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 2

AML cells highly express the PU.1/RUNX1/CEBPA master transcription factor circuit that drives cells to terminal granulomonocytic fates, but the monocyte differentiation program is suppressed.

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AML cells highly express the PU.1/RUNX1/CEBPA master transcription facto...
(A) Expression of granulomonocytic (CEBPA, RUNX1, CEBPA) and HSC (HLF, PBX1, PRDM5) master transcription factors during normal myelopoiesis and in cytogenetically normal AML (CNAML). Gene expression data were integrated and normalized as previously described (48, 49). Boxes indicate median ± IQR, whiskers indicate range. HSCs, n = 6; multipotent progenitors (MPP), n = 2; CMPs, n = 3; GMPs, n = 7; neutrophils (Neut), n = 3; monocytes (Mono), n = 4; CNAML cells, n = 989. (B) Negative (Neg) correlation between myeloid commitment and PU.1 gene expression, but positive correlation between monocyte differentiation and PU.1 gene expression (Pearson’s correlation coefficients). Comparative Marker Selection (Morpheus) analysis of gene expression in HSCs, CMPs, GMPs, CFU monocytes (CFUM), and monocytes from GSE24759 (51) identified ~200 myeloid commitment and ~300 terminal monocytic differentiation genes. MYC target genes identified by others using ChIP-Seq (98), validated by separate analyses (Supplemental Figure 4). Also, Pu.1 localized at monocyte differentiation but not commitment genes by ChIP-Seq (Supplemental Figure 4). Gene sets were also validated in our separate gene expression database of normal hematopoiesis (Supplemental Figure 5). Gene lists are in Supplemental Tables 2–4. (C) CNAML expresses monocyte differentiation genes at levels higher than in normal HSCs, CMPs, or GMPs, but ~4-fold lower than seen in normal monocytes. 100 CNAML shown (truncated from 989 analyzed) (49). P values, 2-sided Mann-Whitney U test. (D) NPM1, RUNX1, and biallelic CEBPA mutations in CNAML cells are highly recurrent but mutually exclusive. n = 101 (analysis of data from The Cancer Genome Atlas [TCGA]).