Abstract 1031: Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest
Authors: Brunetti Lorenzo , Gundry Michael C. , Guzman Anna G. , Gionfriddo Ilaria , Mezzasoma Federica , Milano Francesca , Martelli Maria Paola , Falini Brunangelo , Goodell Margaret A. 
Published: July 2017

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. Mutations in NPM1 result in cytoplasmic protein localization (NPM1c+) through the acquisition of a nuclear export signal (NES) at the C-terminus. The most frequent NPM1 mutation is a heterozygous 4bp insertion in exon 12 (mutA).
The role of NPM1 in leukemogenesis is still a matter of debate and there is no direct proof that cytoplasmic localization of mutant NPM1 is necessary for maintenance of leukemia. We recently developed a CRISPR strategy for highly efficient gene editing in hematopoietic cells. We hypothesized that NPM1 mutA could be specifically targeted due to its 4bp insertion, avoiding the WT allele. Using an sgRNA spanning the insertion site (NPM1c sgRNA), we aimed to introduce indels adjacent to the mutation and disrupt the C-terminal NES. To test our hypothesis, we used the NPM1 mutated OCI-AML3 cell line.
After transfection of OCI-AML3 with NPM1c sgRNA, while NPM1mutA allele showed 70-90% indel frequencies, the NPM1wt allele was intact. To determine whether the novel edited alleles generated NPM1 that was re-localized to the nucleus, we cloned the alleles into a GFP-NPM1 fusion construct and observed nuclear localization after transfection. Consistent with this finding, immunofluorescence revealed that edited OCI-AML3 cells had lost nearly all cytoplasmic NPM1. Return of NPM1 protein to the nucleus was followed by terminal differentiation and cell cycle arrest in G1 phase (controls 45±3%, NPM1c sgRNA 68±1.5%). Cell growth (3.7-4 fold decrease in cell counts in NPM1c sgRNA samples), colony forming ability (16-20 fold reduction in colonies in NPM1c sgRNA samples) and engraftment in xenograft models (significant loss of indels at NPM1mutA allele in engrafted cells) were also significantly impaired after NPM1mutA targeting. Furthermore, RNA sequencing on NPM1mutA-targeted and control OCI-AML3 cells revealed almost complete loss of expression of the HOXA and HOXB cluster genes as well as MEIS1 in treated cells (4.5 average fold reduction of HOXA9-A13; 5.6 average fold reduction of HOXB7-B13; 5 fold reduction of MEIS1).
Allele-specific editing is a powerful tool to probe mechanistic aspects of oncogene dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c+ is necessary for OCI-AML3 cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization such as CRM1 inhibitors are attractive candidates for clinical success in NPM1-mutated AML.
Citation Format: Lorenzo Brunetti, Michael C. Gundry, Anna G. Guzman, Ilaria Gionfriddo, Federica Mezzasoma, Francesca Milano, Maria Paola Martelli, Brunangelo Falini, Margaret A. Goodell. Nuclear relocalization of NPM1c induces terminal differentiation and cell growth arrest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1031. doi:10.1158/1538-7445.AM2017-1031