SUMMARY Somatic loss-of-function mutations in the ten-eleven-translocation-2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in the hematopoietic compartment have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment which leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2+/− mice also displayed increased stem cell self-renewal and extramedulary hematopoiesis, suggesting Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.

SUMMARY Mutations affecting spliceosomal proteins are the most common class of mutations in patients with myelodysplastic syndromes (MDS), yet their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2’s normal sequence-specific RNA binding activity, thereby altering recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2 that triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in splicing of key regulators, and impaired hematopoiesis.

Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types1. Its activity is controlled by the multi-subunit γ–secretase complex (γSE) complex2. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations3. Here we identify novel somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) resulted in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue.

Mutations in spliceosomal genes are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)1–3. These mutations occur at highly recurrent amino acid residues and perturb normal splice site and exon recognition4–6. Spliceosomal mutations are always heterozygous and rarely co-occur with one another, suggesting that cells may only tolerate a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice that express the SRSF2P95H mutation, which commonly occurs in MDS and AML, in an inducible hemizygous manner in hematopoietic cells. These mice developed lethal bone marrow failure, demonstrating that Srsf2-mutant cells depend on the wildtype Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E71077,8 resulted in significant reductions in leukemic burden specifically in isogenic mouse leukemias and patient-derived xenograft (PDX) AMLs carrying spliceosomal mutations. While in vivo E7107 exposure resulted in widespread intron retention and cassette exon skipping regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutant versus wildtype leukemias, consistent with its differential effect on survival in these two genotypes. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal mutations are preferentially susceptible to additional splicing perturbations in vivo compared with wildtype counterparts. Modulation of spliceosome function may provide a novel therapeutic avenue in genetically defined subsets of MDS and AML patients.

Aifantis and colleagues examine the conflicting roles of Notch signaling in various cancer types.

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.

Although transcriptional programs associated with T-cell specification and commitment have been described, the functional hierarchy and the roles of key regulators in structuring/orchestrating these programs remain unclear. Activation of Notch signaling in uncommitted precursors by the thymic stroma initiates the T-cell differentiation program. One regulator first induced in these precursors is the DNA-binding protein T-cell factor 1 (Tcf-1), a T-cellspecific mediator of Wnt signaling. However, the specific contribution of Tcf-1 to early T-cell development and the signals inducing it in these cells remain unclear. Here we assign functional significance to Tcf-1 as a gatekeeper of T-cell fate and show that Tcf-1 is directly activated by Notch signals. Tcf-1 is required at the earliest phase of T-cell determination for progression beyond the early thymic progenitor stage. The global expression profile of Tcf-1-deficient progenitors indicates that basic processes of DNA metabolism are down-regulated in its absence, and the blocked T-cell progenitors become abortive and die by apoptosis. Our data thus add an important functional relationship to the roadmap of T-cell development.

SummaryThe physiological basis and mechanistic requirement for the high immunoreceptor tyrosine activation motifs (ITAM) multiplicity of the T cell receptor (TCR)-CD3 complex remains obscure. Here we show that while low TCR-CD3 ITAM multiplicity is sufficient to engage canonical TCR-induced signaling events that lead to cytokine secretion, high TCR-CD3 ITAM multiplicity is required for TCR-driven proliferation. This is dependent on compact immunological synapse formation, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate Notch1 recruitment and activation and ultimately c-Myc-induced proliferation. Analogous mechanistic events are also required to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and co-ordinated by the multiplicity of phosphorylated TCR-CD3 ITAMs.