Anuhar Chaturvedi

Prognostic Significance of ASXL1 Mutations in Patients With Myelodysplastic Syndromes

PURPOSE To study the incidence and prognostic impact of mutations in Additional sex comb-like 1 (ASXL1) in a large cohort of patients with myelodysplastic syndrome (MDS). PATIENTS, MATERIALS, AND METHODS Overall, 193 patients with MDS and 65 healthy volunteers were examined for ASXL1 mutations by direct sequencing and for expression levels of ASXL1. The prognostic impact of ASXL1 mutation and expression levels was evaluated in the context of other clinical and molecular prognostic markers. RESULTS Mutations in ASXL1 occurred with a frequency of 20.7% in MDS (n = 40 of 193) with 70% (n = 28) of mutations being frameshift mutations and 30% (n = 12) being heterozygous point mutations leading to translational changes. ASXL1 mutations were correlated with an intermediate-risk karyotype (P = .002) but not with other clinical parameters. The presence of ASXL1 mutations was associated with a shorter overall survival for frameshift and point mutations combined (hazard ratio [HR], 1.744; 95% CI, 1.08 to 2.82; P = .024) and for frameshift mutations only (HR, 2.06; 95% CI, 1.21 to 3.50; P = .008). ASXL1 frameshift mutations were associated with a reduced time to progression of acute myeloid leukemia (AML; HR 2.35; 95% CI, 1.17 to 4.74; P = .017). In multivariate analysis, when considering karyotype, transfusion dependence, and IDH1 mutation status, ASXL1 frameshift mutations remained an independent prognostic marker in MDS (overall survival: HR, 1.85; 95% CI, 1.03 to 3.34; P = .040; time to AML progression: HR, 2.39; 95% CI, 1.12 to 5.09; P = .024). CONCLUSION These results suggest that ASXL1 mutations are frequent molecular aberrations in MDS that predict an adverse prognostic outcome. Screening of patients for ASXL1 mutations might be useful for clinical risk stratification and treatment decisions in the future.

Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34(+) bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications

Mutations in the cohesin complex are novel, genetic lesions in acute myeloid leukemia (AML) that are not well characterized. In this study, we analyzed the frequency, clinical, and prognostic implications of mutations in STAG1, STAG2, SMC1A, SMC3, and RAD21, all members of the cohesin complex, in a cohort of 389 uniformly treated AML patients by next generation sequencing. We identified a total of 23 patients (5.9%) with somatic mutations in 1 of the cohesin genes. All gene mutations were mutually exclusive, and STAG1 (1.8%), STAG2 (1.3%), and SMC3 (1.3%) were most frequently mutated. Patients with any cohesin complex mutation had lower BAALC expression levels. We found a strong association between mutations affecting the cohesin complex and NPM1. Mutated allele frequencies were similar between NPM1 and cohesin gene mutations. Overall survival (OS), relapse-free survival (RFS), and complete remission rates (CR) were not influenced by the presence of cohesin mutations (OS: hazard ratio [HR] 0.98; 95% confidence interval [CI], 0.56-1.72 [P = .94]; RFS: HR 0.7; 95% CI, 0.36-1.38 [P = .3]; CR: mutated 83% vs wild-type 76% [P = .45]). The cohesin complex presents a novel pathway affected by recurrent mutations in AML. This study is registered at www.clinicaltrials.gov as #NCT00209833.

Cell of Origin in AML: Susceptibility to MN1-Induced Transformation Is Regulated by the MEIS1/AbdB-like HOX Protein Complex

Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.

Prognostic Importance of Histone Methyltransferase MLL5 Expression in Acute Myeloid Leukemia

