NF-kB inhibitor blocks B cell development at two checkpoints

Members of the NF-kB transcription factor family are differentially expressed in the B cell lineage. Disruption of individual or two NF-kB subunits exhibits distinct defects in B lymphocyte development, activation, and survival. However, the role each NF-kB plays during B cell development has been obscured by molecular compensation. To address this issue, a trans-dominant form of IkBα was transduced into bone marrow cells to act as a pan-inhibitor of NF-kB using a retroviral system. While the development of T-lymphocytes and myeloid cell lineages was not grossly affected by the transduced IkBα gene, a significant reduction in the number and percentage of B lineage cells was apparent in IkBα transduced chimeric mice. IkBα expression decreased the percentage of pre-B and immature B cell subsets in the bone marrow and further impaired the development of follicular mature B cells and marginal zone B cells in the periphery. Introduction of the Bcl-X transgene completely restored the pre-B and immature B cell pool in the bone marrow. However, despite a significant improvement of overall viability of the B cell lineage, Bcl-X expression was insufficient to overcome the maturation block resulting from NF-kB inhibition. Together, our study suggests that NF-kB activity is required for two distinct checkpoints during B cell development: one is for pre-B/immature B cell viability, the other is to provide both survival and maturation signals to ensure the proper development of follicular mature B cells

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells

Notch Simultaneously Orchestrates Multiple Helper T Cell Programs Independently of Cytokine Signals

SummaryTwo models are proposed to explain Notch function during helper T (Th) cell differentiation. One argues that Notch instructs one Th cell fate over the other, whereas the other posits that Notch function is dictated by cytokines. Here we provide a detailed mechanistic study investigating the role of Notch in orchestrating Th cell differentiation. Notch neither instructed Th cell differentiation nor did cytokines direct Notch activity, but instead, Notch simultaneously regulated the Th1, Th2, and Th17 cell genetic programs independently of cytokine signals. In addition to regulating these programs in both polarized and nonpolarized Th cells, we identified Ifng as a direct Notch target. Notch bound the Ifng CNS-22 enhancer, where it synergized with Tbet at the promoter. Thus, Notch acts as an unbiased amplifier of Th cell differentiation. Our data provide a paradigm for Notch in hematopoiesis, with Notch simultaneously orchestrating multiple lineage programs, rather than restricting alternate outcomes

Impaired Notch Signaling Promotes \u3cem\u3eDe novo\u3c/em\u3e Squamous Cell Carcinoma Formation

Signaling through Notch receptors in the skin has been implicated in the differentiation, proliferation, and survival of keratinocytes, as well as in the pathogenesis of basal cell carcinoma (BCC). To determine the composite function of Notch receptor–mediated signaling in the skin and overcome potential redundancies between receptors, conditional transgenic mice were generated that express the pan-Notch inhibitor, dominant-negative Mastermind Like 1 (DNMAML1), to repress all canonical [CBF-1/Suppressor of hairless/LAG-1 (CSL)–dependent] Notch signaling exclusively in the epidermis. Here, we report that DNMAML1 mice display hyperplastic epidermis and spontaneously develop cutaneous squamous cell carcinoma (SCC) as well as dysplastic precursor lesions, actinic keratoses. Mice expressing epidermal DNMAML1 display enhanced accumulation of nuclear ß-catenin and cyclin D1 in suprabasilar keratinocytes and in lesional cells from SCCs, which was also observed in human cutaneous SCC. These results suggest a model wherein CSL-dependent Notch signaling confers protection against cutaneous SCC. The demonstration that inhibition of canonical Notch signaling in mice leads to spontaneous formation of SCC and recapitulates the disease in humans yields fundamental insights into the pathogenesis of SCC and provides a unique in vivo animal model to examine the pathobiology of cutaneous SCC and for evaluating novel therapies

The common oncogenomic program of NOTCH1 and NOTCH3 signaling in T-cell acute lymphoblastic leukemia

Notch is a major oncogenic driver in T cell acute lymphoblastic leukemia (T-ALL), in part because it binds to an enhancer that increases expression of MYC. Here, we exploit the capacity of activated NOTCH1 and NOTCH3 to induce T-ALL, despite substantial divergence in their intracellular regions, as a means to elucidate a broad, common Notch-dependent oncogenomic program through systematic comparison of the transcriptomes and Notch-bound genomic regulatory elements of NOTCH1- and NOTCH3-dependent T-ALL cells. ChIP-seq studies show a high concordance of functional NOTCH1 and NOTCH3 genomic binding sites that are enriched in binding motifs for RBPJ, the transcription factor that recruits activated Notch to DNA. The interchangeability of NOTCH1 and NOTCH3 was confirmed by rescue of NOTCH1-dependent T-ALL cells with activated NOTCH3 and vice versa. Despite remarkable overall similarity, there are nuanced differences in chromatin landscapes near critical common Notch target genes, most notably at a Notch-dependent enhancer that regulates MYC, which correlates with responsiveness to Notch pathway inhibitors. Overall, a common oncogenomic program driven by binding of either Notch is sufficient to maintain T-ALL cell growth, whereas cell-context specific differences appear to influence the response of T-ALL cells to Notch inhibition

Downregulating Notch counteracts KrasG12D-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia

Differential Requirement for LAT and SLP-76 in GPVI versus T Cell Receptor Signaling

Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin αIIbβ3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT−/− platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76–deficient platelets. However, platelets from LAT−/− mice, but not SLP-76−/− mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin αIIbβ3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and αIIbβ3 shows a much greater dependency on SLP-76 than LAT

Trib2 suppresses tumor initiation in Notch-driven T-ALL

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes