DOT1L inhibition does not modify the sensitivity of cutaneous T cell lymphoma to pan-HDAC inhibitors in vitro.

Cutaneous T-cell lymphomas (CTCLs) are a subset of T-cell malignancies presenting in the skin. The treatment options for CTCL, in particular in advanced stages, are limited. One of the emerging therapies for CTCL is treatment with histone deacetylase (HDAC) inhibitors. We recently discovered an evolutionarily conserved crosstalk between HDAC1, one of the targets of HDAC inhibitors, and the histone methyltransferase DOT1L. HDAC1 negatively regulates DOT1L activity in yeast, mouse thymocytes, and mouse thymic lymphoma. Here we studied the functional relationship between HDAC inhibitors and DOT1L in two human CTCL cell lines, specifically addressing the question whether the crosstalk between DOT1L and HDAC1 observed in mouse T cells plays a role in the therapeutic effect of clinically relevant broad-acting HDAC inhibitors in the treatment of human CTCL. We confirmed that human CTCL cell lines were sensitive to treatment with pan-HDAC inhibitors. In contrast, the cell lines were not sensitive to DOT1L inhibitors. Combining both types of inhibitors did neither enhance nor suppress the inhibitory effect of HDAC inhibitors on CTCL cells. Thus our in vitro studies suggest that the effect of commonly used pan-HDAC inhibitors in CTCL cells relies on downstream effects other than DOT1L misregulation.

The histone methyltransferase SETD2 negatively regulates cell size.

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

Signals for antigen-independent differentiation of memory CD8+ T cells.

Conventional CD8+ memory T cells develop upon stimulation with foreign antigen and provide increased protection upon re-challenge. Over the past two decades, new subsets of CD8+ T cells have been identified that acquire memory features independently of antigen exposure. These antigen-inexperienced memory T cells (TAIM) are described under several names including innate memory, virtual memory, and memory phenotype. TAIM cells exhibit characteristics of conventional or true memory cells, including antigen-specific responses. In addition, they show responsiveness to innate stimuli and have been suggested to provide additional levels of protection toward infections and cancer. Here, we discuss the current understanding of TAIM cells, focusing on extrinsic and intrinsic molecular conditions that favor their development, their molecular definitions and immunological properties, as well as their transcriptional and epigenetic regulation.

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B-cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B-cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L indirectly supports the repression of an anti-proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B-cell naivety and GC B-cell differentiation.

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells.

Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

A novel germline variant in the DOT1L gene co-segregating in a Dutch family with a history of melanoma.

A proportion of patients diagnosed with melanoma has a positive family history. Despite increasing knowledge on the genes responsible for familial clustering, the genetic basis in the majority of the families with an inherited predisposition to melanoma remains to be clarified. To identify novel melanoma-susceptibility genes, we applied whole-exome sequencing on DNA from two members of a family with four melanoma cases, not explained by established high penetrance melanoma-susceptibility genes. Whole-exome sequencing identified 10 rare, co-segregating, predicted deleterious missense gene variants. Subsequent co-segregation analysis revealed that only variants in the DOT1L (R409H) and the SLCO4C1 (P597A) genes were present in the other two affected members of this family. DOT1L is a methyltransferase that methylates histone H3 lysine 79 (H3K79). It is involved in maintenance of genomic stability, since mutations in the DOT1L gene have been previously reported to compromise the removal of ultraviolet photoproducts in ultraviolet-irradiated melanocytes, thereby enhancing malignant transformation. We hypothesized that the presence of DOT1L R409H variant might be associated with an increased risk of melanoma, since we found co-segregation of the DOT1L mutation in all four melanoma-affected family members. However, this missense variant did neither lead to detectable loss-of-heterozygosity nor reduction of histone methyltransferase activity in melanoma samples from mutation carriers nor altered ultraviolet-survival of mouse embryonic stem cells containing an engineered homozygous DOT1L R409H mutation. Although functional analysis of this rare co-segregating variant did not reveal compromised histone methyltransferase activity and ultraviolet exposure sensitivity, the role of DOT1L as melanoma susceptibility gene deserves further study.

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma.

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.