T-lymphocyte development branches off from other lymphoid developmental programs through its requirement for sustained environmental signals through the Notch pathway. In the thymus, Notch signaling induces a succession of T-lineage regulatory factors that collectively create the T-cell identity through distinct steps. This process involves both the staged activation of T-cell identity genes and the staged repression of progenitor-cell-inherited regulatory genes once their roles in self-renewal and population expansion are no longer needed. With the recent characterization of innate lymphoid cells (ILCs) that share transcriptional regulation programs extensively with T-cell subsets, T-cell identity can increasingly be seen as defined in modular terms, as the processes selecting and actuating effector function are potentially detachable from the processes generating and selecting clonally unique T-cell receptor structures. The developmental pathways of different classes of T cells and ILCs are distinguished by the numbers of prerequisites of gene rearrangement, selection, and antigen contact before the cells gain access to nearly common regulatory mechanisms for choosing effector function. Here, the major classes of transcription factors that interact with Notch signals during T-lineage specification are discussed in terms of their roles in these programs, the evidence for their spectra of target genes at different stages, and their cross-regulatory and cooperative actions with each other. Specific topics include Notch modulation of PU.1 and GATA-3, PU.1-Notch competition, the relationship between PU.1 and GATA-3, and the roles of E proteins, Bcl11b, and GATA-3 in guiding acquisition of T-cell identity while avoiding redirection to an ILC fate.

To investigate how transcription factor levels impact B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro. The alteration in lineage fate depended on a Notch1-mediated conversion process, whereas no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5(+/-) Ebf1(+/-) pro-B cells were reflected in the transcriptional response. Both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, but only the Pax5(+/-) Ebf1(+/-) pro-B cells down-regulated genes central for the preservation of stable B cell identity. This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling. This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.

Early B-cell factor 1 (Ebf1) is a transcription factor with documented dose-dependent functions in normal and malignant B-lymphocyte development. To understand more about the roles of Ebf1 in malignant transformation, we investigated the impact of reduced functional Ebf1 dosage on mouse B-cell progenitors. Gene expression analysis suggested that Ebf1 was involved in the regulation of genes important for DNA repair and cell survival. Investigation of the DNA damage in steady state, as well as after induction of DNA damage by UV light, confirmed that pro-B cells lacking 1 functional allele of Ebf1 display signs of increased DNA damage. This correlated to reduced expression of DNA repair genes including Rad51, and chromatin immunoprecipitation data suggested that Rad51 is a direct target for Ebf1. Although reduced dosage of Ebf1 did not significantly increase tumor formation in mice, a dramatic increase in the frequency of pro-B cell leukemia was observed in mice with combined heterozygous mutations in the Ebf1 and Pax5 genes, revealing a synergistic effect of combined dose reduction of these proteins. Our data suggest that Ebf1 controls DNA repair in a dose-dependent manner providing a possible explanation to the frequent involvement of EBF1 gene loss in human leukemia.

B-lymphocyte development in the bone marrow is controlled by the coordinated action of transcription factors creating regulatory networks ensuring activation of the B-lymphoid program and silencing of alternative cell fates. This process is tightly connected to malignant transformation because B-lineage acute lymphoblastic leukemia cellsdisplay a pronounced block in differentiation resulting in the expansion of immature progenitor cells. Over the last few years, high-resolution analysis of genetic changes in leukemia has revealed that several key regulators of normal B-cell development, including IKZF1, TCF3, EBF1, and PAX5, are genetically altered in a large portion of the human B-lineage acute leukemias. This opens the possibility of directly linking the disrupted development as well as aberrant gene expression patterns in leukemic cells to molecular functions of defined transcription factors in normal cell differentiation. This review article focuses on the roles of transcription factors in early B-cell development and their involvement in the formation of human leukemia.

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2R-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

Background

NOTCH1 PEST domain mutations in chronic lymphocytic leukemia have recently been shown to be of prognostic relevance. Both NOTCH1 and NOTCH2 are constitutively activated in B-cell CLL but not expressed in normal B cells and may be involved in survival and resistance to apoptosis in CLL. We screened for mutations in different parts of both NOTCH1 and NOTCH2 genes and related the changes to survival and other known risk factors.

Methods

In a cohort of 209 CLL patients, we used single strand conformation analysis to determine which of the samples carrying the NOTCH mutations and direct dideoxy sequencing was used to determine the exact nucleotide changes. Kaplan-Meier curves and log rank test were used to determine overall survival for NOTCH1 mutated cases and Cox regression analysis was used to calculate hazardous ratios.

