ALBERT

Description: Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a−/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a−/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a−/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Description: Self-renewal is a key feature of the cells that initiate acute myeloid leukemia. To identify the mechanisms involved in PML-RARA (PR)-driven self-renewal, we made use of Ctsg-PR mice, which have PR knocked into the UTR of the first exon of Ctsg. Ctsg-PR drives the expression of PR in myeloid progenitor cells and gives these cells the ability to serially replate in methylcellulose-based colony assays. Most Ctsg-PR mice develop acute promyelocytic leukemia (APL) with an average latency of ~300 days. To identify target genes regulated by Ctsg-PR, we performed single cell RNA-seq (scRNA-seq) on whole bone marrow from young, preleukemic Ctsg-PR mice or age-matched littermates. We identified 959 differentially expressed genes (DEGs) within myeloid progenitors (546 upregulated by PR, and 413 downregulated). Gata2 was identified as a DEG in this analysis, and we confirmed this phenotype with bulk RNA-seq of purified promyelocytes from young, preleukemic WT vs. Ctsg-PR mice. All APLs derived from Ctsg-PR mice also expressed high levels of Gata2. To identify the immediate-early target genes of PR, we transduced human CD34+ cord blood cells with MSCV-IRES-GFP retroviruses containing PR, a mutant PR with a RARA DNA binding mutation (C88A, known to abolish PR replating), or an empty vector. ScRNA-seq analysis of these cells after 7 days of ex vivo culture identified 1815 DEGs (1301 upregulated and 514 downregulated by PR) in GFP+ cells expressing PR, compared to GFP+ cells transduced with PRC88A or empty vector. Among the DEGs, Gata2 was upregulated 5-fold in the PR GFP+ cells. Identical short-term retroviral overexpression studies with mouse marrow revealed that PR expression caused an expansion of hematopoietic progenitor cells that overexpressed Gata2. Finally, although normal human promyelocytes do not express GATA2, virtually all primary human APL samples do. Combined, these studies strongly suggest that GATA2 is a target gene of PR, and may therefore play a role in the development of APL. Based on our expression data, we hypothesized that Gata2 inactivation would reduce PR-driven self-renewal. To test this hypothesis, we bred Ctsg-PR mice to Rosa26-Cas9 mice, which ubiquitously express Cas9. Marrow from the resulting Ctsg-PR x Cas9 mice was electroporated with guide RNAs (gRNAs) targeting the zinc finger 1 (ZF1) domain or exon 2 of Gata2, or control loci (Actb intron 5 or Rosa26 intron 1) and serially replated in methylcellulose with SCF, IL-3, and IL-6. CRISPR-Cas9 efficiently induced a wide array of indel mutations at all gRNA target sites. Two days after electroporation, the frequency of Gata2 alleles with indels at the target site ranged from 64% to 93% (n=4); hundreds of different Gata2 indels were generated in each experiment. To our surprise, the Gata2 targeted Ctsg-PR cells replated with dramatically higher efficiency than control locus targeted cells (Figure 1). The enhanced replating efficiency was dependent upon PR, since Gata2 targeted Rosa26-Cas9 bone marrow did not serially replate. Further, cells with Gata2 indels were positively selected for over time. For example, in one experiment, the frequency of a 12 bp deletion in Gata2 (that caused an in-frame deletion in ZF1) rose from an initial variant allele frequency (VAF) of 3% to 70% after 8 weeks of replating. A larger Gata2 deletion was co-selected at a similar frequency, and resulted in the deletion of Gata2 exon 4, which encodes 90% of ZF1. Virtually all of the positively selected cells contained Gata2 indels, demonstrating the competitive advantage of Gata2 loss-of-function mutations in this setting. Control gRNAs did not lead to significant changes in plating efficiency, nor were any indels selected for. Additionally, Ctsg-PR x Cas9 cells with Gata2 indels shifted from a neutrophilic phenotype (Gr1+ CD11b+) to a monocytic one (CD11b+ Gr1-) at late passages. To further investigate the role of Gata2 in Ctsg-PR induced APL, we sequenced the genomes of 16 mouse APLs and found that 2 samples had spontaneous mutations in Gata2 (R330L and N363fs, with VAFs of 74% and 39% respectively). In summary, these data provide evidence that PR positively regulates Gata2, and that Gata2 in turn promotes neutrophilic differentiation of committed, "reprogrammed" myelomonocytic progenitors. Surprisingly, Gata2 appears to contribute to the lineage fate and proliferative capacity of PR-expressing hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

