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 6

The differentiation-restoring effect of selinexor in vivo was saturated at a dose of 2 mg/kg.

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The differentiation-restoring effect of selinexor in vivo was saturated ...
2 mg/kg selinexor (Sel-2) was compared with 5 mg/kg (Sel-5) in a patient-derived xenotransplant model of dual NPM1/FLT3-mutated AML. (A) Experiment schema. After confirmation of bone marrow AML engraftment to ≥20% in 3 randomly selected mice, remaining mice were randomized to vehicle, 2 mg/kg selinexor, or 5 mg/kg selinexor, by oral gavage 4 times per week starting on day 21 (n = 5/group). Treatment (Tx) continued until appearance of signs of distress in vehicle-treated mice (day 75), when the experiment was terminated for analyses. *P < 0.01. (B) IF for PU.1 and NPM1 location in bone marrow AML cells. DAPI was used to stain for nuclei. Images by Leica SP8 inverted confocal microscope; original magnification, ×630. (C) Bone marrow AML burden. Flow cytometry for human (Hu) CD45+ (AML) and murine (Ms) CD45+ (normal) cells. Median ± IQR. P values, Mann-Whitney U test, 2-sided. Significance after Bonferroni’s correction was P < 0.025. (D) Spleen AML burden. Median ± IQR. P values, 2-sided Mann-Whitney U test. Normal NSG spleen weight is ~0.018 g. (E) Serial blood counts. Increasing WBC were circulating myeloblasts. Tail vein phlebotomy, blood counts by HemaVet. Mean ± SD. *P < 0.01 (significant after Bonferroni’s correction), Sel-2 or Sel-5 versus vehicle on day 75, 2-sided t test. (F) CD14 monocyte-lineage differentiation marker expression on bone marrow AML cells. Flow cytometry. Median ± IQR. P values, Mann-Whitney U test, 2-sided. Significance after Bonferroni’s correction was P < 0.025. (G) γ-H2AX apoptosis/DNA damage marker expression on bone marrow AML cells. Flow cytometry. Median ± IQR; P values, 2-sided Mann-Whitney U test (NS, P > 0.025). Hb, hemoglobin; Plts, platelets.