Leukemogenic nucleophosmin mutation disrupts the transcription factor hub that regulates granulomonocytic fates
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
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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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 11

Resistance is by avoidance of selinexor-induced nuclear relocation of mutant NPM1/PU.1. OCI-AML3 NPM1-mutated cells were selected for resistance to selinexor by culture in selinexor, with up to 50 nM added every 3 days.

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Resistance is by avoidance of selinexor-induced nuclear relocation of mu...
(A) NPM1 and PU.1 localization in the resistant cells by IF. DAPI was used to stain for nuclei. Images by Leica SP8 inverted confocal microscope; magnification, ×630. (B) NPM1 and PU.1 cytoplasmic versus nuclear localization in the resistant cells. WB of nuclear and cytoplasmic fractions. (C) Parental and selinexor-resistant OCI-AML3 cells were sensitive to noncytotoxic concentrations of decitabine. Cell counts by automated counter. Mean ± SD for 3 independent experiments. *P < 0.01 (significant after Bonferroni’s correction), selinexor or decitabine versus vehicle on day 7, 2-sided t tests.