Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of functions including ribosome biogenesis, mRNA processing, and maintenance of genomic stability. In acute myeloid leukemia (AML), the terminal exon of NPM1 is often mutated (~30% of adult AMLs), resulting in the change of the nucleolar localization signal into a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA genes, whose overexpression leads to leukemogenic transformation. Recently, it was shown that depletion or re-localization of the NPM1c protein into the nucleus causes downregulation of the HOXA genes, leading to the speculation that NPM1c directly regulates their transcription. Here, we show that NPM1c binds to a subset of active gene promoters marked with high levels of H3K27ac in NPM1c leukemia cell lines and primary leukemia blasts, including well-known leukemia-driving genes such as posterior HOXA, HOXB, and MEIS1/PBX3 genes as well as novel targets IRX5. The binding of NPM1c on chromatin sustains active transcription of key target genes by maintaining high local-concentration of transcriptional complexes, including RNA Pol2, the Super Elongation Complex (SEC) and Menin/MLL1. We also find NPM1c induce this maintenance of high local concentration of transcription complex by multivalent heterotypic interactions. We also found NPM1c could only hijack and amplify the pre-existing active transcription using a NPM1c knock-in HOXB8-immortalized hematopoietic progenitor cell line. Besides the direct modulation of transcription, we found NPM1c also maintains the active chromatin landscape by inhibiting the activity of histone deacetylases (HDACs). Depletion of NPM1c causes histone deacetylation and the silencing of key leukemic genes, leading to cell differentiation and growth arrest. We also found the export protein XPO1 plays a key role by tethering NPM1c onto chromatin. The combination of XPO1 inhibitors (e.g., Selinexor and Eltanexor) with the Menin inhibitor MI-3454 has a synergistic effect on inducing differentiation in both NPM1-mutated leukemia cell lines and PDX model. Together, these findings reveal paradigm-shifting mechanistic insights into NPM1c mediated gene control and open up potential avenues for therapeutic intervention.

Citation Format: Xue Qing David Wang, Dandan Fan, Yiman Liu, Hongzhi Miao, Dong Chen, Xinyu Wang, Haley Gore, Pamela Himadewi, Gerd Pfeifer, Tomasz Cierpicki, Jolanta Su, Liling Wan, Xiaotian Zhang. Mutant NPM1 hijacks active transcriptional machinery to maintain pathogenic gene programs in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A34.