Faculty Bibliography

The erasure of epigenetic modifications across the genome of somatic cells is an essential requirement during their reprogramming into induced pluripotent stem cells (iPSCs). Vitamin C plays a pivotal role in remodeling the epigenome by enhancing the activity of Jumonji-C domain-containing histone demethylases (JHDMs) and the ten-eleven translocation (TET) proteins. By maintaining differentiation plasticity in culture, vitamin C also improves the quality of tissue specific stem cells derived from iPSCs that are highly sought after for use in regenerative medicine. The ability of vitamin C to potentiate the activity of histone and DNA demethylating enzymes also has clinical application in the treatment of cancer. Vitamin C deficiency has been widely reported in cancer patients and has recently been shown to accelerate cancer progression in disease models. Therapies involving high-dose vitamin C administration are currently gaining traction in the treatment of epigenetic dysregulation, by targeting aberrant histone and DNA methylation patterns associated with cancer progression.

TET2 somatic mutations occur in ~10% of DLBCLs but are of unknown significance. Herein we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B-cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation and a pre-neoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence TET2 plays a critical role in the GC reaction and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals.

Vitamin C is an essential dietary requirement for humans. In addition to its known role as an antioxidant, vitamin C is a cofactor for Fe²⁺- and α-ketoglutarate-dependent dioxygenases (Fe²⁺/α-KGDDs) which comprise a large number of diverse enzymes, including collagen prolyl hydroxylases and epigenetic regulators of histone and DNA methylation. Vitamin C can modulate embryonic stem cell (ESC) function, enhance reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs), and hinder the aberrant self-renewal of hematopoietic stem cells (HSCs) through its ability to enhance the activity of either Jumonji C (JmjC) domain-containing histone demethylases or ten-eleven translocation (TET) DNA hydroxylases. Given that epigenetic dysregulation is a known driver of malignancy, vitamin C may play a novel role as an epigenetic anticancer agent.

Glioblastoma (GBM) is the most lethal primary adult brain tumor and its pathology is hallmarked by distorted neovascularization, diffuse tumor-associated macrophage infiltration, and potent immunosuppression. Reconstituting organotypic tumor angiogenesis models with biomimetic cell heterogeneity and interactions, pro-/anti-inflammatory milieu and extracellular matrix (ECM) mechanics is critical for preclinical anti-angiogenic therapeutic screening. However, current in vitro systems do not accurately mirror in vivo human brain tumor microenvironment. Here, we engineered a three-dimensional (3D), microfluidic angiogenesis model with controllable and biomimetic immunosuppressive conditions, immune-vascular and cell-matrix interactions. We demonstrate in vitro, GL261 and CT-2A GBM-like tumors steer macrophage polarization towards a M2-like phenotype for fostering an immunosuppressive and proangiogenic niche, which is consistent with human brain tumors. We distinguished that GBM and M2-like immunosuppressive macrophages promote angiogenesis, while M1-like pro-inflammatory macrophages suppress angiogenesis, which we coin "inflammation-driven angiogenesis." We observed soluble immunosuppressive cytokines, predominantly TGF-β1, and surface integrin (αvβ3) endothelial-macrophage interactions are required in inflammation-driven angiogenesis. We demonstrated tuning cell-adhesion receptors using an integrin (αvβ3)-specific collagen hydrogel regulated inflammation-driven angiogenesis through Src-PI3K-YAP signaling, highlighting the importance of altered cell-ECM interactions in inflammation. To validate the preclinical applications of our 3D organoid model and mechanistic findings of inflammation-driven angiogenesis, we screened a novel dual integrin (αvβ3) and cytokine receptor (TGFβ-R1) blockade that suppresses GBM tumor neovascularization by simultaneously targeting macrophage-associated immunosuppression, endothelial-macrophage interactions, and altered ECM. Hence, we provide an interactive and controllable GBM tumor microenvironment and highlight the importance of macrophage-associated immunosuppression in GBM angiogenesis, paving a new direction of screening novel anti-angiogenic therapies.

Epigenetic heterogeneity is emerging as a feature of tumors. In diffuse large B-cell lymphoma (DLBCL), increased cytosine methylation heterogeneity is associated with poor clinical outcome, yet the underlying mechanisms remain unclear. Activation-induced cytidine deaminase (AICDA), an enzyme that mediates affinity maturation and facilitates DNA demethylation in germinal center (GC) B cells, is required for DLBCL pathogenesis and linked to inferior outcome. Here we show that AICDA overexpression causes more aggressive disease in BCL2-driven murine lymphomas. This phenotype is associated with increased cytosine methylation heterogeneity, but not with increased AICDA-mediated somatic mutation burden. Reciprocally, the cytosine methylation heterogeneity characteristic of normal GC B cells is lost upon AICDA depletion. These observations are relevant to human patients, since DLBCLs with high AICDA expression manifest increased methylation heterogeneity vs. AICDA-low DLBCLs. Our results identify AICDA as a driver of epigenetic heterogeneity in B-cell lymphomas with potential significance for other tumors with aberrant expression of cytidine deaminases.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and alpha-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer.

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1+ (Ph+) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event-free survival in both patient cohorts. Among them are EMP1, which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1, encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease.

Ten-eleven-translocation (TET) proteins oxidize 5-methylcytosine (5mC) to form stable or transient modifications (oxi-mCs) in the mammalian genome. Genome-wide mapping and protein interaction studies have shown that 5mC and oxi-mCs have unique distribution patterns and alternative roles in gene expression. In addition, oxi-mCs may interact with specific chromatin regulators, transcription factors and DNA repair proteins to maintain genomic integrity or alter DNA replication and transcriptional elongation rates. In this review we will discuss recent advances in our understanding of how TETs and 5hmC exert their epigenetic function as tumor suppressors by playing alternative roles in transcriptional regulation and genomic stability.