NPM1 Mutation Reprograms Leukemic Transcription Network Via Reshaping CTCF-Defined TAD Topology

Abstract

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, it remains poorly understood how NPM1C+ mutation results in altered myeloid master transcription program and HOX expression profile leading to AML disease and whether NPM1 mutant associated transcription signature can be targeted to prevent pathogenesis of acute myeloid leukemia. Here, we report that NPM1C+ reprograms MIZ-1/MYC regulatory axis by altering NPM1-associated CTCF-driven topologically associated domains (TADs) that switches the balance of MIZ1 interaction with coactivator NPM1/p300 and corepressors MYC/G9A complexes to control cell cycle progression and myeloid lineage-specific PU.1/CEBPa transcription networks leading to impairment of myeloid differentiation and leukemic transformation. To test the hypothesis that mislocalization of the NPM1C+ mutant to cytoplasm may alter CTCF mediated three-dimensional (3D) TADs, we carried out Hi-C to examine whether NPM1C+ mutation altered hematopoietic TAD integrity by comparing mouse bone marrow (BM) Lin-cKit+ (LK) stem- and progenitor-enriched cells purified from WT and NPM1C+ knock-in animals, which develop a myeloproliferative neoplasm/myelodsplasia-like condition with a long latency characterized by enlarged spleens, leukocytosis, and thrombocytopenia. Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to altered chromatin accessibility and NPM1 mutant associated gene expression signature, which results in disrupted regulation of the cell cycle and myeloid master transcription factors, and eventually myeloid differentiation block. In contract, Retention of NPM1 in OCI-AML3 nucleus, a NPM1C+ mutant driven AML cells, reverses NPM1C+-driven signature TAD formation and gene transcription leading to proper cell cycle control and myeloid differentiation. Mechanistically, we demonstrated that cytoplasmic mislocalization of NPM1C+ mutant exerts two coordinated effects: (a) alteration of CTCF dependent TAD structure, and (b) MYC-MIZ-1 transcription axis. Both of these regulate target genes critical for cell cycle control and myeloid master transcription factors, Pu.1 and CEBPA, mediated myeloid differentiation. Re-activation of Pu.1 or CEBPa within the nucleus by CRISPR-dCas9 mediated endogenous promoter activation restores myeloid differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators, and prevents NPM1C+-driven gene expression. FinalIy, when we transplanted NPM1OE, activated Pu.1, or activated CEBPA OCI-AML3 cell line or primary NPM1C+ AML patient cells into NSG mice along with control cells, NPM1 overexpression or re-activation of PU.1 or CEBPA in OCI-AML3 cells or NPM1C+ primary AML patient cells prolonged transplanted or PDX mice survival and mitigate AML leukemic blast in transplant or PDX mouse models. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation. Restoration of myeloid transcription program in nucleus reversed NPM1C+-driven transcription signature and promotes myeloid differentiation leading to mitigation of AML.