T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic factor TET1 is highly expressed in TALL and is crucial for human TALL cell growth in vivo. Tet1 knockout mice and knockdown in human T-cells did not perturb normal T-cell proliferation, indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of leukemic growth by TET1 was depending on its catalytic property to maintain global 5hydroxymethylcytosine (5hmC) marks, thereby regulating cell cycle, DNA repair genes and TALL associated oncogenes. Furthermore, overexpression of the Tet1 catalytic domain was sufficient to augment global 5hmC levels and leukemic growth of TALL cells in vivo. We demonstrate that PARP enzymes, which are highly expressed in TALL patients, participate in establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. Importantly, the growth related role of TET1 in TALL could be antagonized by the clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 5hmC marks and antagonized leukemic growth of TALL cells, opening a therapeutic avenue for this disease. 10 transplantation (Fig. S2H). Moreover, in a published microarray analysis of TALL cell lines transplanted into xenografts, a similar increase in TET1 expression was observed (Fig. S2I), suggesting that high TET1 expression is associated with leukemic growth in vivo. Indeed, forced expression of Tet1-CD in TALL cell lines significantly augmented leukemic growth in vitro and in vivo (Fig. 3D-E). These data clearly suggest that the growth-promoting role of TET1 in TALL is at least partly dependent on its enzymatic activity. TET1 depletion induces loss of 5hmC marks at promoters and gene bodies of genes involved in cell cycle, DNA repair and NOTCH pathway Next we analyzed 5hmC levels in TALL cells via Intracellular fluorescence (IF) in our knockdown, rescue and overexpression experiments. IF-flow cytometry for 5hmC marks in TET1 depleted JURKAT cells, primary TALL patients and TET1 knockout bulk TALL cell lines revealed a significant decrease in global 5hmC levels (Fig. 4A-B and Fig. S3A-B) and increase in 5mC levels (Fig. S3C). Conversely, overexpression of Tet1-CD in TET1 depleted cells or wild type TALL cells induced a global increase in 5hmC levels (Fig. 4C-D). Furthermore, we performed hydroxymethylated DNA immunoprecipitation (hMeDIP)-seq in TET1 depleted JURKAT cells. In hMeDIP-seq, TET1 depletion resulted in more than a 59% reduction of global 5hmC enrichment at the promoter (-5kbTSS), gene body (GB) and intergenic regions compared to scrambled control (referred to as TET1 dependent 5hmC or T1-5hmC regions from here) (Fig. 4E-F). In detail, a total of 2,404 and 6,115 5hmC enriched promoters and GB were observed in the scrambled arm, respectively, out of which more th...

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Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 39-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero-and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.

RNA-binding proteins (RBPs) form a large and diverse class of factors many members of which are overexpressed in hematological malignancies. RBPs participate in various processes of mRNA metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in acute myeloid leukemia patients and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. It instead largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates human myeloid progenitor signature and LSC-associated genes and upregulates DNA damage signalling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes, and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.