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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.

Mutations in DNA damage regulators and epigenetic modifier genes including those in DNA methyltransferase 3A (DNMT3A) are common in pre-malignant clonal hematopoiesis (CH) and are recurrent in myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While loss-of-function (LOF) mutations in DNMT3A are predominant in CH, single amino-acid substitutions at residue 882, usually from an arginine to a histidine or cysteine (DNMT3A R882H / DNMT3A R882C), are strongly enriched in AML, suggesting a role in promoting pre-malignant clonal evolution. In this study, we compared and contrasted the effects of DNMT3A R882 and LOF on hematopoietic stem cell (HSC) function by investigating self-renewal capacity in vitro and bone marrow repopulation potential in vivo in a genetic mouse model. Bone marrow (BM) from mice conditionally expressing Dnmt3a R878H (equivalent to human DNMT3A R882H) from the endogenous locus, denoted as +/mut, was subjected to colony forming unit (CFU) assays in semisolid media (MethoCult GF M3434, Stem Cell Technologies) to detect hematopoietic progenitors with serial replating every 14 days as a surrogate measure of stem cell self-renewal. Dnmt3a +/mut cells were compared to bone marrow derived from mice with heterozygous or homozygous Dnmt3a knockout (+/- and -/-, respectively), or wild-type control (+/+). Dnmt3a +/- BM cells showed a trend towards a slight increase in the number of replatings compared to wild-type controls (2.83±0.41 in +/+ vs 3.75±0.89 in +/-, n=6,8, Mann-Whitney test p=0.053). In contrast, Dnmt3a +/mut BM occasionally resulted in a significant gain of self-renewal with replating for 6+ passages observed in 4/10 independent trials (6.00±2.06 in +/mut, n=10, p=0.001 vs +/+), yet failed to reach continuous replating of complete Dnmt3a loss that served as a positive control (terminated at passage 10, n=5). Substantial heterogeneity in the Dnmt3a +/mut self-renewal capacity ex vivo was further reflected by increased statistical variance (Welch's F-test p=0.037 compared to +/-). To extend these studies to the in vivo setting, we performed gold-standard serial competitive bone marrow repopulation assays. Here, 250 FACS-sorted Lineage -Sca1 +cKit +CD48 -CD150 + (LSK-SLAM) test cells marked with CD45.2 were mixed with 0.25x10 6 unfractionated wild-type competitor bone marrow cells marked with CD45.1 and engrafted into pre-conditioned congenic recipients (CD45.1). Peripheral blood CD45.2/CD45.1 chimerism is used as a readout for the ability of test cells to reconstitute hematopoiesis; self-renewal potential is assessed via serial retransplantation. In this setting, Dnmt3a +/+ cells showed a decline in their ability to reconstitute hematopoiesis in secondary transplant recipients, while Dnmt3a haploinsufficiency resulted in a competitive advantage over wild-type (CD45.2 chimerism 2.4±2.7% in +/+ vs 75.6±13.0% in +/-, n=4, Welch's t-test p=0.001). In contrast, Dnmt3a +/mut cells displayed a high degree of variability in their long-term stem cell function in some cases yielding high levels of chimerism (CD45.2 >40% in 3/9 recipients) and in other cases not (26.0±21.0%, n=9, p=0.01 vs +/+), in agreement with CFU assays. Collectively, these data suggest that the DNMT3A R882 mutations endow normal HSCs with phenotypic heterogeneity yielding a population of cells with variable stem cell function, some of which may have higher fitness thus accelerating clonal evolution towards malignancy. These findings will be built upon in future experiments aiming to identify underlying molecular mechanisms and to improve our understanding of the pathophysiology and prognostication of clonal hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Clonal hematopoiesis (CH) is a condition wherein hematopoietic stem cells (HSCs) acquire mutations, often in presumed leukemia driver genes such as in DNMT3A, leading to clonal dominance and disproportionate contribution to peripheral blood without overt hematologic abnormalities (Jaiswal et al. 2014). In large cohorts of patients with solid tumors including colorectal cancer, presence of CH is associated with inferior overall survival due to progression of primary malignancy (Coombs et al. 2017). While potential involvement of CH in the pathogenesis of solid tumors driven by unrelated genetic alterations has far-reaching translational implications, the mechanistic understanding of this relationship is lacking. Colorectal cancer is a leading cause of cancer deaths worldwide and can arise from preceding inflammatory bowel disease. More than 20% of patients with colon cancer have detectable CH, which is notably more prevalent than in age-matched general population. We hypothesized that bone marrow-derived cells harboring alterations in DNMT3A, the most commonly disrupted gene in CH (up to 40% of cases), contribute to the pathogenesis of colorectal cancer. To address this, we combined a bone marrow transplantation-based model of CH driven by heterozygous loss of Dnmt3a with a well-established induction of colitis-associated colon cancer (CAC) by azoxymethane (AOM, a carcinogen) and dextran sodium sulfate (DSS, a colitogen). High resolution colonoscopy demonstrated increased colon wall opacity, visible bleeding, numerous fibrin patches and earlier onset of tumor formation with higher penetrance in animals with partial ablation of Dnmt3a in the hematopoietic compartment. Heightened colon pathology was reflected by modified murine endoscopic index of CAC severity (MEICS) (Fung and Putoczki 2018) (MEICS scores 7.5±2.0 in WT vs 10.0±3.2 in Dnmt3a +/-, p=0.05, n=14,13). We observed elevated incidence of tumors >2mm in diameter (40.2±22.2% in WT vs 74.2±15.7% in Dnmt3a +/-, p=0.03, in 5 independent trials) and a higher tumor burden in Dnmt3a +/- chimerae (7.4±7.8 mm 3 in WT vs 14.5±13.9 mm 3 in Dnmt3a +/-, p=0.0017, n=53,55) at end point. Histopathological analysis revealed marked immune infiltration, extensive ulceration and dysplasia of colonic epithelium, and more frequent adenocarcinoma formation with occasional submucosal invasion (histology score 20.2±4.4 in WT vs 24.6±6.9 in Dnmt3a +/-, p=0.03, n=10,9). To uncover signaling pathways deregulated by experimental Dnmt3a-driven CH, we surveyed the auto- and paracrine stimulatory loops by multiplex cytokine/chemokine assays and profiled transcriptomes of colon tumors by bulk RNA-seq. These studies identified gene signatures commonly associated with colon carcinogenesis including accentuated epithelial-to-mesenchymal transition (gene set enrichment analysis (GSEA) NES=2.42, padj=0.0096), Wnt/b-catenin (NES=2.07, padj=0.0096), and Vegf/angiogenesis (NES=1.83, padj=0.0096). Elevated proliferation (Ki67) and increased vascularization (CD31) were validated by immunohistochemistry. These aspects of tumor pathophysiology along with the composition and functional characteristics of the tumor-infiltrating leukocytes derived from animals with and without hematopoietic-specific Dnmt3a partial loss are being further investigated. Preliminary data showed bone marrow-derived macrophages (BMDMs) from Dnmt3a +/- chimera exhibit impaired ability to phagocytose syngeneic MC38 colon tumor cells, compared to WT. Our study provides new insights into the non-tumor-cell-autonomous mechanisms of carcinogenesis effected by alterations in the hematopoietic system and into the interplay between the immune system and the aggressive phenotype of colorectal cancer. Our findings may aid the development of novel therapeutic options to improve outcomes in solid tumor patients with CH. Figure 1 Figure 1. Disclosures Bowman: Mission Bio: Honoraria, Speakers Bureau. Levine: Ajax: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Zentalis: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Roche: Honoraria, Research Funding.

