Hans-Ulrich Klein

Inhibition of the LSD1 (KDM1A) demethylase reactivates the all- trans -retinoic acid differentiation pathway in acute myeloid leukemia

Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4me2) across the genome, but it did increase H3K4me2 and expression of myeloid-differentiation–associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.

Next-Generation Sequencing Technology Reveals a Characteristic Pattern of Molecular Mutations in 72.8% of Chronic Myelomonocytic Leukemia by Detecting Frequent Alterations in TET2, CBL, RAS, and RUNX1

PURPOSE Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Thus far, data on a comprehensive cytogenetic or molecular genetic characterization are limited. PATIENTS AND METHODS Here, we analyzed 81 thoroughly characterized patients with CMML (CMML type 1, n = 45; CMML type 2, n = 36) by applying next-generation sequencing (NGS) technology to investigate CBL, JAK2, MPL, NRAS, and KRAS at known mutational hotspot regions. In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2 aberrations. RESULTS Cytogenetic aberrations were found in 18.2% of patients (14 of 77 patients). In contrast, at least one molecular mutation was observed in 72.8% of patients (59 of 81 patients). A mean of 1.6 mutations per patient was observed by this unprecedented screening. In total, 105 variances were detected by this comprehensive molecular screening. After excluding known polymorphisms or silent mutations, 82 distinct mutations remained (CBL, n = 15; JAK2V617F, n = 8; MPL, n = 0; NRAS, n = 10; KRAS, n = 12; RUNX1, n = 7; and TET2, n = 41). With respect to clinical data, a better outcome was seen for patients carrying TET2 mutations (P = .013). CONCLUSION The number of molecular markers used to categorize myeloid neoplasms is constantly increasing. Here, NGS screening has been demonstrated to support a comprehensive characterization of the molecular background in CMML. A pattern of molecular mutations translates into different biologic and prognostic categories of CMML.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations 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 (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations 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. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations 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 BCOR in CN-AML pathogenesis.

Detection of significantly differentially methylated regions in targeted bisulfite sequencing data

MOTIVATION Bisulfite sequencing is currently the gold standard to obtain genome-wide DNA methylation profiles in eukaryotes. In contrast to the rapid development of appropriate pre-processing and alignment software, methods for analyzing the resulting methylation profiles are relatively limited so far. For instance, an appropriate pipeline to detect DNA methylation differences between cancer and control samples is still required. RESULTS We propose an algorithm that detects significantly differentially methylated regions in data obtained by targeted bisulfite sequencing approaches, such as reduced representation bisulfite sequencing. In a first step, this approach tests all target regions for methylation differences by taking spatial dependence into account. A false discovery rate procedure controls the expected proportion of incorrectly rejected regions. In a second step, the significant target regions are trimmed to the actually differentially methylated regions. This hierarchical procedure detects differentially methylated regions with increased power compared with existing methods. AVAILABILITY R/Bioconductor package BiSeq. SUPPLEMENTARY INFORMATION Supplementary Data are available at Bioinformatics online.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2 , CBL and KRAS mutations by an international consortium involving 10 laboratories

Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1–2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Genome wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

AML with translocation t(8;16)(p11;p13) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features

Balanced chromosomal rearrangements define distinct entities in acute myeloid leukemia (AML). Here, we present 13 AML cases with t(8;16)(p11;p13) with observed low incidence (13/6124 patients), but more frequent presentation in therapy-related AML than in de novo AML (7/438 versus 6/5686, P=0.00001). Prognosis was poor with median overall survival of 4.7 months. Cytomorphology was characterized by parallel positive myeloperoxidase and non-specific esterase staining, therefore, French–American–British (FAB)-classification was impossible and origin of the AML with t(8;16) from an early stem cell with myeloid and monoblastic potential is hypothesized. Erythrophagocytosis was observed in 7/13 cases. Using gene expression profiling on 407 cases, patients with t(8;16) were compared to AML FAB subtypes with normal karyotype. Principal component analyses demonstrated that AML with t(8;16) were distinct from FAB subtypes M1, M4, M5a/b. When further compared to AML showing balanced rearrangements, that is, current WHO categories t(15;17), t(8;21), inv(16) and t(11q23)/MLL, AML with t(8;16) cases were clustered close to t(11q23)/MLL sharing commonly expressed genes. Subsequently, a pairwise comparison discriminated AML with t(8;16) from AML with t(11q23)/MLL, thus defining a highly unique signature for AML with t(8;16). In conclusion, AML with t(8;16) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features and is a specific subtype of AML.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.