Mutations in CCAAT/enhancer binding protein α (CEBPA) are seen in 5% to 14% of acute myeloid leukemia (AML) and have been associated with a favorable clinical outcome. Most AMLs with CEBPA mutations simultaneously carry 2 mutations (CEBPAdouble-mut), usually biallelic, whereas single heterozygous mutations (CEBPAsingle-mut) are less frequently seen. Using denaturing high-performance liquid chromatography and nucleotide sequencing, we identified among a cohort of 598 newly diagnosed AMLs a subset of 41 CEBPA mutant cases (28 CEBPAdouble-mut and 13 CEBPAsingle-mut cases). CEBPAdouble-mut associated with a unique gene expression profile as well as favorable overall and event-free survival, retained in multivariable analysis that included cytogenetic risk, FLT3-ITD and NPM1 mutation, white blood cell count, and age. In contrast, CEBPAsingle-mut AMLs did not express a discriminating signature and could not be distinguished from wild-type cases as regards clinical outcome. These results demonstrate significant underlying heterogeneity within CEBPA mutation-positive AML with prognostic relevance.

© F e r r a t a S t o r t i F o u n d a t i o nR.G.W. Verhaak et al. between both AML sample populations. Mutational analyses to detect recurrent mutations in AML were performed as previously described. [13][14][15][16] All supervised class prediction analyses were performed with Prediction Analysis for Microarrays (PAM) software version 1.28 in R version 2.1.0. 17 Clinical, cytogenetic and molecular information as well as the gene expression profiles of all primary AML cases is available at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo, accession number GSE6891). Results and DiscussionIn this study of 461 clinically and molecularly well-characterized cases of AML (Table 1), we were able to comprehensively validate the application of GEP to predict therapeutically relevant molecular subtypes in AML.We applied PAM to investigate whether karyotypic and mutational abnormalities with prognostic or therapeutic value in AML were accurately predictable based on GEP. PAM allows the selection of the minimal number of genes required for optimal prediction, which may be beneficial in a diagnostic setting. The AML cohort1 (n=247) was used as training set to derive predictive signatures that were subsequently validated on AML cohort2 (n=214). The deduced expression signatures are available in the Online Supplementary Tables S1-18.The cytogenetic status of all AML patients with favorable risk, i.e. those with t(8;21), t(15;17) or inv(16) abnormalities, was predicted with 100 percent accuracy ( Table 2). In fact, among these predicted AML cases, there were cases with favorable cytogenetics that had previously been missed by routine cytogenetics (4 out of 37 inv(16) and 4 out of 25 t(15;17)). The presence of the translocation-related fusion transcripts in these specific cases was confirmed by real-time quantitative PCR. Thus, GEP is a reliable alternative to discriminate these three AML subtypes, 2,3 which represent approximately 20% of all cases.2,3 Prediction of t(15;17) and inv(16) required only few genes, as seen previously. 8 For the t(8;21) cases, 76 probe sets were needed to correctly classify all samples. However, as few as two probe sets, including one associated with the RUNX1T1 (ETO) gene, were sufficient to accurately classify all but one t(8;21) cases, which is also consistent with earlier studies 8 (Online Supplementary Figure S3). AML cases with mutations in the transcription factor CCAAT/enhancer binding protein α (CEBPA), which are associated with a relatively favorable treatment outcome, were predicted with positive and negative predictive values of 100% and 97% respectively. Six out of 15 CEBPA mutant cases were missed in the validation set (sensitivity 60%; Table 2). Of note, the misclassified cases all carried a single heterozygous CEBPA mutation, whereas samples with biallelic mutations (either homo-or heterozygous) were all correctly recognized (data not shown). In the training cohort, all but two (14/16) samples carried biallelic mutations 14,18 and in cross-validation in the training cohort ...

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Tribbles homolog 2 (Trib2) was identified as a downregulated transcript in leukemic cells undergoing growth arrest. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-transduced bone marrow cells exhibited a growth advantage ex vivo and readily established factor-dependent cell lines. In vivo, Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML). In mechanistic studies, we found that Trib2 associated with and inhibited C/EBPalpha. Furthermore, Trib2 expression was elevated in a subset of human AML patient samples. Together, our data identify Trib2 as an oncogene that induces AML through a mechanism involving inactivation of C/EBPalpha.

Highlights d Inhibition of SDMA or ADMA preferentially kills splicing factor (SF)-mutant cells d Combined inhibition of PRMT5 and type I PRMTs has synergistic effects d RNA-binding proteins are the most enriched cellular substrates of PRMTs d Inhibition of RNA splicing underlies the cytotoxic effects of PRMT inhibition