PURPOSE To assess the prognostic importance of mixed lineage leukemia 5 (MLL5) expression in acute myeloid leukemia (AML). PATIENTS AND METHODS MLL5 transcript levels from 509 patients with AML who were treated in multicenter trials AML SHG 0199 and AML SHG 0295 and 48 healthy volunteers were analyzed by real-time reverse-transcription polymerase chain reaction in the context of other molecular markers (NPM1, FLT3, CEBPA, IDH1/IDH2, NRAS, KIT, MN1, BAALC, ERG, and WT1). RESULTS Patients with high (n = 127) compared with low (n = 382) MLL5 expression had a higher complete response rate in multivariate analysis (odds ratio, 1.87; 95% CI, 1.08 to 3.24; P = .026). In multivariate analysis, high MLL5 expression was a favorable prognostic marker for overall survival (OS; hazard ratio [HR], 0.66; 95% CI, 0.49 to 0.89; P = .007) and relapse-free survival (RFS; HR, 0.72; 95% CI, 0.52 to 1.01; P = .057). Patient characteristics, cytogenetic aberrations, and gene mutations were similarly distributed between patients with high and low MLL5 expression except for a higher platelet count in those with high MLL5 expression. MLL5 expression independently predicted prognosis in cytogenetically normal AML patients (n = 268; OS: HR, 0.53; 95% CI, 0.33 to 086; P = .011; RFS: HR, 0.61; 95% CI, 0.38 to 0.99; P = .05) and in patients with core-binding factor leukemias (n = 81; OS: HR, 0.12; 95% CI, 0.02 to 0.91; P = .04; RFS: HR, 0.18; 95% CI, 0.04 to 0.77; P = .02). The prognostic importance of high MLL5 expression was independently validated in 167 patients treated in the AMLSG 07/04 trial (OS: HR, 0.5; 95% CI, 0.27 to 0.92; P = .023; RFS: HR, 0.49; 95% CI, 0.25 to 0.96; P = .033). CONCLUSION High MLL5 expression levels are associated with a favorable outcome and may improve risk and treatment stratification in AML.

Genetic Deletion of SEPT7 Reveals a Cell Type-Specific Role of Septins in Microtubule Destabilization for the Completion of Cytokinesis

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

Prognostic effect of calreticulin mutations in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation

Prognostic effect of calreticulin mutations in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation

Acute myeloid leukemia derived from lympho-myeloid clonal hematopoiesis.

We studied acute myeloid leukemia (AML) patients with lympho-myeloid clonal hematopoiesis (LM-CH), defined by the presence of DNA methyltransferase 3A (DNMT3A) mutations in both the myeloid and lymphoid T-cell compartment. Diagnostic, complete remission (CR) and relapse samples were sequenced for 34 leukemia-related genes in 171 DNMT3A mutated adult AML patients. AML with LM-CH was found in 40 patients (23%) and was associated with clonal hematopoiesis of indeterminate potential years before AML, older age, secondary AML and more frequent MDS-type co-mutations (TET2, RUNX1 and EZH2). In 82% of AML patients with LM-CH, the preleukemic clone was refractory to chemotherapy and was the founding clone for relapse. Both LM-CH and non-LM-CH MRD-positive AML patients who achieved CR had a high risk of relapse after 10 years (75% and 75%, respectively) compared with patients without clonal hematopoiesis in CR with negative MRD (27% relapse rate). Long-term survival of patients with LM-CH was only seen after allogeneic hematopoietic stem cell transplantation (HSCT). We define AML patients with LM-CH as a distinct high-risk group of AML patients that can be identified at diagnosis through mutation analysis in T cells and should be considered for HSCT.

Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia

Hypomethylating agents are widely used in patients with myelodysplastic syndromes and unfit patients with acute myeloid leukemia. However, it is not well understood why only some patients respond to hypomethylating agents. We found previously that the effect of decitabine on hematopoietic stem cell viability differed between Mll5 wild-type and null cells. We, therefore, investigated the role of MLL5 expression levels on outcome of acute myeloid leukemia patients who were treated with decitabine. MLL5 above the median expression level predicted longer overall survival independent of DNMT3A mutation status in bivariate analysis (median overall survival for high vs. low MLL5 expression 292 vs. 167 days; P=0.026). In patients who received three or more courses decitabine, high MLL5 expression and wild-type DNMT3A independently predicted improved overall survival (median overall survival for high vs. low MLL5 expression 468 vs. 243 days; P=0.012). In transformed murine cells, loss of Mll5 was associated with resistance to low-dose decitabine, less global DNA methylation in promoter regions, and reduced DNA demethylation upon decitabine treatment. Together, these data support our clinical observation of improved outcome in decitabine-treated patients who express MLL5 at high levels, and suggest a mechanistic role of MLL5 in the regulation of DNA methylation.

Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate

Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.