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Purpose:In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations.Experimental Design:Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression.Results:We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism.Conclusions:Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress.See related commentary by Viny, p. 573

Supplementary Data from DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Purpose: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Experimental Design: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. Results: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Conclusions: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

Clonal hematopoiesis (CH) is a nonmalignant condition characterized by clonal expansion of one or few hematopoietic stem cells. CH is associated with advanced age and presence of presumed leukemic driver mutations, such as in the DNMT3A gene. Clinical studies show that presence of CH mutations in the bone marrow in patients with solid tumors is associated with unfavorable prognosis and shorter survival. Moreover, CH mutations are enriched in tumor-infiltrating leukocytes compared to peripheral blood. While these observations have profound clinical implications, the understanding of the relationship between mutations in the blood system and aggressive phenotype of solid tumors is still lacking. We hypothesized that bone marrow-derived cells harboring alterations in DNMT3A contribute to tumor-promoting microenvironment through immune imbalance. To this end, we established a mouse model of CH driven by Dnmt3a alterations and are investigating its effect on a well-established colitis-associated colon cancer model (CAC) as a prototypical solid tumor. Wild-type recipient mice were transplanted with bone marrow from animals with: a) heterozygous loss of Dnmt3a (Dnmt3a+/−); b) Dnmt3a R878H point-mutation (Dnmt3aRH); c) wild-type controls (Dnmt3a+/+). Since inflammation is known to promote tumorigenesis, we are testing the sensitivity of engrafted mice to experimental colitis induced by 2.5% dextran sulfate sodium salt (DSS) in drinking water. Animals of all 3 experimental groups will be co-housed to control for cage-to-cage variability in microbiota. We will use total body weight loss, histopathology of harvested colons (swiss roll method)/colitis scores, colon size, and appearance of intestinal contents as a readout for colitis severity. Next, we will investigate AOM/DSS-induced tumorigenesis in CH model, including tumor burden and extent of inflammation between experimental groups. Finally, we will characterize tumor-infiltrating leukocytes that contribute to aggressive CAC phenotype to identify immune populations that engender this tumor-modulating function, including epigenetic and gene expression profiles. Our goal is to understand the relationship between mutations in the blood system and aggressive phenotype of nonhematologic cancers and to nominate potential targets for therapeutic intervention. Citation Format: Yang Feng, Daniil E. Shabashvili, Luisa M. Posada, Kathryn I. Krajcik, Chamara Gunaratne, Olga A. Guryanova. Investigating the role of the hematopoietic-specific DNMT3A mutations in the aggressive phenotype of colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B93.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novoacute myeloid leukemia (AML) and are associated with poor prognosis. Although studies demonstrated survival benefit of induction chemotherapy dose intensification, outcomes remain unsatisfactory in most patients due to advanced age, comorbidities, and hence inability to tolerate treatment. Clinical trials of low-intensity regimens combining cytarabine and cladribine, nucleoside analog chain terminators that stall DNA replication, appear to be safe and effective, and tend to particularly benefit patients with DNMT3Amutations. Consistently, we observe increased sensitivity to cytarabine, fludarabine, and cladribine in multiple cellular systems harboring mutant DNMT3Ain vitro (Figure 1A, B). Differential sensitivity to cytarabine was confirmed in normal and leukemic primary bone marrow cells derived from mice with and without Dnmt3a mutations ex vivo (Figure 1C). Dynamic chromatin organization plays a pivotal role in DNA-associated cellular processes including DNA replication and damage repair. We previously found altered chromatin remodeling in cells expressing mutant DNMT3A after genotoxic stress. Gene expression studies by us and others demonstrated negative enrichment of cell cycle related signatures including G2/M checkpoint adaptation, in cells with DNMT3A mutations. These signatures are implicated in DNA damage response and replication fork integrity and suggest sensitivity to replication stress. To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed wildtype (WT) or R882 mutant (MUT) forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. Analysis of the DNA damage signaling proficiency in response to cytarabine revealed persistent intra-S phase checkpoint activation (phospho-CHK1), accompanied by accumulation of DNA damage visualized by γH2A.X foci and by Comet assay in the DNMT3A(mut) overexpression cells (Figure 1D). This damage was only partially resolved after drug had been removed and cells were allowed to repair the DNA (Figure 1E), and was carried through mitosis, resulting in increased rate of micronucleation.At the same time, DNMT3A mutant cells remained proficient in initiating homology-directed repair (HDR) and non-homologous end joining (NHEJ) pathways, evidenced by RAD51 and 53BP-1 foci formation, respectively. These data demonstrate enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, rather than defects in double-strand DNA break repair. In support of this, cells with mutant DNMT3Awere characterized by accentuated replication stress as evidenced by high levels of phospho-RPA, which persisted after drug wash-out (Figure 1F). Consistently, DNMT3A-mutant cells treated with cytarabine were characterized by a higher number and a larger area of PCNA foci. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that while the overall kinetics of replication restart remained unchanged, cells with DNMT3A(mut) showed higher rate of fork collapse and increased reliance on latent replication origins (Figure 1G). Gene expression profiling by RNA-seq identified dysregulation of pathways associated with cell cycle progression, specifically G1/S phase transition, DNA replication, DNA integrity checkpoint, and chromatin. Our studies show cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication and suggest that cytarabine-induced replication fork stalling may further synergize with other agents aimed at DNA damage and replication. Figure 1 Disclosures No relevant conflicts of interest to declare.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) (20-35%) and are associated with poor prognosis. Although studies demonstrated survival benefit after daunorubicin dose intensification for DNMT3A mutant AML, these patients tend to be older (median age at diagnosis 67 y.o. in DNMT3A-mutated cases compared to 60 y.o. in DNMT3A-WT) and more likely to be unfit for high-dose chemotherapy. In older patients, cytarabine monotherapy, a nucleoside analog chain terminator that stalls DNA replication, may be preferred due to lower toxicity. However, its efficacy in DNMT3A mutant AML has not been evaluated. Previous gene expression studies in large AML cohorts and in a mouse model of Dnmt3a-mutant hematopoiesis demonstrated negative enrichment of the cell cycle related signatures, including G2/M checkpoint and E2F targets. Hence, we hypothesized cell cycle-specific drugs cytarabine and fludarabine may be effective in DNMT3A-mutant context. DNMT3A mutant AML cell lines (MUT) SET-2 and KO-52 were more sensitive to cytarabine and fludarabine than DNMT3A wild-type (WT) cells KU-812 and K-562 in vitro (cytarabine IC50 46.1 and 165.8 μM in MUT SET-2 and KO-52 compared to 465.2 μM and not reached in WT KU-812 and K-562, respectively; fludarabine IC50 1.1 and 1.3 μg/ml in MUT vs 16 μg/ml and not reached in WT, Figure 1A). Higher proportion of cells with mutant DNMT3A were undergoing apoptosis 24 hours after cytarabine exposure (26.5±2.3% and 13.1±2.3% Annexin V+ cells in SET-2 and KO-52 vs 6.9±2.6% and 4.6±2.5% in KU-812 and K-562, respectively, p To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed WT or R882 mutant forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. MUT cells were more sensitive to cytarabine compared to WT (IC50 38 μM vs 213 μM after 48 hours of exposure, Figure 1B), which was accompanied by increased apoptosis (22.3±7.6% in MUT vs 7.2±0.3% in WT Annexin V+ cells, p In conclusion, our studies show cells with DNMT3A mutations may be sensitive to DNA damage induced by clinically relevant nucleoside analogs such as cytarabine. Our data support continuous infusion of cytarabine rather than bolus administration, and establish its mechanistic basis. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication. Figure 1. Figure 1. Disclosures Licht: Celgene: Research Funding.