The CRM1 Nuclear Export Receptor Activates HOXA Gene Expression in Leukemogenesis

HOXA genes are effectors of oncogenic transformation that are frequently upregulated in myeloid and T-cell acute leukemias. Chromosomal translocation-derived oncoproteins, including MLL fusions, NUP (NUP98 or NUP214) fusions or CALM-AF10, bind to HOXA genes and result in their overexpression. We have previously demonstrated that a CRM1-dependent Nuclear Export Signal (NES) within CALM is essential for CALM-AF10’s ability to upregulate HOXA genes and cause leukemia in mice. Interfering with the CRM1/CALM-AF10 interaction by either genetic or pharmacologic inhibition abolishes CALM-AF10’s ability to bind to and activate HOXA gene expression. Furthermore, we showed that CRM1 binds to HOXA loci, suggesting that CRM1 recruits CALM-AF10 to its target genes.

To explore whether CRM1 is also involved in the upregulation of Hoxa genes associated with MLL- and NUP98-fusion genes, we measured Hoxa transcript levels in murine leukemia cells treated with the CRM1 inhibitor Leptomycin B (LMB). LMB is a small molecule that covalently binds to the NES binding domain of CRM1 and blocks its ability to interact with NES partner proteins. We found that treatment of MLL-AF10, MLL-ENL, NUP98-HOXA9 or NUP98-AF10 leukemia cells with LMB (1 nM, 2 hours) causes a 50% reduction of Hoxa7, Hoxa9, Hoxa10 and Hoxa11 levels, similar to what is observed in CALM-AF10 leukemia cells. This suggests that in addition to its ability to interact with CALM-AF10, CRM1 may also participate in the transcriptional activation of Hoxa genes caused by MLL- or NUP98-fusion proteins.

To demonstrate the importance of the CRM1/CALM interaction in CALM-AF10-dependent oncogenesis, we studied the biological activity of an artificial CRM1-AF10 fusion protein. Using a murine bone marrow clonogenic progenitor replating assay, we found that while native CRM1 overexpression did not result in transformation, the CRM1-AF10 fusion significantly increased the self-renewal of clonogenic progenitors. This effect was even more pronounced when CRM1 was fused to the MLL partner ENL: transduction with a CRM1-ENL fusion gene caused the immortalization of clonogenic bone marrow progenitors. Both CRM1-AF10- and CRM1-ENL-transduced progenitors displayed overexpression of Hoxa genes. To investigate the leukemogenic potential of CRM1-AF10in vivo, we transplanted mice with retrovirally transduced bone marrow progenitors and found that CRM1-AF10 induces myeloid neoplasms with a low penetrance and long latency (after more than a year of observation, 5 of 15 mice developed myeloid neoplasms between 160 and 220 days). These primary CRM1-AF10 leukemias could be transplanted to secondary recipients and cause myeloid leukemias with a shorter latency. Experiments to determine the leukemogenic potential of CRM1-ENLin vivo are ongoing. In contrast to CRM1-AF10, CRM1-ENL-transduced progenitors displayed a marked proliferative advantage in all transplanted mice (assessed by the elevation in the percentage of GFP-expressing CRM1-ENL-transduced cells in the peripheral blood over time); mice transplanted 74 days ago will be followed to determine survival curves.

In summary, our results demonstrate that CRM1 regulates the expression of Hoxa genes in mouse leukemia cells, and alteration of CRM1’s activity can drive murine leukemogenesis. This has implications for understanding the mechanisms of HOXA deregulation in human leukemias induced by various fusion oncoproteins. It is noteworthy that in addition to interacting directly with CALM-AF10 through the NES, CRM1 physiologically interacts with NUP98 and NUP214 to facilitate transport through the nuclear pore. Our data also suggest that the anti-tumor effects of CRM1 inhibitors (Selective Inhibitors of Nuclear Export, SINEs) currently undergoing clinical trials, could be mediated, at least in part, by their ability to block the transcriptional activation of tumor-promoting genes by CRM1.