Results

In the present study, we found NOTCH1 PEST domain mutations in 6.7% of the cases. A shorter overall survival was found in patients with NOTCH1 mutations compared to wildtype (p = 0.049). Further, we also examined the extracellular and the heterodimerisation domains of the NOTCH1 gene and the PEST domain and heterodimerisation domain of the NOTCH2 gene, but no mutations were found in these regions. NOTCH1 mutations were most commonly observed in patients with unmutated IGHV gene (10/14), and associated with a more aggressive disease course. In addition, NOTCH1 mutations were almost mutually exclusive with TP53 mutations. In the combined group of NOTCH1 (6.7%) or TP53 (6.2%) mutations, a significant difference in overall survival compared to the wildtype NOTCH1 and TP53 was found (p = 0.002).

Conclusions

Both NOTCH1 and TP53 mutations seem to be independent predictive markers for worse outcome in CLL-patients and this study emphasizes the contention that NOTCH1 mutations is a novel risk marker.

Colorectal tumors are continuously exposed to an inflammatory environment, which together with mitogenic signals sustain several cancer hallmarks. Nuclear factor-kappa B (NFκB) is a major regulator of inflammation and variation in NFκB-associated genes could potentially be used as biomarkers to identify patients with increased risk of colorectal cancer (CRC) development, and/or a rapidly progressing disease. In this study, 348 CRC cases and 806 randomly selected healthy individuals from southeastern Sweden were examined with regard to seven polymorphisms in NFκB pathway-associated genes. Log-rank-tests and Cox proportional hazard regression analysis examined the association between the polymorphisms and CRC-specific survival, whereas chi-square tests and logistic regression analysis were used to test for associations between the polymorphisms and CRC susceptibility. Gene expression and loss of heterozygosity analyses of TNFAIP3 were carried out in a subset of tumors to assess its role as a tumor suppressor in CRC. Heterozygous and polymorphic TNFAIP3 (rs6920220), heterozygous NLRP3 (Q705K) and polymorphic NFκB -94 ATTG ins/del genotypes were found to be associated with poorer survival in patients diagnosed with invasive CRC (aHR = 5.2, 95% CI: 2.5-10.9, P < 0.001). TNFAIP3 mRNA levels were significantly decreased in tumors compared with adjacent non-neoplastic mucosa (P < 0.0001) and loss of heterozygosity of 6q23.3 (TNFAIP3) was detected in 17% of cases, whereas only 2.5% of the investigated specimens displayed TNFAIP3 gene mutations. We propose that TNFAIP3 (rs6920220), NLRP3 (Q705K) and NFκB -94 ATTG ins/del polymorphisms are associated with poor survival in patients with advanced CRC and may be used as prognostic markers. Experimental results indicate that TNFAIP3 may act as a tumor suppressor in CRC.

Colorectal cancer (CRC) is a global ‘killer’ and every year more than 1.2 million new individuals are affected and approximately 600 000 succumb to the disorder. Several mechanisms such as inactivation of tumor suppressor genes, activation of oncogenes and dysregulation of cell fate determinating pathways e.g. Wnt and Notch can initiate a cancerous cell growth and promote colorectal tumorigenesis. In addition, most tumors are exposed to an inflammatory environment, which together with the presence of mitogenic and angiogenic signals may sustain several hallmarks of cancer. Genetic alterations in inflammatory genes are associated with chronic inflammatory bowel disease, which is a strong risk factor of developing CRC. Scientists have for a long time looked for ‘the Key’ that would unlock the ‘cancer door’ but more likely cancer should be considered as not one but many diseases where almost every single patient is genetically and clinically unique. Hence recent research has turned to identify such inter-individual discrepancies and to find disease markers and strategies for guiding clinicians when tailoring individual management and optimized therapy. A deeper understanding of the regulation and genetic variation of inflammation and intestinal-homeostasis associated genes is pivotal to find potential targets for future therapies.

The present thesis focuses on genetic variation and alterations in inflammatory genes as well as genes specifically involved in maintaining intestinal homeostasis. The most common anti-inflammatory drugs, NSAIDs, inhibit the prostanoid-generating COX-enzymes and are associated with decreased CRC risk when administered for a long time. Unfortunately, continuous NSAID treatment may lead to severe side-effects such as gastrointestinal bleeding, possibly through the ablation of non-PGE2 prostanoids. Therefore, a more specific inhibition of PGE2 has been suggested to be superior to classical NSAIDs. In papers I and II, the terminal PGE2 generating enzyme mPGES1 was studied in the context of intestinal cancer. Unexpectedly, ApcMin/+ mice with a targeted deletion of the mPGES1 encoding gene displayed significantly more and larger intestinal adenomas as compared to their wilde-type (wt) littermates. Probably this was due to the redirected generation of PGE2 towards non-PGE2 prostanoids seen in the murine tumors, resulting in enhanced pro-tumorigenic activity of these transmitter substances. Next, with a battery of functional and descriptive assays we investigated whether the outcome of mPGES1 expression and activity could depend on the genetic profile of the tumor e.g. the Apc mutational status. Indeed, high expression of mPGES1 was associated with the presence of wt-Apc, both in vitro and in vivo, most likely depending on mPGES1 mRNA stabilization rather than upregulation through β–catenin/Lef/Tcf4 signaling.

