Evidence supporting a dominant negative mechanism for DNMT3A hotspot mutation-mediated leukemic cell transformation
Ontology highlight
ABSTRACT: Mutation of DNA methyltransferase 3A at arginine 882 (DNMT3AR882mut) is prevalent in various hematological cancers. DNMT3AR882mut was recently shown to carry partially defective, dominant-negative or gain-of-function activities under different in vitro contexts. However, the causal roles for such a multifaceted effect of DNMT3AR882mut on leukemogenesis remain undefined. Here we report TF-1 leukemia cells as a robust system for modeling DNMT3AR882mut-dependent cell transformation phenotypes and for performing structure-function relationship studies of DNMT3AR882mut. We show that expression of DNMT3AR882mut and not its wildtype counterpart promotes TF-1 cell transformation and induces CpG hypomethylation predominantly at enhancers. Such effect by DNMT3AR882mut is dose-dependent, acts synergistically with that of IDH1 mutation, and resembles what was seen in human leukemia patients with DNMT3AR882mut. Both transformation- and hypomethylation-inducing capacities of DNMT3AR882mut rely on a motif involved in heterodimerization whereas its various chromatin-binding and enzymatic domains were dispensable. We also show bromodomain inhibition as a therapeutic means for treatment of murine leukemia carrying DNMT3AR882mut. Collectively, this study describes a useful model system for studying DNMT3AR882mut and supports a requirement for the dominant-negative effect of DNMT3AR882mut during leukemogenesis.
Project description:Infant and adult MLL-rearranged (MLLr) leukemia represents a disease with few treatment options and a dismal prognosis. Here, we present an in-depth proteomic characterization of in utero-initiated and adult-onset MLLr leukemia in a mouse model of MLL-ENL-mediated leukemogenesis. We characterize early proteomic events of MLL-ENL-mediated transformation in fetal and adult progenitors.
Project description:Somatic mutations in DNA Methyltransferase 3A (DNMT3A) with a hotspot in exon 23 at Arginine 882 (DNMT3AR882mut) are the most frequent single mutations in clonal hematopoiesis. Here we analyze the expression of genes and endogenous retrovirus (ERV) sequences in a murine model carrying human DNMT3A-R882H mutation in one allele of the endogenous DNMT3A with respect to normal condition and azacitidine treatment.
Project description:CHAF1B is the p60 subunit of the chromatin assembly factor (CAF1) complex, which is responsible for assembly of H3.1/H4 heterodimers at the replication fork during S phase. Here we report that CHAF1B is required for normal hematopoiesis while its overexpression promotes leukemia. CHAF1B has a pro-leukemia effect by binding chromatin at discrete sites and interfering with occupancy of transcription factors that promote myeloid differentiation, such as CEBPA. Reducing Chaf1b activity by either heterozygous deletion or overexpression of a CAF1 dominant negative allele was sufficient to suppress leukemogenesis in vivo without impairing normal hematopoiesis.
Project description:DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
Project description:Connectivity map analysis using an AML-specific cMYB gene signature (Walf-Vorderwulbecke et al. Leukemia. 2018;32(4):882-889. 10.1038/leu.2017.317) identified withaferin A (WFA) as a candidate cMYB deregulating agent in AML. In order to validate the predicted effect of WFA on cMYB function, RNAseq analysis was performed on THP1 cells following exposure to DMSO or 1mM WFA for 6 hours.
Project description:Genetic studies have identified recurrent somatic mutations in Acute Myeloid Leukemia (AML) patients, including in WT1 (Wilms’ tumor gene 1). The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, with increased stem cell function over time and age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including in known leukemia disease alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 haploinsufficiency cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss in leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.
Project description:The AML1/ETO fusion protein is essential to the development of acute myeloid leukemia (AML), and is well recognized for its dominant-negative effect on the co-existing wild-type protein AML1. However, the involvement of wild-type AML1 in AML1/ETO-driven leukemogenesis remains elusive. Through chromatin immunoprecipitation sequencing, computational analysis plus a series of experimental validations, we report here that AML1 is able to orchestrate the expression of AML1/ETO targets regardless of being activated or repressed, via forming a complex with AML1/ETO and via recruiting the cofactor.
Project description:T lymphocyte acute lymphoblastic leukemia (T-ALL) is frequently associated with increased expression of the E protein transcription factor inhibitors TAL1 and LYL1. In mouse models, ectopic expression of Tal1 or Lyl1 in T cell progenitors, or inactivation of E2a, is sufficient to predispose mice to develop T-ALL. How E2a suppresses thymocyte transformation is currently unknown. Here, we show that early deletion of E2a, prior to the DN3 stage, was required for robust leukemogenesis and was associated with alterations in thymus cellularity, T cell differentiation, and gene expression in immature CD4+CD8+ thymocytes. Introduction of wild-type thymocytes into mice with early deletion of E2a prevented leukemogenesis, or delayed disease onset, and impacted the expression of multiple genes associated with transformation and genome instability. Our data indicate that E2a suppresses leukemogenesis by promoting T cell development and enforcing inter-thymocyte competition, a mechanism that is emerging as a safeguard against thymocyte transformation. These studies have implications for understanding how multiple essential regulators of T cell development suppress T-ALL and support the hypothesis that thymus cellularity is a determinant of leukemogenesis
Project description:ZNF384-rearranged fusion oncoproteins (FO) define a subset of lineage ambiguous leukemias, but the mechanistic role of ZNF384 FO in leukemogenesis and lineage ambiguity is poorly understood. Here, using viral expression in mouse and human hematopoietic stem and progenitor cells (HSPCs) and a Ep300-Zfp384 mouse model we show that ZNF384 FO promote hematopoietic expansion, myeloid lineage skewing, and self-renewal. In mouse HSPCs, concomitant lesions such as NRASG12D, were required for fully penetrant leukemia, whereas expression of ZNF384 FO drove development of B/myeloid leukemia in human HSPCs, with sensitivity of human ZNF384r leukemia to FLT3 inhibition in vivo. Mechanistically, ZNF384 FO occupy a subset of predominantly intragenic/enhancer regions with increased histone 3 lysine acetylation suggesting enhancer function. These data define a paradigm for FO-driven lineage ambiguous leukemia, in which expression in HSPCs results in deregulation of lineage-specific genes and hematopoietic skewing, progressing to full leukemic transformation in the presence of proliferative stress.
Project description:Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemia stage in which B-cell precursors display self-renewal ability, initiating precursor B-ALL that resembles PAX5 P80R or Ph-like human leukemia. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving both JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are novel treatment avenues for IL-7R-related cases. Our model, a unique resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.