Description: The gene that encodes DNA methyltransferase 3A (DNMT3A) is mutated in nearly 40% of normal karyotype acute myeloid leukemia (AML) patients. More importantly, DNMT3A mutations are almost invariably the initiating event for the disease; however, the mechanisms by which they contribute to AML initiation are not yet clear [1, 2]. Regardless, ancestral clones containing the DNMT3A mutation are hard to eradicate, and often persist in clinical remission [3, 4]. We have recently shown that the most common DNMT3A mutation in AML patients (R882H; R878H in mice) is a dominant negative mutation that causes a focal, canonical pattern of DNA hypomethylation that is present in pre-leukemic hematopoietic cells, and appears to evolve further in fully transformed AML cells [5]. However, it is not yet clear how the DNMT3AR882H mutation can cause the clonal expansion of HSPCs, or how it makes HSPCs more susceptible to transformation by secondary mutations. We have utilized the conditional Dnmt3aR878H knock-in model described by Guryanova et. al. [6] and established an hematopoietic-specific model of clonal hematopoiesis driven by DNMT3AR882 mutations. With this model, we set out to investigate the epigenetic and functional consequences of Dnmt3aR878H expression in murine hematopoietic cells. The bone marrow cells of 6-week-old Dnmt3aR878H x Vav1-CREmice are 〉95% floxed; although these mice have a distinct focal hypomethylation phenotype, it is not as striking as that of Dnmt3a deficient bone marrow cells (this is expected, since the dominant negative R882H mutation reduces DNMT3A activity by 80%, not 100%). Importantly, Dnmt3aR878H and WT RNA are expressed at a precise 50:50 ratio, mimicking the ratio found in human patients with this mutation [7]. To establish the "baseline" methylation status of Dnmt3a+/+ vs. Dnmt3aR878H/+ bone marrow cells, we harvested marrow from 6-week-old littermates and performed whole genome bisulfite sequencing on 7 independent mice from each genotype. The R878H bone marrow cells have focal, canonical regions of DNA hypomethylation that strongly resemble those seen in human patients with DNMT3AR882 mutations. 2,621 differentially methylated regions (DMRs) exist in the Dnmt3aR878H/+ marrow samples, and 〉99% are hypomethylated. Dnmt3a-/- bone marrow cells have 〉 8,000 DMRs compared to Dnmt3a+/+ bone marrow cells, highlighting the intermediate methylation phenotype in Dnmt3aR878H marrow (Figure 1A). Further, the hypomethylation phenotype is stable with aging, and does not progress between weeks 2 and 52 of life (data not shown). Previous studies utilizing bulk RNA sequencing demonstrated very few differentially expressed genes (DEGs) in primary human AML samples with DNMT3AR882H. Therefore, we utilized single cell RNA sequencing to identify DEGs in the progenitor populations of Dnmt3aR878H hematopoietic cells. In addition to the methylation phenotype outlined above, we have identified a set of 117 DEGs (30 genes up and 87 genes down) in lineage negative, c-KIT positive cells from Dnmt3aR878H bone marrow, including Hspa1a and Hspa1b, Cxcl2 and Cxcl12, Fosb and C1q complement genes, with pathway analyses suggesting dysregulation of the proteasome, chemokine and inflammatory signaling, and apoptotic and proliferative pathways (Figure 1B). These pathways have all been implicated in cancer, and may provide a rationale for the Dnmt3aR878H -driven pre-leukemic phenotype. Extensive hematopoietic immunophenotyping has revealed subtle population changes in progenitor and stem populations of 6-week old Dnmt3aR878H mice; we identified significantly lower granulocyte-monocyte progenitors (GMP) and multipotent progenitors (MPP), and a significant increase in SLAM cells. Although these differences are significant, they do not result in any gross lineage population skewing in the bone marrow or peripheral blood. In sum, these data define some of the epigenetic consequences of the Dnmt3aR878H mutation in hematopoietic cells, and establish this mouse model as a valid one for the study of this mutation. This is the first report of the pre-leukemic state mediated by Dnmt3aR878H and these data have important implications in the design of future pharmacological agents targeting this mutation in patients with AML. Disclosures No relevant conflicts of interest to declare.

Description: Bacterial leaf streak (BLS) caused by Xanthomonas campestris pv. translucens is one of the major bacterial diseases threatening wheat production in the United States Northern Great Plains (NGP) region. It is a sporadic but widespread wheat disease that can cause significant loss in grain yield and quality. Identification and characterization of genomic regions in wheat that confer resistance to BLS will help track resistance genes/QTLs in future wheat breeding. In this study, we evaluated a hard winter wheat association mapping panel (HWWAMP) containing 299 hard winter wheat lines from the US hard winter wheat growing region for their reactions to BLS. We observed a range of BLS responses among the lines, importantly, we identified ten genotypes that showed a resistant reaction both in greenhouse and field evaluation. ­Genome-wide association analysis with 15,990 SNPs was conducted using an exponentially compressed mixed linear model. Five genomic regions (p 

Description: DNA hypomethylation is a feature of epidermal cells from aged and sun-exposed skin, but the mechanisms responsible for this methylation loss are not known. Dnmt3a is the dominant de novo DNA methyltransferase in the skin; while epidermal Dnmt3a deficiency creates a premalignant state in which keratinocytes are more easily transformed by topical mutagens, the conditions responsible for this increased susceptibility to transformation are not well understood. Using whole genome bisulfite sequencing, we identified a focal, canonical DNA hypomethylation phenotype in the epidermal cells of Dnmt3a-deficient mice. Single-cell transcriptomic analysis revealed an increased proportion of cells with a proliferative gene expression signature, while other populations in the skin were relatively unchanged. Although total DNMT3A deficiency has not been described in human disease states, rare patients with an overgrowth syndrome associated with behavioral abnormalities and an increased risk of cancer often have heterozygous, germline mutations in DNMT3A that reduce its function (Tatton-Brown Rahman syndrome [TBRS]). We evaluated the DNA methylation phenotype of the skin from a TBRS patient with a germline DNMT3AR882H mutation, which encodes a dominant-negative protein that reduces its methyltransferase function by ∼80%. We detected a focal, canonical hypomethylation phenotype that revealed considerable overlap with hypomethylated regions found in Dnmt3a-deficient mouse skin. Together, these data suggest that DNMT3A loss creates a premalignant epigenetic state associated with a hyperproliferative phenotype in the skin and further suggest that DNMT3A acts as a tumor suppressor in the skin.