NFκB is a major regulator of inflammation e.g. through the production of inflammatory cytokines. Variations in genes controlling inflammation and angiogenesis could potentially be used as biomarkers to identify patients with increased risk of CRC development, and/or to identify those with high risk of a rapidly progressing disease. Further, such analyzes have been suggested to select patients, which may benefit from specific anti-inflammatory or anti-angiogenic therapies. In paper III, genetic alterations in NFκB associated genes were studied among CRC patients and healthy controls. The NFκB negative regulator TNFAIP3 was found to exert tumor suppressive functions in CRC and moreover, homozygous mutant TNFAIP3 (rs6920220), homozygous mutant NFκB -94 ATTG ins/del and heterozygous NLRP3 (Q705K) were identified as prognostic markers for identifying CRC patients with a high risk of rapid progression. Further studies, which focus on the potential to treat such patients with anti-inflammatory IL-1β targeting therapies, are warranted.

In the intestinal epithelium, Notch and Wnt signaling function in synergy to maintain homeostasis and together these pathways promote stem cell renewal and drive proliferation. Thus, dysregulation and/or overactivation of one of the two pathways could potentially lead to simultaneous activation of the other. While the genetic mechanisms explaining aberrant Wnt signaling in CRC are well-known, the reasons for the Notch pathway activation are less so. Further, relatively little is known about the mechanisms linking the two pathways in CRC. In paper IV, we addressed this question with a set of experimental in vitro assays, hereby identifying Notch2 together with several additional genes classically belonging to the Notch pathway, as putative targets for canonical and non-canonical Wnt signaling. We therefore suggest that aberrant Notch signaling in colon cancer cells may be the result of dysregulated Wnt signaling.

In summary, the results here presented add a couple of pieces to the immensely complex jigsaw puzzle connecting intestinal homeostasis, inflammation and CRC. These results may aid in identifying future biomarkers or potential drug targets that could take us to the next level in the war against cancer.

Background: Notch and Wnt pathways are key regulators of intestinal homeostasis and alterations in these pathways may lead to the development of colorectal cancer (CRC). In CRC the Apc/beta-catenin genes in the Wnt signaling pathway are frequently mutated and active Notch signaling contributes to tumorigenesis by keeping the epithelial cells in a proliferative state. These pathways are simultaneously active in proliferative adenoma cells and a crosstalk between them has previously been suggested in normal development as well as in cancer. Principal Findings: In this study, in silico analysis of putative promoters involved in transcriptional regulation of genes coding for proteins in the Notch signaling pathway revealed several putative LEF-1/TCF sites as potential targets for beta-catenin and canonical Wnt signaling. Further results from competitive electrophoretic mobility-shift assay (EMSA) studies suggest binding of several putative sites in Notch pathway gene promoters to in vitro translated beta-catenin/Lef-1. Wild type (wt)-Apc negatively regulates beta-catenin. By induction of wt-Apc or beta-catenin silencing in HT29 cells, we observed that several genes in the Notch pathway, including Notch-2, were downregulated. Finally, active Notch signaling was verified in the Apc(Min/+) mouse model where Hes-1 mRNA levels were found significantly upregulated in intestinal tumors compared to normal intestinal mucosa. Luciferase assays showed an increased activity for the core and proximal Notch-2 promoter upon co-transfection of HCT116 cells with high expression recombinant Tcf-4, Lef-1 or beta-catenin. Conclusions: In this paper, we identified Notch-2 as a novel target for beta-catenin-dependent Wnt signaling. Furthermore our data supports the notion that additional genes in the Notch pathway might be transcriptionally regulated by Wnt signaling in colorectal cancer.

BACKGROUND: The single nucleotide polymorphism SNP309 with a change from T to G in the promoter region of the MDM2 gene is shown to increase the MDM2 protein levels and attenuate the p53 levels and associates with disease progression in several tumors. OBJECTIVE: In this study, the role of the polymorphism was investigated with regard to the clinical outcome in B-cell chronic lymphocytic leukemia (B-CLL). PATIENTS: A total of 210 patients with B-CLL were followed for up to 19 yr. RESULTS: The overall survival (OS) of patients with at least one G-allele was significantly shorter when compared with those with two T-alleles (P = 0.024) with a more pronounced difference in patients below the median age. Age at onset of B-CLL was similar irrespective of MDM2 status. The presence of a G-allele in combination with TP53 mutations or unmutated IgVH gene status resulted in an additive risk of death. CONCLUSION: In this report, with a high proportion of B-CLL patients with an advanced Binet stage and with an unmutated IgVH gene, MDM2 SNP309 was found to be independently associated with OS. The survival difference was more pronounced in younger patients.