Nuclear relocalization of mutant NPM1 induces downregulation of Hox/Meis1, terminal differentiation, and cell cycle arrest

Abstract

NPM1 mutated acute myeloid leukemia (AML) is a distinct entity in the WHO classification of hematopoietic cancers. It displays a specific phenotype characterized by favorable prognosis and upregulation of HOX cluster genes. NPM1 is a multifunctional nucleolar chaperone. All the mutations in NPM1 described so far in result in cytoplasmic protein localization (NPM1c) through the acquisition of a nuclear export signal (NES) at the C-terminus. We hypothesized that NPM1 mutA, the most frequent NPM1 mutation, could be specifically targeted using CRISPR to disrupt the C-terminal NES. After transfection of two NPM1 mutated cell lines (OCI-AML3 and IMS-M2) with NPM1c sgRNA, we found that the NPM1mutA allele was edited in 70-90% of cells, while the NPM1wt allele was not targeted. We cloned the edited alleles into a GFP-NPM1 fusion construct, observing nuclear localization in all tested alleles. Nuclear re-localization was followed by significant impairment of cell growth (3.7-4 fold decrease), colony forming ability (16-20 fold reduction in colonies) and engraftment in xenograft models (significant loss of indels at NPM1mutA allele in engrafted cells). The observed immunophenotype and growth defects were preceded by significant downregulation (5 fold) of HOXA genes, HOXB genes and MEIS1. Chromatin profiling revealed that the mechanism of downregulation appeared to involve loss of active promoters and enhancers, rather than the deposition of repressive chromatin marks. We reasoned that the dependency of NPM1-mutated cells on the cytoplasmic localization of NPM1c would sensitize them to nuclear export inhibitors. Indeed, treatment of OCI-AML3 cells with 50nM Selinexor (KPT-330) induced both morphologic and immunophenotypic differentiation beginning as early as day 5. Furthermore, the transcriptional and chromatin changes phenocopied those observed with CRISPR-targeting of the mutant allele. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1 is necessary for maintenance of the leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization are attractive candidates for clinical success in NPM1-mutated AML.