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Nucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (∼30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-transretinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)–retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics.

The NPM1mutation is the most frequent genetic alteration thus far identified in acute myeloid leukemia (AML). Despite progress in the clinical and biological characterization of NPM1-mutated AML, the role of NPM1mutation in leukemogenesis in vivo has not been fully elucidated. We report a novel mouse model that conditionally expresses the most common human NPM1mutation (type A) in the hematopoietic compartment. In Npm1-TCTG/WT;Cre+mice, the NPM1 mutant localized in the cytoplasm (NPMc+) of bone marrow (BM) cells. The mutant mice developed no AML after 1.5-year follow-up. However, NPMc+expression determined a significant platelet count reduction and an expansion of the megakaryocytic compartment in the BM and spleen. Serum thrombopoietin levels overlapped in mutant vs control mice, and BM cells from Npm1-TCTG/WT;Cre+mice formed more megakaryocytic colonies in vitro. Moreover, we demonstrated the up-regulation of microRNAs (miRNAs; miR-10a, miR-10b,and miR-20a) inhibiting megakaryocytic differentiation along with increased expression of HOXBgenes. Notably, these findings mimic those of human NPM1-mutated AML, which also exhibits a similar miRNA profile and expansion of the megakaryocytic compartment. Our mouse model provides evidence that the NPM1 mutant affects megakaryocytic development, further expanding our knowledge of the role of NPM1 mutant in leukemogenesis.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1and CEBPAmutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR(BCL6corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCORmutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCORsomatic mutations were: (1) disruptive events similar to the germline BCORmutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCORmRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1mutations; and (4) frequently associated with DNMT3Amutations, suggesting cooperativity among these genetic alterations. Finally, BCORmutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P= .032). Our results for the first time implicate BCORin CN-AML pathogenesis.

Acute myeloid leukemia (AML) with mutated NPM1shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34−cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Nucleophosmin(NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but sporadically also exon 9 and 11, all causing changes at protein C-terminal end (loss of tryptophans and creation of a nuclear export signal-NES motif) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 AML patients, we found non-exon 12 NPM1mutations in 5/387 (1.3%) NPM1c+ cases. Besides mutations in exon 9 (n=1) and exon 11 (n=1), novel mutations in exon 5 were discovered (n=3). One more exon 5 mutation was identified in additional 141 AML patients selected for wild-type NPM1exon 12. Furthermore, 3 NPM1rearrangements (i.e. NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detectedand characterized among 13,979AML samples screened by cytogenetic/FISH and RNA sequencing. Functional studies demonstrated that in AML cases the new NPM1 proteins harboured an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting any AML-associated NPM1genetic lesions. Also, this study highlights the need for developing new specific assays for molecular diagnosis and monitoring of NPM1-mutated AML.

Introduction.Nucleophosmin (NPM1) gene mutations occur in 50-60% of adult AML with normal karyotype (Falini et al, NEJM 2005). About 50 NPM1mutations have been so far identified, all clustering in exon-12 (Falini et al, Blood 2007) but few sporadic cases involving either exon-9 (one) (Mariano et al, Oncogene 2006) or exon-11 (two) (Albiero et al, Leukemia 2007). In spite of molecular heterogeneity, all mutations cause common changes at the C-terminus of NPM1 mutants, i.e. loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES) motif (Falini et al, Blood 2006a).As a consequence, all NPM1 mutants aberrantly accumulates in the cytoplasm of leukemic cells and can be detected by immunohistochemistry, which is fully predictive of NPM1gene mutations (Falini et al, Blood 2006b).

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