Faculty Bibliography

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the T cell receptor alpha (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

Over the past decade, it has become clear that both genetics and epigenetics play pivotal roles in cancer onset and progression. The importance of epigenetic regulation in proper maintenance of cellular state is highlighted by the frequent mutation of chromatin modulating factors across cancer subtypes. Identification of these mutations has created an interest in designing drugs that target enzymes involved in DNA methylation and posttranslational modification of histones. In this review, we discuss recurrent genetic alterations to epigenetic modulators in both myeloid and lymphoid leukemias. Furthermore, we review how these perturbations contribute to leukemogenesis and impact disease outcome and treatment efficacy. Finally, we discuss how the recent advances in our understanding of chromatin biology may impact treatment of leukemia.

Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of leukaemia and lymphoma. Nonetheless, mistakes can happen, leading to the generation of aberrant rearrangements, such as Tcra/d-Igh inter-locus translocations that are a hallmark of ataxia telangiectasia-mutated (ATM) deficiency. Current evidence indicates that these translocations arise from the persistence of unrepaired breaks converging at different stages of thymocyte differentiation. Here we show that a defect in feedback control of RAG2 activity gives rise to bi-locus breaks and damage on Tcra/d and Igh in the same T cell at the same developmental stage, which provides a direct mechanism for generating these inter-locus rearrangements. Both the RAG2 C-terminus and ATM prevent bi-locus RAG-mediated cleavage through modulation of three-dimensional conformation (higher-order loops) and nuclear organization of the two loci. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

Summary: In this issue of Cancer Discovery, Geng and colleagues report on their use of a combination of promoter cytosine methylation profiling with gene expression and ChIP sequencing to elucidate molecular signatures of adult B-acute lymphoblastic leukemia patient samples with BCR-ABL1, E2A-PBX1, and MLL rearrangements. The unique epigenetic and gene expression signatures of these clinically unfavorable B-ALL subtypes identify novel biomarkers and provide a strong rationale for repurposing existing therapies to treat these molecularly distinct diseases. Cancer Discov; 2(11); 976-8. (c)2012 AACR.

The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPalpha-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPalpha binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPalpha rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.