Faculty Bibliography

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) ce!

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche.

Accumulating evidence suggests that Notch signaling is active at multiple points during hematopoiesis. Until recently, the majority of such studies focused on Notch signaling in lymphocyte differentiation and knowledge of individual Notch receptor roles has been limited due to a paucity of genetic tools available. In this manuscript we generate and describe animal models to identify and fate-map stem and progenitor cells expressing each Notch receptor, delineate Notch pathway activation, and perform in vivo gain- and loss-of-function studies dissecting Notch signaling in early hematopoiesis. These models provide comprehensive genetic maps of lineage-specific Notch receptor expression and activation in hematopoietic stem and progenitor cells. Moreover, they establish a previously unknown role for Notch signaling in the commitment of blood progenitors toward the erythrocytic lineage and link Notch signaling to optimal organismal response to stress erythropoiesis.

Recent studies have identified recurrent mutations in SETBP1, the gene that encodes SET-binding protein 1, in several types of myeloid malignancies, including chronic myeloid and acute myeloid leukemias. The identified mutations frequently target the SKI-homologous domain, although the exact pathogenic mechanisms remain unknown.

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

The molecular mechanisms regulating leukemia-initiating cell (LIC) function are of important clinical significance. We use chronic myelogenous leukemia (CML) as a model of LIC-dependent malignancy and identify the interaction between the ubiquitin ligase Fbw7 and its substrate c-Myc as a regulator of LIC homeostasis. Deletion of Fbw7 leads to c-Myc overexpression, p53-dependent LIC-specific apoptosis, and the eventual inhibition of tumor progression. A decrease of either c-Myc protein levels or attenuation of the p53 response rescues LIC activity and disease progression. Further experiments showed that Fbw7 expression is required for survival and maintenance of human CML LIC. These studies identify a ubiquitin ligase:substrate pair regulating LIC activity, suggesting that targeting of the Fbw7:c-Myc axis is an attractive therapy target in refractory CML.

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.