemotherapeutics and in some cases reverse drug resistance. Listed in this section are several studies where CRM1 inhibitors were used in combination with other cancer therapeutics and in drug-resistant/relapsed cancers. A table of current abstracts (Table 1) summarizes the latest findings in CRM1 combination therapies. In addition, we include a table on drug sequencing in the treatment of multiple myeloma (Table 2).
    Funding Our study received valuable assistance from the Flow Cytometry Core Facility at the H. Lee Moffitt Cancer Center & Research Institute; an NCI designated Comprehensive Cancer Center, supported under NIH grant P30-CA76292. We especially thank Jodi Kroeger for her expert assistance with flow cytometry. This work was partially supported by the State of Florida Bankhead-Coley Team Science Project Grant 2BT03-43424. Experimental data presented in Table 2 contain lab results that were supported by Karyopharm Therapeutics (Natick, MA).