In this study, we have synthesized analogs of LMB, the prototypical nuclear export inhibitor, which show potential as novel anticancer therapeutics. LMB itself has significant in vitro
potency but is poorly tolerated in vivo
). The covalent nature of the interaction with CRM1 needs to be considered when comparing compounds. In vitro
values for covalent inhibitors determined after prolonged drug exposure cannot be interpreted as indicators of target binding affinity, as they would be for reversible inhibitors, since covalent inhibitors have essentially infinite binding affinity for their target. The IC50
values for NEIs determined after short drug exposure serve as an indirect measure of the rate of CRM1 inactivation and thereby reflect the relative efficiencies of a series of compounds at inactivating CRM1. The observation that our series of NEIs retain essentially equivalent cytotoxicities to LMB after a one hour exposure () demonstrates that they retain the potency of LMB.
In order to define the kinetics of nuclear export inhibition for this class of compounds, we wanted to find an endogenous protein dependent on CRM1 for nuclear export and which is ubiquitously expressed. Such a protein could serve as a useful biomarker for nuclear export in all cell types without the need for additional inducers of expression or nuclear import. We chose RanBP1, an accessory protein for RanGAP-mediated dissociation of CRM1-bound cargo proteins upon export to the cytoplasm. The rapid shuttling of RanBP1 into the nucleus makes it a particularly useful tool for the study of NEIs. Other CRM1-dependent cargo proteins that are imported more slowly, not ubiquitously expressed, or that require inducing conditions for their nuclear import are not as useful for the purpose of defining the kinetics of CRM1 inhibition, although they could undoubtedly play a role in mediating downstream cytotoxic effects. Using RanBP1 as a biomarker, our in vitro experiments show that exposure of cancer cells to NEIs leads to a rapid and prolonged block of nuclear export in all cell types tested.
The observation that the induction of p53 is detectable at 24 or 48 hours () despite the fact that nuclear export has recovered in the majority of the cell population by 24 hours ( and data not shown) shows that the downstream effects of nuclear export inhibition can persist longer than the actual nuclear export block. Nuclear entrapment of p53 turns on the p53 transcriptional program, including activation of transcription of p53 itself. As the data show, this leads to a significant increase in p53 protein levels in the cell (, (9
)). Once this program is turned on, since it is part of an auto-activation loop, its regulation need no longer depend on nuclear export block and will instead depend on factors such as the stability of p53 and the dynamic point at which p53 levels drop to the range in which the positive feedback loop is no longer engaged. Thus, a downstream consequence of nuclear export inhibition, in this case p53 induction, can persist independently of the original signal which was the nuclear export block.
The inhibition of nuclear export is associated with an increase in multiple markers of apoptosis in cancer cells. In contrast to this, NEIs induce cell cycle arrest, but not apoptosis, in normal lung fibroblasts. Thus, although NEIs cause the inhibition of CRM1 in both tumor and normal cell types, a difference is observed in the downstream consequences of this inhibition. The basis of this difference in response remains under investigation.
We have synthesized novel NEIs that are up to 16-fold better tolerated than LMB in mouse models while retaining significant potency. These results suggest that the limited in vivo efficacy of LMB was likely due to off-target effects since our NEIs retain the potent inhibition of CRM1, but are clearly better tolerated in vivo. The reasons why the novel NEIs are better tolerated are currently under investigation. Areas of exploration include the tissue distribution profile of these molecules as well an investigation of their in vivo on-target and off-target protein binding properties. The better tolerance enables these novel NEIs to be dosed at higher levels in vivo. As shows, 10mg/kg of compound 3, which is four-fold higher than the MTD of leptomycin B, demonstrates only modest efficacy whereas a dose of 40mg/kg results in regression. This supports the conclusion that leptomycin’s low MTD limits its efficacy and higher doses are required for robust efficacy for compounds such as these, which show comparable activity in vitro. Doses above 30mg/kg of compound 3 are associated with significant efficacy in multiple mouse xenograft models, thereby validating nuclear export as a potentially useful therapeutic target in cancer.
In this study, we have focused on p53 wild type cancer models including an HCT-116 colon model and a SiHa cervical cancer model. In these models, the p53 tumor suppressor becomes trapped in the nucleus upon inhibition of CRM1. In HPV-positive cancer types, such as SiHa, this prevents the aberrant cytoplasmic localization and degradation of p53 and leads to activation of pathways that cause cell-cycle arrest and apoptotic cell death. We show that such effects are not limited to HPV-positive cancers, as NEIs induce p53 activation () and show anti-tumor efficacy in the HCT-116 colon cancer model (). Furthermore, we have also tested for anti-tumor activity of compound 3
in a variety of other tumor models, including NCI-H460 (non-small cell lung cancer), A375 (melanoma), and K562 (chronic myelogenous leukemia) (Supplementary Table 2
). Compound 3
demonstrated anti-tumor activity in all of these models ranging from induction of tumor regression to tumor growth inhibition. Thus, in contrast to the poor in vivo
activity of LMB, the NEI analog compound 3
shows robust efficacy in all xenograft models examined to date and is of great interest as a potential cancer therapeutic.
Although we have focused here on p53 wild type cancer types, CRM1 mediates the nuclear export of numerous other proteins that are also important therapeutic targets. Various lines of evidence provide a strong biological rationale for the use of NEIs in multiple cancer types. For example, in chronic myelogenous leukemia (CML), the oncogenic BCR-Abl tyrosine kinase is found in the cytoplasm where it activates a number of mitogenic signaling pathways (20
). Treatment of CML cells with the tyrosine kinase inhibitor imatinib not only inhibits BCR-Abl but also promotes its shuttling into and out of the nucleus (23
). Co-administration of LMB with imatinib was demonstrated to cause nuclear accumulation of BCR-Abl, ultimately resulting in the activation of programmed cell death both in vitro
) and in ex vivo
). Given the poor in vivo
tolerance of LMB, a significant therapeutic benefit could be gained by combining a potent but better tolerated CRM1 inhibitor with an inhibitor of BCR-Abl. This combination has the potential to overcome the problem of drug resistance by eradicating rather than inhibiting the growth of CML cells.
Similarly, LMB inhibition of CRM1 has been shown to promote the nuclear buildup of the Forkhead family of transcription factors (FOXOs) (25
). These transcription factors are regulated by multiple signaling pathways that play critical roles in tumorigenesis including the PI3K/PTEN/Akt pathway (reviewed in (27
)). In PTEN-deficient cells, the Akt pathway is activated and FOXO transcription factors are rendered inactive by localization to the cytoplasm. Restoring PTEN function in these cells blocks Akt activity and restores nuclear localization of FOXO and, therefore, its ability to activate downstream factors. Prolonged FOXO residence in the nucleus leads to the induction of pro-apoptotic genes and ultimately to growth arrest and death in PTEN-null tumor cells (29
). In addition, it has recently been shown that localization of FOXO to the nucleus in PTEN-null cells inhibits Hif1 transcriptional activity(31
), thereby potentially interfering with the ability of PTEN-null cells to survive under hypoxic conditions. It will thus be of great interest to examine the efficacy of NEIs in PTEN-deficient cancers.
In conclusion, the NEIs we have synthesized have enabled us to validate CRM1 as a target for anti-cancer therapeutics. These data show that the limited in vivo efficacy observed for LMB was a result of its poor tolerance. The identification of NEIs that are significantly better tolerated has demonstrated that molecules with this mechanism of action can show robust in vivo efficacy. These results provide strong evidence supporting the development of NEIs as a novel anticancer therapy.