L1CAM regulates DNA damage checkpoint response of glioblastoma stem cells through NBS1.

Glioblastomas (GBMs) are highly lethal brain tumours with current therapies limited to palliation due to therapeutic resistance. We previously demonstrated that GBM stem cells (GSCs) display a preferential activation of DNA damage checkpoint and are relatively resistant to radiation. However, the molecular mechanisms underlying the preferential checkpoint response in GSCs remain undefined. Here, we show that L1CAM (CD171) regulates DNA damage checkpoint responses and radiosensitivity of GSCs through nuclear translocation of L1CAM intracellular domain (L1-ICD). Targeting L1CAM by RNA interference attenuated DNA damage checkpoint activation and repair, and sensitized GSCs to radiation. L1CAM regulates expression of NBS1, a critical component of the MRE11-RAD50-NBS1 (MRN) complex that activates ataxia telangiectasia mutated (ATM) kinase and early checkpoint response. Ectopic expression of NBS1 in GSCs rescued the decreased checkpoint activation and radioresistance caused by L1CAM knockdown, demonstrating that L1CAM signals through NBS1 to regulate DNA damage checkpoint responses. Mechanistically, nuclear translocation of L1-ICD mediates NBS1 upregulation via c-Myc. These data demonstrate that L1CAM augments DNA damage checkpoint activation and radioresistance of GSCs through L1-ICD-mediated NBS1 upregulation and the enhanced MRN-ATM-Chk2 signalling.

Cutaneous tumors cease CXCL9/Mig production as a result of IFN-γ-mediated immunoediting.

During growth in the host, tumor cells are subjected to the stresses of innate and adaptive immunity (immunoediting), which provoke epigenetic changes in the tumor and increase tumor resistance to these immune responses. Our recent studies in methylcholanthrene-induced fibrosarcomas have indicated the appearance and rapid growth of tumor variants deficient in producing the T cell chemoattractant chemokine CXCL9/Mig, an important component of antitumor immunity. In the current report, we demonstrate that highly tumorigenic Mig-deficient tumor variants arise in both cutaneous fibrosarcoma and melanoma as a result of immune stress imposed by IFN-γ and T cells. The consequence of the loss of tumor-derived Mig expression is the increased resistance of Mig-deficient tumors to T cell-mediated immunity, which promotes the accelerated growth of these tumor variants. Remarkably, the ability of Mig-deficient tumor cells to express another CXCR3 ligand, CXCL10/IFN-γ-inducible protein, does not compensate for the absent antitumor functions of Mig, suggesting a nonredundant role for this chemokine in the suppression of tumor growth. To our knowledge, these studies report for the first time that IFN-γ-mediated stress leads to the loss of specific chemokine expression by tumor cells, which in turn promotes tumor growth and evasion of the immune response.

DR5 disulfide bonding as a sensor and effector of protein folding stress.

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

Epigenetic Mechanisms in Hematologic Aging and Premalignant Conditions.

Hematopoietic stem cells (HSCs) are essential for maintaining overall health by continuously generating blood cells throughout an individual's lifespan. However, as individuals age, the hematopoietic system undergoes significant functional decline, rendering them more susceptible to age-related diseases. Growing research evidence has highlighted the critical role of epigenetic regulation in this age-associated decline. This review aims to provide an overview of the diverse epigenetic mechanisms involved in the regulation of normal HSCs during the aging process and their implications in aging-related diseases. Understanding the intricate interplay of epigenetic mechanisms that contribute to aging-related changes in the hematopoietic system holds great potential for the development of innovative strategies to delay the aging process. In fact, interventions targeting epigenetic modifications have shown promising outcomes in alleviating aging-related phenotypes and extending lifespan in various animal models. Small molecule-based therapies and reprogramming strategies enabling epigenetic rejuvenation have emerged as effective approaches for ameliorating or even reversing aging-related conditions. By acquiring a deeper understanding of these epigenetic mechanisms, it is anticipated that interventions can be devised to prevent or mitigate the rates of hematologic aging and associated diseases later in life. Ultimately, these advancements have the potential to improve overall health and enhance the quality of life in aging individuals.

NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology.

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, molecular mechanisms underlying NPM1C+-driven leukemogenesis remain elusive. Here, we report that NPM1C+ activates signature HOX genes and reprograms cell cycle regulators by altering CTCF-driven topologically associated domains (TADs). Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to disrupted regulation of the cell cycle as well as aberrant chromatin accessibility and homeotic gene expression, which results in myeloid differentiation block. Restoration of NPM1 within the nucleus re-establishes differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators that switch the oncogenic MIZ1/MYC regulatory axis in favor of interacting with coactivator NPM1/p300, and prevents NPM1C+-driven leukemogenesis. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation.

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer.

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer (CAC) with experimental CH driven by Dnmt3a+/Δ, we found higher tumor penetrance and increased tumor burden compared with controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia, and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency and rebalanced hematopoiesis. This study provides conceptually novel insights into non-tumor-cell-autonomous effects of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies.

Actin cytoskeleton remodeling by the alternatively spliced isoform of PDLIM4/RIL protein.

RIL (product of PDLIM4 gene) is an actin-associated protein that has previously been shown to stimulate actin bundling by interacting with actin-cross-linking protein α-actinin-1 and increasing its affinity to filamentous actin. Here, we report that the alternatively spliced isoform of RIL, denoted here as RILaltCterm, functions as a dominant-negative modulator of RIL-mediated actin reorganization. RILaltCterm is regulated at the level of protein stability, and this protein isoform accumulates particularly in response to oxidative stress. We show that the alternative C-terminal segment of RILaltCterm has a disordered structure that directs the protein to rapid degradation in the core 20 S proteasomes. Such degradation is ubiquitin-independent and can be blocked by binding to NAD(P)H quinone oxidoreductase NQO1, a detoxifying enzyme induced by prolonged exposure to oxidative stress. We show that either overexpression of RILaltCterm or its stabilization by stresses counteracts the effects produced by full-length RIL on organization of actin cytoskeleton and cell motility. Taken together, the data suggest a mechanism for fine-tuning actin cytoskeleton rearrangement in response to stresses.

Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations.

Cytarabine and other chain-terminating nucleoside analogs that damage replication forks in rapidly proliferating cells are a cornerstone of leukemia chemotherapy, yet the outcomes remain unsatisfactory because of resistance and toxicity. Better understanding of DNA damage repair and downstream effector mechanisms in different disease subtypes can guide combination strategies that sensitize leukemia cells to cytarabine without increasing side effects. We have previously found that mutations in DNMT3A, one of the most commonly mutated genes in acute myeloid leukemia and associated with poor prognosis, predisposed cells to DNA damage and cell killing by cytarabine, cladribine, and other nucleoside analogs, which coincided with PARP1 dysfunction and DNA repair defect (Venugopal K, Feng Y, Nowialis P, et al. Clin Cancer Res 2022;28:756-769). In this article, we first overview DNA repair mechanisms that remove aberrant chain-terminating nucleotides as determinants of sensitivity or resistance to cytarabine and other nucleoside analogs. Next, we discuss PARP inhibition as a rational strategy to increase cytarabine efficacy in cells without DNMT3A mutations, while considering the implications of PARP inhibitor resistance for promoting clonal hematopoiesis. Finally, we examine the utility of p53 potentiators to boost leukemia cell killing by cytarabine in the context of mutant DNMT3A. Systematic profiling of DNA damage repair proficiency has the potential to uncover subtype-specific therapeutic dependencies in AML.

miR-196b-TLR7/8 Signaling Axis Regulates Innate Immune Signaling and Myeloid Maturation in DNMT3A-Mutant AML.

PURPOSE: DNMT3A mutations confer a poor prognosis in acute myeloid leukemia (AML), but the molecular mechanisms downstream of DNMT3A mutations in disease pathogenesis are not completely understood, limiting targeted therapeutic options. The role of miRNA in DNMT3A-mutant AML pathogenesis is understudied. EXPERIMENTAL DESIGN: DNA methylation and miRNA expression was evaluated in human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. The treatment efficacy and molecular mechanisms of TLR7/8-directed therapies on DNMT3A-mutant AML were evaluated in vitro on human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. RESULTS: miR-196b is hypomethylated and overexpressed in DNMT3A-mutant AML and is associated with poor patient outcome. miR-196b overexpression in DNMT3A-mutant AML is important to maintain an immature state and leukemic cell survival through repression of TLR signaling. The TLR7/8 agonist resiquimod induces dendritic cell-like differentiation with costimulatory molecule expression in DNMT3A-mutant AML cells and provides a survival benefit to Dnmt3a/Flt3-mutant AML mice. The small molecule bryostatin-1 augments resiquimod-mediated AML growth inhibition and differentiation. CONCLUSIONS: DNMT3A loss-of-function mutations cause miRNA locus-specific hypomethylation and overexpression important for mutant DNMT3A-mediated pathogenesis and clinical outcomes. Specifically, the overexpression of miR-196b in DNMT3A-mutant AML creates a novel therapeutic vulnerability by controlling sensitivity to TLR7/8-directed therapies.

