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 1

The NPM1 interactome includes the master transcription factor PU.1, which is cytoplasmically dislocated along with mutant NPM1 in NPM1-mutated AML cells.

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The NPM1 interactome includes the master transcription factor PU.1, whic...
(A) Transcription factors pulled down with NPM1 and mutant NPM1 (mNMP1) from nuclear (N) and cytoplasmic (C) fractions of WT and NPM1-mutated (mut) AML cells. Endogenous NPM1 and mutant NPM1 were immunoprecipitated from nuclear and cytoplasmic fractions of WT (THP1) and NPM1-mutated AML cells (OCI-AML3), and protein interactions were analyzed by LC-MS/MS. Only interactome transcription factors are shown (additional data in Supplemental Table 1). Individual protein enrichment is presented as total spectral counts, a semiquantitative method for estimating the abundance of a specific protein in the coimmunoprecipitate; larger circle size indicates higher number of total spectral counts for the protein. (B) NPM1 and PU.1 interaction in nuclei of WT AML cells, and in cytoplasm of NPM1-mutated AML cells, was also evident by IP-WB. Blue boxes indicate expected locations of NPM1 and PU.1 if in nuclear fractions of NPM1-mutated AML cells. WB with mutant NPM1-specific antibody also shown. (C) IF for NPM1 and PU.1 in WT (OCI-AML2, THP1, NB4) and NPM1-mutated (OCI-AML3, IMS-M2) AML cell lines. Nuclei were stained with DAPI. Images by Nikon Eclipse 400 microscope; original magnification, ×630. Secondary antibody–alone controls are shown in Supplemental Figure 2. (D) IF for NPM1 and PU.1 in WT and NPM1-mutated AML primary cells from patients’ bone marrow. Images by Nikon Eclipse 400 microscope; original magnification, ×630. Secondary antibody–alone controls are shown in Supplemental Figure 2. (E) WB for PU.1, RUNX1, CEBPA, and NPM1 in nuclear and cytoplasmic fractions of WT and NPM1-mutated AML cell lines. Blue boxes indicate expected locations of NPM1 and PU.1 in nuclear fractions of NPM1-mutated AML cells; red boxes highlight location in cytoplasm of these cells instead.