In vivo kinetics of early, non-random methylome and transcriptome changes induced by DNA-hypomethylating treatment in primary AML blasts.

Despite routine use of DNA-hypomethylating agents (HMAs) in AML/MDS therapy, their mechanisms of action are not yet unraveled. Pleiotropic effects of HMAs include global methylome and transcriptome changes. We asked whether in blasts and T-cells from AML patients HMA-induced in vivo demethylation and remethylation occur randomly or non-randomly, and whether gene demethylation is associated with gene induction. Peripheral blood AML blasts from patients receiving decitabine (20 mg/m2 day 1-5) were serially isolated for methylome analyses (days 0, 8 and 15, n = 28) and methylome-plus-transcriptome analyses (days 0 and 8, n = 23), respectively. T-cells were isolated for methylome analyses (days 0 and 8; n = 16). We noted massive, non-random demethylation at day 8, which was variable between patients. In contrast, T-cells disclosed a thousand-fold lesser, random demethylation, indicating selectivity of the demethylation for the malignant blasts. The integrative analysis of DNA demethylation and transcript induction revealed 87 genes displaying a significant inverse correlation, e.g. the tumor suppressor gene IFI27, whose derepression was validated in two AML cell lines. These results support HMA-induced, non-random early in vivo demethylation events in AML blasts associated with gene induction. Larger patient cohorts are needed to determine whether a demethylation signature may be predictive for response to this treatment.

Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients.

BACKGROUND: Mutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co-occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation. RESULTS: EZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co-occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2-mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co-mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a significant decrease in EZH2 expression (p = 0.0002), which was similar in patients with chromosome 7 aberrations and patients with intact chromosome 7. An association between EZH2 protein expression and H3K27 trimethylation was observed in EZH2-unmutated patients (R2 = 0.2, p = 0.01). The monoallelic state of EZH2 was not associated with EZH2 promoter hypermethylation. In multivariable analyses, EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confidence interval 0.87-7.25], p = 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confidence interval 1.07-6.04], p = 0.04). CONCLUSIONS: Perturbations of EZH2 activity in AML/MDS occur on different, genetic and non-genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy.

Decitabine Induces Gene Derepression on Monosomic Chromosomes: In Vitro and In Vivo Effects in Adverse-Risk Cytogenetics AML.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813.

Psychophysiological insomnia and respiratory tract infections: results of an infection-diary-based cohort study.

STUDY OBJECTIVES: The immune theory of sleep suggests an important role of sleep for a functioning immune system. Insomnia has been associated with heightened risk for infections. The aim of the study was to test whether psychophysiological insomnia (PI) is associated with subsequent respiratory tract infections (RTIs) in the context of an infection-diary-based cohort study. METHODS: We recruited 674 adults from a cross-sectional survey on airway infections into the airway infection susceptibility (AWIS) cohort and invited them to self-report in diaries incident RTIs experienced during 7097 months (mean of 11.9 months of completed infection diaries per individual). The Regensburg Insomnia Scale (RIS) was assessed at baseline to measure PI. As outcome, we considered an infection diary score summing up prospectively reported RTIs. RESULTS: The RIS score correlated significantly with the infection diary score summarizing reported RTIs (correlation coefficient = 0.265, p < 0.001). Adjustments by putative confounders did only marginally affect this relationship. No significant differences in the relationship between RIS score and diary score were found for subgroups including those by gender, body mass index, perceived stress, and comorbidity. People affected by a combination of high PI and obesity were eight times more likely to belong to the group reporting the highest 10% of RTIs compared to the nonobese group with low RIS score (p < 0.001). A high RIS score in men was associated with a higher neutrophil-to-lymphocyte ratio, an indicator of inflammation. CONCLUSIONS: Our data support the relevance of adequate sleep for an immune system ready to fight pathogens and prevent airway infections.

Combination treatment of acute myeloid leukemia cells with DNMT and HDAC inhibitors: predominant synergistic gene downregulation associated with gene body demethylation.

