Cytoplasmic Localization Of Pu.1 With Mutated Npm1 Causes Myeloid Differentiation Arrest

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LAThe most frequently mutated gene in de novo acute myeloid leukemia (AML) is nucleophosmin (NPM1). How mutated NPM1 (mNPM1) confers clonal advantage is unknown. We hypothesized that mNPM1 interferes with the function of one or more master transcription factors (TF) essential for myeloid differentiation. We used mass-spectrometry to comprehensively analyze the protein interactome of endogenous NPM1 and mNPM1 in AML cell nuclear and cytoplasmic fractions, and examined the cell fate implications of observed interactions. NPM1 was found in abundance in both nuclear and cytoplasmic fractions of wtNPM1 AML cells, but only in the cytoplasm of mNPM1 AML cells. In the nucleus of wtNPM1- and cytoplasm of mNPM1-AML cells, the NPM1/mNPM1 interactome was enriched for the master TF driver of monocytic differentiation PU.1 and the RARA co-factors STAT1 and CNOT1. Accordingly, by both Western blot (WB) of cell fractions and immunofluorescence (IF) microscopy, PU.1 was localized in the cytoplasm of mNPM1 AML cells together with mNPM1. Another master differentiation-driving TF highly expressed in AML cells, CEBPA, was not in the NPM1 interactome nor localized to cytoplasm. Nuclear export of NPM1 is dependent on the nuclear export protein XPO1 (CRM1). Selinexor (KPT-330) is an anti-cancer drug that inhibits protein trafficking to cytoplasm by covalently binding to the XPO1 cargo binding pocket. Selinexor 20-50nM relocated both mNPM1 and PU.1 into the nucleus in mNPM1 AML cells, demonstrated by IF and cell fraction WB. Importantly, this nuclear relocation prompted terminal monocytic differentiation of mNPM1 AML cells. The RARA ligand ATRA at 10nM transferred RARA and STAT1 but not mNPM1/PU.1 into the nucleus and triggered terminal granulocytic rather than monocytic differentiation, presumably enabled by high baseline intra-nuclear CEBPA. mNPM1 AML is distinguished by high HOX gene expression. To evaluate a potential cause-effect relationship, Hox expression was examined in Pu.1 knock-out hematopoietic precursors retrovirally transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER). Addition of estrogen to translocate Pu.1-ER into the nucleus substantially decreased Hox expression accompanied by terminal monocytic differentiation. Thus, nuclear export of PU.1 and RARA cofactors with mNPM1 causes the differentiation arrest that defines AML. Low nanomolar concentrations of selinexor transfer PU.1 to the nucleus and trigger terminal monocytic differentiation, while low nanomolar concentrations of ATRA transfer RARA/STAT1 to the nucleus and trigger terminal granulocytic differentiation. These are p53-independent pathways of cell cycle exit distinct from p53-dependent apoptosis that mediates chemotherapy effect. Thus selinexor and/or ATRA differentiation therapy may offer the ∼50% of mNPM1 AML patients with chemo-refractory disease a needed novel, mechanistically rational treatment option.Citation Format: Xiaorong Gu, Reda Mahfouz, Ji Zhang, Francis Enane, Zhenbo Hu, Tomas Radivoyevitch, David Wald, Maria Paola Martelli, Brunangelo Falini, Yosef Landesman, Jaroslaw Maciejewski, Yogen Saunthararajah. Cytoplasmic localization of PU.1 with mutated NPM1 causes myeloid differentiation arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2872.