Leukemogenic nucleophosmin mutation disrupts the transcription factor hub that regulates granulomonocytic fates
Xiaorong Gu, … , Babal K. Jha, Yogen Saunthararajah
Xiaorong Gu, … , Babal K. Jha, Yogen Saunthararajah
Published July 17, 2018
Citation Information: J Clin Invest. 2018;128(10):4260-4279. https://doi.org/10.1172/JCI97117.
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Research Article Hematology Oncology

Leukemogenic nucleophosmin mutation disrupts the transcription factor hub that regulates granulomonocytic fates

Abstract

Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules.

Authors

Xiaorong Gu, Quteba Ebrahem, Reda Z. Mahfouz, Metis Hasipek, Francis Enane, Tomas Radivoyevitch, Nicolas Rapin, Bartlomiej Przychodzen, Zhenbo Hu, Ramesh Balusu, Claudiu V. Cotta, David Wald, Christian Argueta, Yosef Landesman, Maria Paola Martelli, Brunangelo Falini, Hetty Carraway, Bo T. Porse, Jaroslaw Maciejewski, Babal K. Jha, Yogen Saunthararajah

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Figure 5

Nuclear retention of mutant NPM1 and PU.1 by selinexor triggered terminal monocytic differentiation of NPM1-mutated, but not WT, AML cells.

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Nuclear retention of mutant NPM1 and PU.1 by selinexor triggered termina...
(A) Cell counts of NPM1-mutated (OCI-AML3, IMS-M2) and NPM1-WT (OCI-AML2, THP1) AML cells. Decitabine was used to deplete DNMT1. Cell counts by automated counter. Mean ± SD of 3 independent experiments. *P < 0.01 (significant after Bonferroni’s correction), t test, 2-sided, selinexor or decitabine versus vehicle on day 5; NS, P > 0.025. (B) Protein levels of MYC (master transcription factor driver of proliferation) and p27/CDKN1B (cyclin-dependent kinase inhibitor mediating cell cycle exits by differentiation). WB. Dec, decitabine; Sel, selinexor. (C) Monocyte lineage marker CD14 and granulocyte lineage marker CD11b expression. Flow cytometry. (D) Cell morphology, day 5. Giemsa stain. Leica DMR microscope; original magnification, ×630. Quantified in Supplemental Figure 8. (E) MCSFR/CSF1R or GCSFR/CSF3R expression. QRT-PCR, multiple primer sets were used for each gene (#1–3/4). Mean ± SD 3 independent experiments. *P < 0.01 (significant after Bonferroni’s correction), 2-sided t test, selinexor versus vehicle (Veh); NS, P > 0.0125.