DNA methyltransferase inhibitors (DNMTi) approved for older AML patients are clinically tested in combination with histone deacetylase inhibitors (HDACi). The mechanism of action of these drugs is still under debate. In colon cancer cells, 5-aza-2'-deoxycytidine (DAC) can downregulate oncogenes and metabolic genes by reversing gene body DNA methylation, thus implicating gene body methylation as a novel drug target. We asked whether DAC-induced gene body demethylation in AML cells is also associated with gene repression, and whether the latter is enhanced by HDACi.Transcriptome analyses revealed that a combined treatment with DAC and the HDACi panobinostat or valproic acid affected significantly more transcripts than the sum of the genes regulated by either treatment alone, demonstrating a quantitative synergistic effect on genome-wide expression in U937 cells. This effect was particularly striking for downregulated genes. Integrative methylome and transcriptome analyses showed that a massive downregulation of genes, including oncogenes (e.g., MYC) and epigenetic modifiers (e.g., KDM2B, SUV39H1) often overexpressed in cancer, was associated predominantly with gene body DNA demethylation and changes in acH3K9/27. These findings have implications for the mechanism of action of combined epigenetic treatments, and for a better understanding of responses in trials where this approach is clinically tested.

Evaluating the impact of genetic and epigenetic aberrations on survival and response in acute myeloid leukemia patients receiving epigenetic therapy.

Treatment with hypomethylating agents such as decitabine, which results in overall response rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy. However, there still exists a lack of prognostic and predictive molecular biomarkers that enable selection of patients who are likely to benefit from epigenetic therapy. Here, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα, and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331) to identify potential biomarkers for patients receiving hypomethylating therapy. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, patients harboring DNMT3A R882 mutations showed a non-significant association towards shorter overall survival (hazard ratio (HR) 2.15, 95% confidence interval (CI) 0.91-5.12, p = 0.08). Promoter DNA methylation analyses using pyrosequencing also revealed a non-significant association towards shorter overall survival of patients with higher levels of methylation of ERα (HR 1.50, CI 0.97-2.32, p = 0.07) and OLIG2 CpG4 (HR 1.52, CI 0.96-2.41, p = 0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR 1.76, CI 1.01-3.06, p = 0.05 and HR 1.67, CI 0.91-3.08, p = 0.10, respectively). In contrast, none of the investigated genetic and epigenetic markers was associated with response to treatment. Additional to the previously reported adverse prognostic clinical parameters such as patients' age, reduced performance status, and elevated lactate dehydrogenase levels, DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status, may prove to be molecular markers in older AML patients prior to hypomethylating therapy.

Single cell genotyping of exome sequencing-identified mutations to characterize the clonal composition and evolution of inv(16) AML in a CBL mutated clonal hematopoiesis.

We recently described the development of an inv(16) acute myeloid leukemia (AML) in a CBL mutated clonal hematopoiesis. Here, we further characterized the clonal composition and evolution of the AML based on the genetic information from the bulk specimen and analyses of individual bone marrow cells for mutations in CAND1, PTPRT, and DOCK6. To control for allele dropout, heterozygous polymorphisms located close to the respective mutation loci were assessed in parallel. The clonal composition concluded from exome sequencing suggested a proliferation advantage associated with the acquisition of mutations in CAND1, PTPRT, and DOCK6. Out of 102 single cell sequencing reactions on these mutations and the respective polymorphisms, analyses yielded conclusive results for at least 2 mutation sites in 12 cells. The single cell genotyping not only confirmed the co-occurrence of the PTPRT, CAND1 and DOCK6 mutations in the same AML clone but also revealed a clonal hierarchy, as the PTPRT mutation was likely acquired after the CAND1 and DOCK6 mutations. This insight had not been possible based solely on the exome sequencing data and suggests that the mutation in PTPRT, which encodes a STAT3-inhibiting protein tyrosine phosphatase, contributed to the AML development at a later stage by enhancing proliferation.

Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy.

Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible.

Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells.

Decitabine (5-aza-2'-deoxycytidine) is a DNA methyltransferase inhibitor and an archetypal epigenetic drug for the therapy of myeloid leukemias. The mode of action of decitabine strictly depends on the incorporation of the drug into DNA. However, DNA incorporation and ensuing genotoxic effects of decitabine have not yet been investigated in human cancer cell lines or in models related to the approved indication of the drug. Here we describe a robust assay for the quantitative determination of decitabine incorporation rates into DNA from human cancer cells. Using a panel of human myeloid leukemia cell lines we show appreciable amounts of decitabine incorporation that closely correlated with cellular drug uptake. Decitabine incorporation was also detectable in primary cells from myeloid leukemia patients, indicating that the assay is suitable for biomarker analyses to predict drug responses in patients. Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level. Interestingly, this approach failed to reveal significant changes in the rates of point mutations and genome rearrangements in myeloid leukemia cell lines. These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.

Tracing the development of acute myeloid leukemia in CBL syndrome.