The Impact of Inflammation-Induced Tumor Plasticity during Myeloid Transformation.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated preleukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation frequently found in patients with CH and leukemia. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single-cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated with poor prognosis in patients with AML. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms. SIGNIFICANCE: Progression from a preleukemic state to transformation, in the presence of TET2 mutations, is coupled with the emergence of inflammation and a novel population of inflammatory monocytes. Genes characteristic of this inflammatory population are associated with the worst prognosis in patients with AML. These studies connect inflammation to progression to leukemia. See related commentary by Pietras and DeGregori, p. 2234 . This article is highlighted in the In This Issue feature, p. 2221.

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.

Elevated invasive potential of glioblastoma stem cells.

Glioblastomas (GBMs) are the most lethal and common types of primary brain tumors. The hallmark of GBMs is their highly infiltrative nature. The cellular and molecular mechanisms underlying the aggressive cancer invasion in GBMs are poorly understood. GBM displays remarkable cellular heterogeneity and hierarchy containing self-renewing glioblastoma stem cells (GSCs). Whether GSCs are more invasive than non-stem tumor cells and contribute to the invasive phenotype in GBMs has not been determined. Here we provide experimental evidence supporting that GSCs derived from GBM surgical specimens or xenografts display greater invasive potential in vitro and in vivo than matched non-stem tumor cells. Furthermore, we identified several invasion-associated proteins that were differentially expressed in GSCs relative to non-stem tumor cells. One of such proteins is L1CAM, a cell surface molecule shown to be critical to maintain GSC tumorigenic potential in our previous study. Immunohistochemical staining showed that L1CAM is highly expressed in a population of cancer cells in the invasive fronts of primary GBMs. Collectively, these data demonstrate the invasive nature of GSCs, suggesting that disrupting GSCs through a specific target such as L1CAM may reduce GBM cancer invasion and tumor recurrence.

Alterations to DNMT3A in Hematologic Malignancies.

In the last decade, large-scale genomic studies in patients with hematologic malignancies identified recurrent somatic alterations in epigenetic modifier genes. Among these, the de novo DNA methyltransferase DNMT3A has emerged as one of the most frequently mutated genes in adult myeloid as well as lymphoid malignancies and in clonal hematopoiesis. In this review, we discuss recent advances in our understanding of the biochemical and structural consequences of DNMT3A mutations on DNA methylation catalysis and binding interactions and summarize their effects on epigenetic patterns and gene expression changes implicated in the pathogenesis of hematologic malignancies. We then review the role played by mutant DNMT3A in clonal hematopoiesis, accompanied by its effect on immune cell function and inflammatory responses. Finally, we discuss how this knowledge informs therapeutic approaches for hematologic malignancies with mutant DNMT3A.

Repurposing Tranexamic Acid as an Anticancer Agent.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.

Disulfide bond-disrupting agents activate the tumor necrosis family-related apoptosis-inducing ligand/death receptor 5 pathway.

Disulfide bond-disrupting agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. Blockade of tumor growth is associated with downregulation of EGFR, HER2, and HER3 and reduced Akt phosphorylation, as well as the induction of endoplasmic reticulum stress. However, it is not known how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through transcriptional and post-transcriptional mechanisms to activate the extrinsic cell death pathway. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor Lapatinib. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression. DDA-induced cytotoxicity is strongly amplified by MYC overexpression. This is consistent with the known potentiation of TRAIL-mediated cell death by MYC. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation, and protein stabilization, and that DDAs have activity against drug-resistant cancer cells. Our results indicate that DDAs are unique in causing DR5 accumulation and oligomerization and inducing downstream caspase activation and cancer cell death through mechanisms involving altered DR5 disulfide bonding. DDAs thus represent a new therapeutic approach to cancer therapy.

Loss of Dnmt3a Immortalizes Hematopoietic Stem Cells In Vivo.

