SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells.

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

Autophagic signaling promotes systems-wide remodeling in skeletal muscle upon oncometabolic stress by D2-HG.

OBJECTIVES: Cachexia is a metabolic disorder and comorbidity with cancer and heart failure. The syndrome impacts more than thirty million people worldwide, accounting for 20% of all cancer deaths. In acute myeloid leukemia, somatic mutations of the metabolic enzyme isocitrate dehydrogenase 1 and 2 cause the production of the oncometabolite D2-hydroxyglutarate (D2-HG). Increased production of D2-HG is associated with heart and skeletal muscle atrophy, but the mechanistic links between metabolic and proteomic remodeling remain poorly understood. Therefore, we assessed how oncometabolic stress by D2-HG activates autophagy and drives skeletal muscle loss. METHODS: We quantified genomic, metabolomic, and proteomic changes in cultured skeletal muscle cells and mouse models of IDH-mutant leukemia using RNA sequencing, mass spectrometry, and computational modeling. RESULTS: D2-HG impairs NADH redox homeostasis in myotubes. Increased NAD+ levels drive activation of nuclear deacetylase Sirt1, which causes deacetylation and activation of LC3, a key regulator of autophagy. Using LC3 mutants, we confirm that deacetylation of LC3 by Sirt1 shifts its distribution from the nucleus into the cytosol, where it can undergo lipidation at pre-autophagic membranes. Sirt1 silencing or p300 overexpression attenuated autophagy activation in myotubes. In vivo, we identified increased muscle atrophy and reduced grip strength in response to D2-HG in male vs. female mice. In male mice, glycolytic intermediates accumulated, and protein expression of oxidative phosphorylation machinery was reduced. In contrast, female animals upregulated the same proteins, attenuating the phenotype in vivo. Network modeling and machine learning algorithms allowed us to identify candidate proteins essential for regulating oncometabolic adaptation in mouse skeletal muscle. CONCLUSIONS: Our multi-omics approach exposes new metabolic vulnerabilities in response to D2-HG in skeletal muscle and provides a conceptual framework for identifying therapeutic targets in cachexia.

Hematologic DNMT3A reduction and high-fat diet synergize to promote weight gain and tissue inflammation.

During aging, blood cell production becomes dominated by a limited number of variant hematopoietic stem cell (HSC) clones. Differentiated progeny of variant HSCs are thought to mediate the detrimental effects of such clonal hematopoiesis on organismal health, but the mechanisms are poorly understood. While somatic mutations in DNA methyltransferase 3A (DNMT3A) frequently drive clonal dominance, the aging milieu also likely contributes. Here, we examined in mice the interaction between high-fat diet (HFD) and reduced DNMT3A in hematopoietic cells; strikingly, this combination led to weight gain. HFD amplified pro-inflammatory pathways and upregulated inflammation-associated genes in mutant cells along a pro-myeloid trajectory. Aberrant DNA methylation during myeloid differentiation and in response to HFD led to pro-inflammatory activation and maintenance of stemness genes. These findings suggest that reduced DNMT3A in hematopoietic cells contributes to weight gain, inflammation, and metabolic dysfunction, highlighting a role for DNMT3A loss in the development of metabolic disorders.

SOD1 is a synthetic lethal target in PPM1D-mutant leukemia cells.

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase, Mg2+/Mn2+ dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacologic target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate the protective role of SOD1 against oxidative stress in PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells.

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.

The disordered N-terminal domain of DNMT3A recognizes H2AK119ub and is required for postnatal development.

DNA methyltransferase 3a (DNMT3A) plays a crucial role during mammalian development. Two isoforms of DNMT3A are differentially expressed from stem cells to somatic tissues, but their individual functions remain largely uncharacterized. Here we report that the long isoform DNMT3A1, but not the short DNMT3A2, is essential for mouse postnatal development. DNMT3A1 binds to and regulates bivalent neurodevelopmental genes in the brain. Strikingly, Dnmt3a1 knockout perinatal lethality could be partially rescued by DNMT3A1 restoration in the nervous system. We further show that the intrinsically disordered N terminus of DNMT3A1 is required for normal development and DNA methylation at DNMT3A1-enriched regions. Mechanistically, a ubiquitin-interacting motif embedded in a putative α-helix within the N terminus binds to mono-ubiquitinated histone H2AK119, probably mediating recruitment of DNMT3A1 to Polycomb-regulated regions. These data demonstrate an isoform-specific role for DNMT3A1 in mouse postnatal development and reveal the N terminus as a necessary regulatory domain for DNMT3A1 chromatin occupancy and functions in the nervous system.

Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder caused by germline heterozygous mutations in the DNA methyltransferase DNMT3A. DNMT3A is a critical regulator of hematopoietic stem cell (HSC) differentiation and somatic DNMT3A mutations are frequent in hematologic malignancies and clonal hematopoiesis. Yet, the impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is undefined. In order to establish how constitutive mutation of DNMT3A impacts blood development in TBRS we gathered clinical data and analyzed blood parameters in 18 individuals with TBRS. We also determined the distribution of major peripheral blood cell lineages by flow cytometric analyses. Our analyses revealed non-anemic macrocytosis, a relative decrease in lymphocytes and increase in neutrophils in TBRS individuals compared to unaffected controls. We were able to recapitulate these hematologic phenotypes in multiple murine models of TBRS and identified rare hematological and non-hematological malignancies associated with constitutive Dnmt3a mutation. We further show that loss of DNMT3A in TBRS is associated with an altered DNA methylation landscape in hematopoietic cells affecting regions critical to stem cell function and tumorigenesis. Overall, our data identify key hematopoietic effects driven by DNMT3A mutation with clinical implications for individuals with TBRS and DNMT3A-associated clonal hematopoiesis or malignancies.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice.

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor-/- knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor-/-/Dnmt3a-/- knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

Tissue-Biased Expansion of DNMT3A-Mutant Clones in a Mosaic Individual Is Associated with Conserved Epigenetic Erosion.

DNA methyltransferase 3A (DNMT3A) is the most commonly mutated gene in clonal hematopoiesis (CH). Somatic DNMT3A mutations arise in hematopoietic stem cells (HSCs) many years before malignancies develop, but difficulties in comparing their impact before malignancy with wild-type cells have limited the understanding of their contributions to transformation. To circumvent this limitation, we derived normal and DNMT3A mutant lymphoblastoid cell lines from a germline mosaic individual in whom these cells co-existed for nearly 6 decades. Mutant cells dominated the blood system, but not other tissues. Deep sequencing revealed similar mutational burdens and signatures in normal and mutant clones, while epigenetic profiling uncovered the focal erosion of DNA methylation at oncogenic regulatory regions in mutant clones. These regions overlapped with those sensitive to DNMT3A loss after DNMT3A ablation in HSCs and in leukemia samples. These results suggest that DNMT3A maintains a conserved DNA methylation pattern, the erosion of which provides a distinct competitive advantage to hematopoietic cells.

A combination strategy targeting enhancer plasticity exerts synergistic lethality against BETi-resistant leukemia cells.

Primary and acquired drug resistance imposes a major threat to achieving optimized clinical outcomes during cancer treatment. Aberrant changes in epigenetic modifications are closely involved in drug resistance of tumor cells. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on the CRISPR interference technology, we identified the second intron of IncRNA, PVT1, as a unique bona fide gained enhancer that restored MYC transcription independent of BRD4 recruitment in leukemia. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYC locus in BETi-resistant leukemia cells. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies.

Antibiotic treatment ameliorates Ten-eleven translocation 2 (TET2) loss-of-function associated hematological malignancies.

TET2 is among the most frequently mutated genes in hematological malignancies, as well as in healthy individuals with clonal hematopoiesis. Inflammatory stress is known to promote the expansion of Tet2-deficient hematopoietic stem cells, as well as the initiation of pre-leukemic conditions. Infection is one of the most frequent complications in hematological malignancies and antibiotics are commonly used to suppress infection-induced inflammation, but their application in TET2 mutation-associated cancers remained underexplored. In this study, we discovered that Tet2 depletion led to aberrant expansion of myeloid cells, which was correlated with elevated serum levels of pro-inflammatory cytokines at the pre-malignant stage. Antibiotics treatment suppressed the growth of Tet2-deficient myeloid and lymphoid tumor cells in vivo. Transcriptomic profiling further revealed significant changes in the expression of genes involved in the TNF-α signaling and other immunomodulatory pathways in antibiotics-treated tumor cells. Pharmacological inhibition of TNF-α signaling partially attenuated Tet2-deficient tumor cell growth in vivo. Therefore, our findings establish the feasibility of targeting pro-inflammatory pathways to curtail TET2 inactivation-associated hematological malignancies.

Mutant NPM1 Maintains the Leukemic State through HOX Expression.

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.

Genome-Wide Analysis of DNA Methylation in Hematopoietic Cells: DNA Methylation Analysis by WGBS.

DNA methylation is a major epigenetic modification that regulates gene expression, genome imprinting, and development and has a role in diseases including cancer. There are various methods for whole-genome methylation profiling that differ in cost and resolution. Recent advances in high-throughput sequencing technologies coupled with bisulfite treatment enable absolute DNA methylation quantification and genome-wide single-nucleotide resolution analysis. In this chapter, we provide detailed protocols for whole-genome bisulfite sequencing (WGBS), which captures the complete methylome. Using WGBS, we are able to generate a reference DNA methylome for normal or malignant hematopoietic cells.