We describe the development of acute myeloid leukemia (AML) in an adult with CBL syndrome caused by a heterozygous de novo germline mutation in CBL codon D390. In the AML bone marrow, the mutated CBL allele was homozygous after copy number-neutral loss-of-heterozygosity and amplified through a chromosomal gain; moreover, an inv(16)(p13q22) and, as assessed by whole-exome sequencing, 12 gene mutations (eg, in CAND1, NID2, PTPRT, DOCK6) were additionally acquired. During complete remission of the AML, in the presence of normal blood counts, the hematopoiesis stably maintained the homozygous CBL mutation, which is reminiscent of the situation in children with CBL syndrome and transient juvenile myelomonocytic leukemia. No additional mutations were identified by whole-exome sequencing in granulocytes during complete remission. The study highlights the development of AML in an adult with CBL syndrome and, more generally, in genetically aberrant but clinically inconspicuous hematopoiesis.

Epigenetic priming of AML blasts for all-trans retinoic acid-induced differentiation by the HDAC class-I selective inhibitor entinostat.

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARß2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARß2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARß2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARß2 was not necessarily required for the differentiation effect, and pharmacological RARß2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARß2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARß2 gene silencing by DNA methylation.

Quantitative analyses of DAPK1 methylation in AML and MDS.

Aberrant DNA methylation and concomitant transcriptional silencing of death-associated protein kinase 1 (DAPK1) have been demonstrated to be key pathogenic events in chronic lymphocytic leukemia (CLL). In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), however, the presence of elevated DNA methylation levels has been a matter of continued controversy. Several studies demonstrated highly variable frequencies of DAPK1 promoter methylation by the use of methylation-specific PCR (MSP). By quantitative high-resolution assessment, we demonstrate that aberrant DNA methylation is an extremely rare event in this region. We observed elevated levels just in one out of 246 (0.4%) AML patients, all 42 MDS patients were unmethylated. In conclusion, we present a refined DAPK1 methylation analysis in a large representative patient cohort of AML and MDS patients proofing almost complete absence of elevated DNA methylation. Our results highlight the importance of quantitative measurements for translational research questions on primary patient specimens, particularly.

The DNA demethylating agent 5-aza-2'-deoxycytidine induces expression of NY-ESO-1 and other cancer/testis antigens in myeloid leukemia cells.

Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic syndromes and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are not expressed in almost all normal tissues and many tumor types, but are consistently derepressed by epigenetically active agents in various cancer cell lines. Since the expression of CTA genes is usually very low or absent in myeloid leukemias, we treated various AML cell lines with 5-aza-2'-deoxycytidine (DAC) and quantified mRNA expression of the CTAs NY-ESO-1, MAGEA1, MAGEA3 and MAGEB2. Consistent time- and dose-dependent reactivation of all 4 CTA genes was observed, with maximum mRNA levels 72-144h after treatment start. As determined by RNA microarray analyses, numerous other CTA genes (all located on the X-chromosome) were also derepressed in a time-dependent fashion by DAC. NY-ESO-1 derepression was confirmed at the protein level. By Elispot and chromium release assays we showed that the de novo expressed NY-ESO-1 protein was naturally processed and presented in a time- and dose-dependent fashion up to 8 days after the start of DAC treatment, and converted the cell lines susceptible to antigen-specific recognition by CD8+ T-cell clones. In conclusion, NY-ESO-1 and numerous other CTAs localized on the X-chromosome are readily and transiently derepressed in AML cell lines treated with DAC. The susceptibility of DAC-treated AML cell lines to antigen-specific T-cell recognition has clear implications for future clinical trials combining DAC and specific immunotherapy in AML.

Decitabine.

The pyrimidine analogs, 5-azacytidine (azacitidine, Vidaza) and its deoxy derivative, 5-aza-2'-deoxycytidine (decitabine, Dacogen, are the most widely used inhibitors of DNA methylation which trigger demethylation leading to a consecutive reactivation of epigenetically silenced tumor suppressor genes in vitro and in vivo. Although the antileukemic capacity of decitabine has been known for almost 40 years, its therapeutic potential in hematologic malignancies is still under intensive investigation. Multiple clinical trials have shown the promising activity of low-dose decitabine in AML, MDS, CML, and hemoglobinopathies, whereas its efficacy in solid tumors is rather limited.Clinical responses appear to be induced by both epigenetic alterations and the induction of cell-cycle arrest and/or apoptosis. Recent clinical trials have been investigating new dosing schedules, routes of administration, and combination of decitabine with other agents, including histone deacetylase (HDAC) inhibitors.