Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.

Molecular Minimal Residual Disease in Acute Myeloid Leukemia.

BACKGROUND: Patients with acute myeloid leukemia (AML) often reach complete remission, but relapse rates remain high. Next-generation sequencing enables the detection of molecular minimal residual disease in virtually every patient, but its clinical value for the prediction of relapse has yet to be established. METHODS: We conducted a study involving patients 18 to 65 years of age who had newly diagnosed AML. Targeted next-generation sequencing was carried out at diagnosis and after induction therapy (during complete remission). End points were 4-year rates of relapse, relapse-free survival, and overall survival. RESULTS: At least one mutation was detected in 430 out of 482 patients (89.2%). Mutations persisted in 51.4% of those patients during complete remission and were present at various allele frequencies (range, 0.02 to 47%). The detection of persistent DTA mutations (i.e., mutations in DNMT3A, TET2, and ASXL1), which are often present in persons with age-related clonal hematopoiesis, was not correlated with an increased relapse rate. After the exclusion of persistent DTA mutations, the detection of molecular minimal residual disease was associated with a significantly higher relapse rate than no detection (55.4% vs. 31.9%; hazard ratio, 2.14; P<0.001), as well as with lower rates of relapse-free survival (36.6% vs. 58.1%; hazard ratio for relapse or death, 1.92; P<0.001) and overall survival (41.9% vs. 66.1%; hazard ratio for death, 2.06; P<0.001). Multivariate analysis confirmed that the persistence of non-DTA mutations during complete remission conferred significant independent prognostic value with respect to the rates of relapse (hazard ratio, 1.89; P<0.001), relapse-free survival (hazard ratio for relapse or death, 1.64; P=0.001), and overall survival (hazard ratio for death, 1.64; P=0.003). A comparison of sequencing with flow cytometry for the detection of residual disease showed that sequencing had significant additive prognostic value. CONCLUSIONS: Among patients with AML, the detection of molecular minimal residual disease during complete remission had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent mutations that are associated with clonal hematopoiesis did not have such prognostic value within a 4-year time frame. (Funded by the Queen Wilhelmina Fund Foundation of the Dutch Cancer Society and others.).

Acquired mutations in ASXL1 in acute myeloid leukemia: prevalence and prognostic value.

Somatic mutations in the additional sex comb-like 1 (ASXL1) gene have been described in various types of myeloid malignancies, including acute myeloid leukemia. Analysis of novel markers, such as ASXL1 mutations, in independent clinical trials is indispensable before considering them for clinical decision-making. We analyzed 882 well-characterized acute myeloid leukemia cases to determine the prevalence and prognostic impact of ASXL1 exon12 mutations. Truncating ASXL1 mutations were present in 46 cases (5.3%). ASXL1 mutations were inversely associated with FLT3 internal tandem duplications and mutually exclusive with NPM1 mutations. ASXL1 mutations were an unfavorable prognostic factor as regards survival (median overall survival 15.9 months vs. 22.3 months; P=0.019), with a significantly lower complete response rate (61% vs. 79.6%; P=0.004). In multivariate analyses, ASXL1 mutations were independently associated with inferior poor overall survival (HR 1.52, P=0.032). In conclusion, ASXL1 mutations are common mutations in acute myeloid leukemia and indicate a poor therapy outcome.

Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome.

Mutations in CCAAT/enhancer binding protein alpha (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 (CEBPA(double-mut)), usually biallelic, whereas single heterozygous mutations (CEBPA(single-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 CEBPA(double-mut) and 13 CEBPA(single-mut) cases). CEBPA(double-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, CEBPA(single-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.

Genome-wide epigenetic analysis delineates a biologically distinct immature acute leukemia with myeloid/T-lymphoid features.

Acute myeloid leukemia is a heterogeneous disease from the molecular and biologic standpoints, and even patients with a specific gene expression profile may present clinical and molecular heterogeneity. We studied the epigenetic profiles of a cohort of patients who shared a common gene expression profile but differed in that only half of them harbored mutations of the CEBPA locus, whereas the rest presented with silencing of this gene and coexpression of certain T-cell markers. DNA methylation studies revealed that these 2 groups of patients could be readily segregated in an unsupervised fashion based on their DNA methylation profiles alone. Furthermore, CEBPA silencing was associated with the presence of an aberrant DNA hypermethylation signature, which was not present in the CEBPA mutant group. This aberrant hypermethylation occurred more frequently at sites within CpG islands. CEBPA-silenced leukemias also displayed marked hypermethylation compared with normal CD34(+) hematopoietic cells, whereas CEBPA mutant cases showed only mild changes in DNA methylation compared with these normal progenitors. Biologically, CEBPA-silenced leukemias presented with a decreased response to myeloid growth factors in vitro.

Gene transactivation without direct DNA binding defines a novel gain-of-function for PML-RARalpha.

PML-RARalpha is the causative oncogene in 5% to 10% of the cases of acute myeloid leukemia. At physiological concentrations of retinoic acid, PML-RARalpha silences RARalpha target genes, blocking differentiation of the cells. At high concentrations of ligand, it (re)activates the transcription of target genes, forcing terminal differentiation. The study of RARalpha target genes that mediate this differentiation has identified several genes that are important for proliferation and differentiation control in normal and malignant hematopoietic cells. In this paper, we show that the PML-RARalpha fusion protein not only interferes with the transcription of regular RARalpha target genes. We show that the ID1 and ID2 promoters are activated by PML-RARalpha but, unexpectedly, not by wild-type RARalpha/RXR. Our data support a model in which the PML-RARalpha fusion protein regulates a novel class of target genes by interaction with the Sp1 and NF-Y transcription factors, without directly binding to the DNA, defining a gain-of-function for the oncoprotein.

Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1.

Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.

The E3 ubiquitin-protein ligase Triad1 inhibits clonogenic growth of primary myeloid progenitor cells.

Protein ubiquitination plays important roles in a variety of basic cellular processes. Proteins are ubiquitinated by E2-E3 ubiquitin ligase complexes. Depending on the type of ubiquitin chain conjugated, proteins are either targeted for degradation by the proteasome or their activity is specifically altered. We describe a novel conserved nuclear protein, Triad1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1), which is strongly induced during myeloid differentiation. Triad1 contains a TRIAD motif that harbors 2 RING finger structures. Triad1 binds the E2 ubiquitin-conjugating enzyme UbcH7 as well as ubiquitinated proteins and supports the formation of ubiquitin chains that are recognized by the proteasome. The biologic function of Triad1 in myelopoiesis was studied by performing granulocyte-macrophage colony-forming unit (CFU-GM) assays using retrovirally transduced primary murine bone marrow cells. Triad1 severely inhibited myeloid colony formation. In contrast, 2 Triad1 RING finger point mutants that failed to bind UbcH7 did not affect colony formation. Moreover, proteasome inhibition counteracted the inhibition of colony formation exerted by wild-type Triad1. In liquid cultures, Triad1 did not influence differentiation but strongly inhibited proliferation resulting in a G0/G1 accumulation. We conclude that proteasomal degradation of proteins that are ubiquitinated by Triad1 affects the clonogenic growth of primary myeloid progenitor cells.