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.

The rapidly increasing energy consumption by computing and communications equipment is a significant economic and environmental problem that needs to be addressed. Ethernet network interface controllers (NICs) in the US alone consume hundreds of millions of US dollars in electricity per year. Most Ethernet links are underutilized and link energy consumption can be reduced by operating at a lower data rate. In this paper, we investigate adaptive link rate (ALR) as a means of reducing the energy consumption of a typical Ethernet link by adaptively varying the link data rate in response to utilization. Policies to determine when to change the link data rate are studied. Simple policies that use output buffer queue length thresholds and fine-grain utilization monitoring are shown to be effective. A Markov model of a state-dependent service rate queue with rate transitions only at service completion is used to evaluate the performance of ALR with respect to the mean packet delay, the time spent in an energy-saving low link data rate, and the oscillation of link data rates. Simulation experiments using actual and synthetic traffic traces show that an Ethernet link with ALR can operate at a lower data rate for over 80 percent of the time, yielding significant energy savings with only a very small increase in packet delay.

The IT equipment comprising the Internet in the USA uses about $6 billion of electricity every year. Much of this electricity use is wasted on idle, but fully powered-up, desktop PCs and network links. We show how to recover a large portion of the wasted electricity with improved power management methods that are focused on network issues.

Storage, memory, processor, and communications bandwidth are all relatively plentiful and inexpensive. However, a growing expense in the operation of computer networks is electricity usage. Estimates place devices connected to the Internet as consuming about 2%, and growing, of the total electricity produced in the USA-much of this power consumption is unnecessary. Power management is needed to reduce this large and growing energy consumption of the Internet. We see power management as the 'next frontier' in research in computer networks. In this paper, we propose methods for reducing energy consumption of networked desktop computers. Using traffic characterization of university dormitory computers, we show that there is significant idle time that can be exploited for power management. However, current Ethernet adapters in desktop computers lack the capabilities needed to allow existing system power management features to be enabled. We address this problem with a proxying Ethernet adapter that handles routine network tasks for a desktop computer when it is in a low-power sleep mode. This proxying adapter can allow existing power management features in desktop computers to remain enabled and have the computer be 'on the network' at all times. The energy that we expect can be saved is in the range of 0.8-2.7 billion US dollars/year.

Rapidly increasing energy use by computing and communications equipment is a significant problem that needs to be addressed. Ethernet network interface controllers (NICs) consume hundreds of millions of US$ in electricity per year. Most Ethernet links are underutilized and link power consumption can be reduced by operating at lower data rates. An output buffer threshold policy to change link data rate in response to utilization is investigated. Analytical and simulation models are developed to evaluate the performance of Adaptive Link Rate (ALR) with respect to mean packet delay and time spent in low data rate with Poisson traffic and 100 Mb/s network traces as inputs. A Markov model of a state-dependent service rate queue with rate transitions only at service completion is developed. For the traffic traces, it is found that a link can operate at 10 Mb/s for over 99% of the time yielding energy savings with no user-perceivable increase in packet delay.