Somatic mutations in DNMT3A are recurrent events across a range of blood cancers. Dnmt3a loss of function in hematopoietic stem cells (HSCs) skews divisions toward self-renewal at the expense of differentiation. Moreover, DNMT3A mutations can be detected in the blood of aging individuals, indicating that mutant cells outcompete normal HSCs over time. It is important to understand how these mutations provide a competitive advantage to HSCs. Here we show that Dnmt3a-null HSCs can regenerate over at least 12 transplant generations in mice, far exceeding the lifespan of normal HSCs. Molecular characterization reveals that this in vivo immortalization is associated with gradual and focal losses of DNA methylation at key regulatory regions associated with self-renewal genes, producing a highly stereotypical HSC phenotype in which epigenetic features are further buttressed. These findings lend insight into the preponderance of DNMT3A mutations in clonal hematopoiesis and the persistence of mutant clones after chemotherapy.

Ibrutinib inactivates BMX-STAT3 in glioma stem cells to impair malignant growth and radioresistance.

Glioblastoma (GBM) is the most lethal primary brain tumor and is highly resistant to current treatments. GBM harbors glioma stem cells (GSCs) that not only initiate and maintain malignant growth but also promote therapeutic resistance including radioresistance. Thus, targeting GSCs is critical for overcoming the resistance to improve GBM treatment. Because the bone marrow and X-linked (BMX) nonreceptor tyrosine kinase is preferentially up-regulated in GSCs relative to nonstem tumor cells and the BMX-mediated activation of the signal transducer and activator of transcription 3 (STAT3) is required for maintaining GSC self-renewal and tumorigenic potential, pharmacological inhibition of BMX may suppress GBM growth and reduce therapeutic resistance. We demonstrate that BMX inhibition by ibrutinib potently disrupts GSCs, suppresses GBM malignant growth, and effectively combines with radiotherapy. Ibrutinib markedly disrupts the BMX-mediated STAT3 activation in GSCs but shows minimal effect on neural progenitor cells (NPCs) lacking BMX expression. Mechanistically, BMX bypasses the suppressor of cytokine signaling 3 (SOCS3)-mediated inhibition of Janus kinase 2 (JAK2), whereas NPCs dampen the JAK2-mediated STAT3 activation via the negative regulation by SOCS3, providing a molecular basis for targeting BMX by ibrutinib to specifically eliminate GSCs while preserving NPCs. Our preclinical data suggest that repurposing ibrutinib for targeting GSCs could effectively control GBM tumor growth both as monotherapy and as adjuvant with conventional therapies.

Epigenetic Identity in AML Depends on Disruption of Nonpromoter Regulatory Elements and Is Affected by Antagonistic Effects of Mutations in Epigenetic Modifiers.

We performed cytosine methylation sequencing on genetically diverse patients with acute myeloid leukemia (AML) and found leukemic DNA methylation patterning is primarily driven by nonpromoter regulatory elements and CpG shores. Enhancers displayed stronger differential methylation than promoters, consisting predominantly of hypomethylation. AMLs with dominant hypermethylation featured greater epigenetic disruption of promoters, whereas those with dominant hypomethylation displayed greater disruption of distal and intronic regions. Mutations in IDH and DNMT3A had opposing and mutually exclusive effects on the epigenome. Notably, co-occurrence of both mutations resulted in epigenetic antagonism, with most CpGs affected by either mutation alone no longer affected in double-mutant AMLs. Importantly, this epigenetic antagonism precedes malignant transformation and can be observed in preleukemic LSK cells from Idh2R140Q or Dnmt3aR882H single-mutant and Idh2R140Q/Dnmt3aR882H double-mutant mice. Notably, IDH/DNMT3A double-mutant AMLs manifested upregulation of a RAS signaling signature and displayed unique sensitivity to MEK inhibition ex vivo as compared with AMLs with either single mutation.Significance: AML is biologically heterogeneous with subtypes characterized by specific genetic and epigenetic abnormalities. Comprehensive DNA methylation profiling revealed that differential methylation of nonpromoter regulatory elements is a driver of epigenetic identity, that gene mutations can be context-dependent, and that co-occurrence of mutations in epigenetic modifiers can result in epigenetic antagonism. Cancer Discov; 7(8); 868-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

UNLABELLED: The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. IMPLICATIONS: A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis.