Selective Inhibitors of Nuclear Export (SINEs) are a novel class of anti-cancer drugs showing very promising efficacy in ongoing clinical trials. SINEs selectively bind to the nuclear transport receptor CRM1 and abolish its ability to export cargo proteins from the nucleus to the cytoplasm. Thus, the therapeutic activity of SINEs is thought to result from nuclear sequestration of tumor promoting proteins and/or tumor suppressors such as p53. Our laboratory has recently discovered a completely unexpected function of CRM1 that suggests a novel mechanism by which SINEs exert their anti-tumor effects. We propose to explore this unanticipated function of CRM1 to shed insight on the mechanism of action of this important class of antitumor drugs. We discovered that CRM1 binds to chromatin and activates the transcription of pro-leukemia genes in cells transformed by the CALM-AF10 fusion oncoprotein. CALM-AF10 chromosomal translocations have been identified in aggressive myeloid and T-lymphoid acute leukemias. These leukemias are characterized by over- expression of HOXA cluster genes, which are critical effectors of leukemic transformation. CALM-AF10 has a nuclear export signal (NES) that mediates interaction with CRM1. We determined that transcriptional activation of HOXA genes by CALM-AF10 is mediated by CRM1: abrogation of the NES-CRM1 interaction by either genetic mutation of the NES or pharmacologic inhibition with SINEs impairs the transcription of HOXA genes and abolishes CALM-AF10's oncogenicity. Furthermore, we determined that CRM1 binds HOXA gene loci and that the NES-CRM1 interaction enables CALM-AF10 to be recruited to HOXA genes and induce their transcription. Our discovery that CRM1 binds to leukemogenic genes and induces their transcription suggests a novel mechanism by which SINEs inhibit tumor growth. Our long-term goal is to elucidate the molecular mechanisms by which SINEs block the proliferation of leukemia cells while sparing normal hematopoietic progenitors. The objective of this application is to characterize the effect of SINEs on gene expression by identifying the array of genes bound and regulated by CRM1. Our central hypothesis is that the anti- cancer efficacy of SINEs is mediated, at least in part, by their ability to interfere with CRM1's direct effect on the transcription of genes that control the behavior of cancer cells. Here we propose to identify the array of genes regulated by CRM1 in CALM-AF10 leukemia cells; chromatin immunoprecipitation followed by sequencing (ChIP-Seq) and RNA-Seq will be used to identify genes regulated by CRM1 (Aim #1). The subset of CRM1 targets regulated by CALM-AF10 will be determined and validated by quantitative PCR and ChIP (Aim #2). The rationale for the proposed research is that a better understanding of the molecular mechanisms of the anti-cancer activity of SINEs will optimize their therapeutic efficacy by guiding their use in selected indications and by identifying potential synergies with adjunctive pharmacological approaches.
Selective Inhibitors of Nuclear Export (SINEs) are promising novel anticancer therapies with reduced toxicity towards normal cells, but the molecular basis of their activity remains poorly understood. Our preliminary work is the first to show that SINEs act through CRM1 to alter the transcription of pro-leukemia genes. The identification of previously undefined CRM1 transcriptional targets will advance our understanding of the selective toxicity of SINEs toward neoplastic cells and, ultimately, allow for optimization of the therapeutic efficacy of this novel class of anti-cancer agents.
