Germline pathogenic variants in DNMT3A were recently described in patients with overgrowth, obesity, behavioral, and learning difficulties (DNMT3AOvergrowth Syndrome/DOS). Somatic mutations in the DNMT3A gene are also the most common cause of clonal hematopoiesis, and can initiate acute myeloid leukemia (AML). Using whole genome bisulfite sequencing, we studied DNA methylation in peripheral blood cells of 11 DOS patients and found a focal, canonical hypomethylation phenotype, which is most severe with the dominant negative DNMT3AR882H mutation. A germline mouse model expressing the homologous Dnmt3aR878H mutation phenocopies most aspects of the human DOS syndrome, including the methylation phenotype and an increased incidence of spontaneous hematopoietic malignancies, suggesting that all aspects of this syndrome are caused by this mutation.

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.

CD19-targeted immunotherapies that stimulate a cytotoxic T-cell response have revolutionized the management of B-cell malignancies, specifically relapsed or refractory acute lymphoblastic leukemia (R/ R ALL). Blinatumomab is an anti-CD19 bispecific T-cell engager approved for the treatment of patients with R/R ALL. 1 KTE-X19 is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy under investigation in the ZUMA-3 (XXX) phase 1/2 clinical trial (registered at www.clinicaltrials.gov as #NCT02614066) for adult patients with R/R ALL. 2 The emergence of leukemic clones that have lost surface expression of CD19 is a cause of disease relapse in 10% to 25% of patients treated with either blinatumomab or CAR T cells. [1][2][3][4][5] Several distinct mechanisms have been documented, including truncating mutations in the transmembrane domain of CD19 that prevent surface expression, 5 as well as alternative messenger RNA splicing that results in loss of the antigenic epitope and prevents CAR binding. 3,4 Here we describe the discovery of a novel genomic modification outside of the transmembrane and antigenic domains of CD19 that enable resistance to blinatumomab and KTE-X19.A 60-year-old woman with B-cell ALL was treated with chemotherapy followed by 2 cycles of blinatumomab as initial therapy. She experienced disease relapse after the second cycle of blinatumomab and was enrolled in the ZUMA-3 investigational study of KTE-X19. She received a single infusion of anti-CD19 CAR T cells at the target dose but had no disease response.