Mutant NPM1 Is Recruited to MLL Target Genes Via Its Acidic Stretch and Nuclear Export Factor CRM1 and Regulates Oncogenic Transcription in Acute Myeloid Leukemia

Mutations that generate cytoplasmic NPM1 (NPM1c) were first described in acute myeloid leukemia (AML) almost two decades ago and represent one of the most frequently mutated genes in these leukemias. Much effort has been focused on the cytoplasmic functions of mutant NPM1, however, its mechanistic role in leukemia development remains elusive. It is especially unclear how NPM1c expression in hematopoietic cells leads to its characteristic gene expression pattern, including many MLL target genes, such as HOXA9 and MEIS1. We have recently shown that NPM1c AMLs are highly sensitive to the disruption of the MLL1 histone methyltransferase complex. Small molecule inhibitors that block the interaction between MLL1 and its adaptor protein Menin have been shown to impair binding of MLL1 to a subset of its target genes and to inhibit leukemia cell proliferation and self-renewal. Several MLL1-Menin inhibitors are currently in Phase I/II clinical trials and show promising activity in patients with NPM1c AML. The effectiveness of these molecules in NPM1c AML prompts the question whether NPM1c and the wildtype MLL complex cooperate directly on chromatin to drive leukemic self-renewal.

Here, we show that a fraction of mutant NPM1c is localized to the nucleus of leukemia cells and can be recruited to chromatin. We used an endogenously tagged degradable NPM1c leukemia cell line that allows rapid small molecule induced degradation to show that endogenous NPM1c binds to chromatin at specific target genes, which are highly expressed in NPM1c leukemias and are co-occupied by high levels of MLL1. The loss of NPM1c from its chromatin target leads to specific alterations in active histone modifications, such as H3K27 acetylation as well as RNA Polymerase II chromatin occupancy, and this is accompanied by rapid changes in gene expression. The genome-wide recruitment of NPM1c to chromatin is dependent on the nuclear exporter CRM1 and its interaction with the nuclear export signal (NES) of NPM1c. NPM1c can also be dissociated from chromatin using the CRM1 inhibitor Selinexor. Using a CRISPR domain screen, we further showed that an acidic region, which has previously been demonstrated to be important for histone binding of wildtype NPM1, is required for the chromatin recruitment of mutant NPM1. In addition, we demonstrated that NPM1c and CRM1 are lost from a subset of NPM1c target loci after treatment with the small molecule Menin-MLL interaction inhibitor VTP-50469, which can be enhanced by the addition of CRM1 inhibitor Selinexor.

Overall, we demonstrate that NPM1c chromatin binding is mediated by a combination of factors, including the CRM1-NES interaction, the acidic region of NPM1c, and the presence of the MLL complex. We further show that NPM1c acts in collaboration with the MLL1 complex to enhance oncogenic transcription and define the mechanism by which MLL1-Menin small molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.

Armstrong:Cyteir Therapeutics: Consultancy, Other: Shareholder; C4 Therapeutics: Consultancy, Other: SHareholder; Accent Therapeutics: Consultancy, Other: Shareholder; Twenty eight-seven Therapeutics: Consultancy, Other: Shareholder; Mana Therapeutics: Consultancy, Other: Shareholder; Janssen: Research Funding; Novartis: Research Funding; Syndax: Research Funding; -: Patents & Royalties: MENIN inhibition WO/2017/132398A1; Neomorph Inc: Consultancy; Imago Biosciences: Consultancy, Other: Shareholder.