SUMMARY 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 cofactor of Fe2+ and α-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.

T cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy with dismal overall prognosis, exhibiting up to a 25% relapse rate, mainly due to the absence of non-cytotoxic targeted therapy options. Despite the fact that drugs targeting the function of key epigenetic factors have been approved in the context of hematopoietic disorders1 and the recent identification of mutations affecting chromatin modulators in a variety of leukemias2,3, “epigenetic” drugs are not currently used for TALL treatment. Recently, we described a tumor suppressor role of the polycomb repressive complex 2 (PRC2) in this tumor4. Here we sought out to delineate the role of histone 3 lysine 27 (H3K27) demethylases, JMJD3 and UTX. We show that JMJD3 is essential for initiation and maintenance of disease, as it controls important oncogenic gene targets through the modulation of H3K27 methylation. In contrast, UTX acts a tumor suppressor and frequently genetically inactivated in T-ALL. Moreover, we demonstrate that the small molecule inhibitor GSKJ45 affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with similar enzymatic function can play opposing roles in the context of the same disease and pave the way for the use of a new category of epigenetic inhibitors in hematopoietic malignancies.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.

The TET methylcytosine dioxygenase 1 (TET1) enzyme is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. Decreased expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we show that deletion of Tet1 promoted the development of B cell lymphoma in mice. Tet1 was required for maintaining normal content of 5hmC, preventing DNA hypermethylation and in the regulation of B cell lineage, chromosome maintenance and DNA repair genes. Whole-exome sequencing of Tet1-deficient tumors revealed mutations frequently found in Non-Hodgkin B cell lymphoma, where TET1 was hypermethylated and transcriptionally silenced. These findings provide in vivo evidence of TET1 function as a tumor suppressor of hematopoietic malignancy.

Three-dimensional (3D) chromatin architectural differences can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to human disease. Here, we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) using primary human leukemia specimens and its dynamic responses to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD “fusion” event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.