Nichole M. Helton

Epigenomic analysis of the HOX gene loci reveals mechanisms that may control canonical expression patterns in AML and normal hematopoietic cells

HOX genes are highly expressed in many acute myeloid leukemia (AML) samples, but the patterns of expression and associated regulatory mechanisms are not clearly understood. We analyzed RNA sequencing data from 179 primary AML samples and normal hematopoietic cells to understand the range of expression patterns in normal versus leukemic cells. HOX expression in AML was restricted to specific genes in the HOXA or HOXB loci, and was highly correlated with recurrent cytogenetic abnormalities. However, the majority of samples expressed a canonical set of HOXA and HOXB genes that was nearly identical to the expression signature of normal hematopoietic stem/progenitor cells. Transcriptional profiles at the HOX loci were similar between normal cells and AML samples, and involved bidirectional transcription at the center of each gene cluster. Epigenetic analysis of a subset of AML samples also identified common regions of chromatin accessibility in AML samples and normal CD34+ cells that displayed differences in methylation depending on HOX expression patterns. These data provide an integrated epigenetic view of the HOX gene loci in primary AML samples, and suggest that HOX expression in most AML samples represents a normal stem cell program that is controlled by epigenetic mechanisms at specific regulatory elements.

Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

Genetic heterogeneity of induced pluripotent stem cells: Results from 24 clones derived from a single C57BL/6 mouse

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs “captures” the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically “fit” for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0–45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40–60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.

Cellular stressors contribute to the expansion of hematopoietic clones of varying leukemic potential

Hematopoietic clones harboring specific mutations may expand over time. However, it remains unclear how different cellular stressors influence this expansion. Here we characterize clonal hematopoiesis after two different cellular stressors: cytotoxic therapy and hematopoietic transplantation. Cytotoxic therapy results in the expansion of clones carrying mutations in DNA damage response genes, including TP53 and PPM1D. Analyses of sorted populations show that these clones are typically multilineage and myeloid-biased. Following autologous transplantation, most clones persist with stable chimerism. However, DNMT3A mutant clones often expand, while PPM1D mutant clones often decrease in size. To assess the leukemic potential of these expanded clones, we genotyped 134 t-AML/t-MDS samples. Mutations in non-TP53 DNA damage response genes are infrequent in t-AML/t-MDS despite several being commonly identified after cytotoxic therapy. These data suggest that different hematopoietic stressors promote the expansion of distinct long-lived clones, carrying specific mutations, whose leukemic potential depends partially on the mutations they harbor.Cellular stressors can impact clonal hematopoiesis. Here, the authors explore the impact of cytotoxic therapy and hematopoietic transplantation on clonal expansion, suggesting different stressors can promote expansion of distinct long-lived clones.

DNMT3A R882 -associated hypomethylation patterns are maintained in primary AML xenografts, but not in the DNMT3A R882C OCI-AML3 leukemia cell line

DNMT3A mutations act as dominant negative alleles in vitro and are associated with focal regions of DNA hypomethylation in primary acute myeloid leukemia (AML) samples and non-leukemic hematopoietic cells. In primary AML cells, this hypomethylation manifests both as methylation loss and attenuated CpG island hypermethylation relative to normal hematopoietic stem/ progenitor cells. Although DNMT3A mutations have a clear effect on DNA methylation in AML cells, the functional consequences of these changes are not yet clear. Future study of the downstream effects of mutant DNMT3A-associated hypomethylation will require model systems to investigate the genomic targets that are affected, and to understand whether these changes alter gene regulation in ways that promote leukemogenesis. Examples of model systems include genetically modified mice, patient-derived xenografts, and human cell lines containing DNMT3A mutations. The methylation phenotypes of mice lacking Dnmt3a, or expressing mutant Dnmt3a alleles, have been reported previously, but much less is known about whether alterations in methylation caused by DNMT3A alleles are retained in either patient-derived xenografts or human AML cell lines, and whether these models could therefore be used to accurately represent DNMT3A-dependent methylation changes in AML cells. To address this question, we performed whole-genome bisulfite sequencing (WGBS) using DNA from OCIAML3 cells, which is the only leukemia cell line currently known to have a native DNMT3A mutation. We also evaluated four xenografts derived from a primary AML sample containing the DNMT3A mutation. The OCI-AML3 line was obtained from the DSMZ cell collection and cultured via recommended conditions before DNA extraction at two independent passages for WGBS. The presence of the DNMT3A allele in these cells was verified via targeted sequencing prior to methylation analysis (Supplementary Figure S1), as were the recurrent NPM1 exon 12 insertion (NPMc) and the NRAS mutation. No functional mutations were identified in other recurrently mutated AML genes with roles in epigenetic modification, such as IDH1, IDH2, ASXL1, EZH2, or TET2. Two missense variants of unknown significance were present in TET1 (Supplementary Table S2), which is not frequently mutated in AML samples. Importantly, we saw no evidence for amplification of the wild-type DNMT3A allele in this cell line (data not shown). We also extracted two replicate DNA samples from comparator AML cell lines that are wild-type for DNMT3A, including Kasumi-1 and NB4, which have a t(8;21) translocation (creating the RUNX1RUNX1T1 fusion gene) and a t(15;17) translocation (resulting in a PML-RARA fusion), respectively. Patientderived AML xenografts were generated in two independent humanized NSG mice (NSG-SGM3) from a primary AML sample with the DNMT3A mutation (along with NPM1 and FLT3-ITD mutations; AML 721214, described as AML88 in ref. ; Supplementary Table S1) via tail vein injection of 1 million cells. Mice

Expression profiling of snoRNAs in normal hematopoiesis and AML

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis.

Comprehensive discovery of noncoding RNAs in acute myeloid leukemia cell transcriptomes

To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses.