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 10

Combination differentiation-restoring treatment in vivo.

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Combination differentiation-restoring treatment in vivo. (A) Experiment ...
(A) Experiment schema. Immunodeficient mice were xenotransplanted with NPM1/FLT3-mutated primary AML cells (55). After bone marrow engraftment to ≥20% AML was confirmed in 3 randomly selected mice, mice were randomly assigned to treatment with (i) vehicle; (ii) nuclear export inhibition — 2 mg/kg selinexor 4 times per week by oral gavage; (iii) DNMT1 depletion — 0.1 mg/kg decitabine, 3 times per week alternating with 1 mg/kg 5-azacytidine 3 times per week subcutaneously, combined with THU 10 mg/kg intraperitoneally (to inhibit in vivo degradation of decitabine/5-azacytidine (Dec/5Aza) by cytidine deaminase); or (iv) combination nuclear export inhibition/DNMT1 depletion. Mice were euthanized after appearance of signs of distress. (B) Serial blood counts. The increase in WBC was due to myeloblasts (right). Tail vein phlebotomy; blood counts by HemaVet. Mean ± SD. (C) Survival (time to distress). P values, log-rank test. (D) Spleen AML burden at euthanasia. Median ± IQR. NS, P > 0.01, 2-sided Mann-Whitney U test. Photos show a spleen from a normal NSG mouse versus a vehicle-treated mouse, with H&E-stained spleen sections showing AML infiltration (yellow arrow) and necrosis (white arrow) (original magnification, ×400). Normal NSG spleen weight is ~0.018 g. (E) Cell cycle distribution of marrow AML cells at euthanasia. Mean ± SD for percentage of cells in each cell cycle phase. P value, unpaired t test 2-sided. Raw data are shown in Supplemental Figure 14. (F) Monocyte (CD14) and granulocyte (CD11b) lineage differentiation marker expression in marrow AML cells at euthanasia. Flow cytometry. Median ± IQR. *P < 0.01 (significant after Bonferroni’s correction), NS, P > 0.01, Mann-Whitney U test, 2-sided. Raw data are shown in Supplemental Figures 15 and 16. (G) Morphology of marrow AML cells at euthanasia. Giemsa stain. Leica DMR microscope; original magnification, ×630. (H) Apoptosis/DNA damage marker γ-H2AX expression in marrow AML cells at euthanasia. Flow cytometry. Median ± IQR. *P < 0.01 (significant after Bonferroni’s correction), NS, P > 0.01, Mann-Whitney U test, 2-sided. Raw data including positive control are shown in Supplemental Figure 17. Veh, vehicle; Sel, selinexor; TDA, THU-Dec/5Aza; STDA, selinexor + THU-Dec/Aza.