The SQSTM1-NUP214 Fusion Protein Cooperates with Crm1 to Activate Hoxa Genes and Drives Leukemogenesis in Mice

Background: The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the NUP phenylalanine-glycine (FG) repeat motifs that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we studied the SQSTM1-NUP214 fusion. Methods: We synthesized a SQSTM1-NUP214 fusion protein which retains only a short C-terminal portion of NUP214 containing FG motifs that mediate interaction with Crm1, and then introduced point mutations targeting these FG motifs (SQSTM1-NUP214^FGmut). We compared the activity of these two fusion proteins using co-immunoprecipitation with CRM1, methylcellulose colony assays, murine transplantation, RT-qPCR, and chromatin immunoprecipitation. Results: We found that the ability of the SQSTM1-NUP214^FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Further, mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214^FGmut was greatly diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice (6/6), whereas none of the SQSTM1-NUP214^FGmut reconstituted mice developed leukemia (0/7). These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214^FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impairing the interaction of SQSTM1-NUP214 with Crm1 reduced binding of the fusion protein to Hoxa and Meis1 loci. Conclusions: These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.