A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage

ML, CD, IvL, GP, TM, SD, MS, APF, CT, DL, MAH, KL and SL: project grants from the Swedish Research Council, the Swedish Cancer Society and the Swedish Childhood Cancer Foundation. MHi and JC: Cancer Research UK (C8/A6613). MC, EP and WE: Wellcome Trust (073915). MN and BV: projects MEYS-NPS-LO1413 and GACR P206/12/G151. EMC, MP, MMS, ZF and PG: Norwegian Cancer Society (182735, 732200) and Helse Vest (911884, 911789). RB and SC: NIH (R01 CA95684), the Leukemia and Lymphoma Society and the Waxman Foundation. NW, AH, Ad’H: Cancer Research UK (C21383/A6950) and Engineering and Physical Sciences Research Council Doctoral Training Program. JL and YZ: Cancer Research UK (C240/A15751). MH and BW: SARomics Biostructures ABUY, KF: DDDP SciLife, Sweden. LJ, MHa, RS and A-LG: CBCS, Sweden. VP: SciLife fellowship. AT: Breast Cancer Research Scotland.The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.Publisher PDFPeer reviewe

A study on the role of genes of innate immunity in type 1 diabetes

Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic beta-cells. It is a polygenic disease in which maximal genetic susceptibility is conferred by the presence of MHC class II genes. Circulating autoantibodies against islet cell antigens and mononuclear cell infiltrates, especially CD4+ and CD8+ T cells, are characteristic of the disease. Recent evidence suggests innate immunity may be involved in the disease pathogenesis. The aim of this thesis is to identify the genes of innate immunity associated with T1D. In papers I and II, we tested the association of Killer immunoglobulin-like receptor (KIR) genes with T1D in two Caucasian populations (Swedish and Latvian). In the Swedish population, we demonstrated a negative association of KIRs 2DL1 and 3DS1 with T1D. Extensive stratification and interaction analyses showed that the frequency of KIRs 2DL2 and 3DS1 were higher in HLA-DR3/DR4 heterozygous T1D patients, but the difference was not significant. The presence of KIRs 2DL5 and 2DS5 conferred an increased risk to coxsackie B virus infection in T1D patients. In the Latvian cohort, we demonstrated that the presence of KIR 2DL2 and its ligand HLA-C1 conferred susceptibility to the disease. To test the overall association of KIR with T1D we performed a meta-analysis of all published data and found that KIRs 2DL5 and 3DS1 were negatively associated with T1D. These findings indicate that KIR genes are important in regulating innate immune responses and KIR-HLA-C ligand interactions can confer susceptibility to T1D. In papers III and IV, we tested the association of the small ubiquitin-related modifier 4 (SUMO4) M55V polymorphism with T1D in a Swedish (Caucasian) and an Asian-Indian population. SUMO4 was not directly associated with T1D in both populations. However, in the Swedish population, presence of the SUMO4 M55V SNP increased the susceptibility (Odds ratio) caused by HLA-DR3/DR4. In paper V, we tested the association of SNPs in the exons of Toll-like receptor (TLR) genes with T1D. SNPs in TLR-1 (rs4833095), TLR-6 (rs5743808) and TLR-8 (rs2159377) were associated with T1D in subjects belonging to the 0-14 year age group. No significant association was observed in the 15-34 year age group. These findings suggest that dysregulation of the immune response, especially in children below the age of 15 years, may contribute to the development of autoimmunity. In conclusion, this thesis reports novel associations of three innate immunity components with T1D

Regulation of Subunit-Specific Germinal Center B Cell Responses to the HIV-1 Envelope Glycoproteins by Antibody-Mediated Feedback

The regulation of germinal center (GC) B cell responses to single epitopes is well investigated. How monoclonal B cells are regulated within the polyclonal B cell response to protein antigens is less so. Here, we investigate the primary GC B cell response after injection of mice with HIV-1 envelope glycoproteins. We demonstrate that single GCs are seeded by a diverse number of B cell clones shortly after a single immunization and that the presence of Env-specific antibodies can inhibit the development of early GC B cells. Importantly, the suppression was dependent on the GC B cells and the infused antibodies to target the same subunit of the injected HIV-1 envelope glycoproteins. An affinity-dependent antibody feedback has previously been shown to regulate GC B cell development. Here, we propose that this antibody-based feedback acts on GC B cells only if they target the same or overlapping epitopes. This study provides important basic information of GC B cell regulation, and for future vaccine designs with aim to elicit neutralizing antibodies against HIV-1

Risk Conferred by HLA-DR and DQ for Type 1 Diabetes in 0-35-Year Age Group in Sweden

HLA DR4-DQ8 and DR3-DQ2 haplotypes account for 89% of newly diagnosed cases of type 1 diabetes (T1D) in Sweden. The presence of a single copy of DQ6 confers protection. The aim of the present study is to evaluate whether the risk conferred by high risk HLA DR and DQ to T1D is similar in all regions of Sweden and see whether there are any significant regional differences. The subjects comprised 799 consecutively diagnosed T1D patients and 585 age-, sex-, and geography-matched healthy controls in the age group 0-35 years. HLA typing for high-risk haplotypes was previously performed using PCR-SSOP and RFLP The results showed that HLA DR3-DR4 gave an odds ratio of 8.14 for the whole of Sweden. However, when the study group was divided into six geographical regions, subjects from Stockholm had the highest OR, followed by those from Lund, Linkoping, Gothenburg, Umea, and Uppsala. Absolute protection was conferred by the presence of DQ6 in subjects from the Linkoping region, but varied in the other regions. The frequency of DR3 and DQ2, DR4 and DQ8, DR15, and DQ6 in patients showed high linkage for each region, but were different between regions. In conclusion: The risk conferred by high-risk HLA varies in different regions for a homogenous population in Sweden. The results highlight the important role played by the various environmental factors in the precipitation of T1D

PPAR-γ promotes type 2 immune responses in allergy and nematode infection

A hallmark of immunity to worm infections and many allergies is a strong type 2 immune response. This is characterized by the production of cytokines interleukin-5 (IL-5) and IL-13 by adaptive T helper 2 (TH2) cells and/or type 2 innate lymphoid cells. Peroxisome proliferator activated receptor-γ (PPAR-γ) is typically regarded as an anti-inflammatory factor. We report that TH2 cells express high levels of PPAR-γ in response to the allergen house dust mite and after infection with the parasite Heligmosomoides polygyrus Mice lacking PPAR-γ in T cells failed to effectively differentiate into IL-5- and IL-13-secreting cells and, hence, did not develop TH2 cell-associated pathologies, including goblet cell metaplasia and eosinophilia, in response to allergen challenge. Furthermore, these mice could not mount protective immune responses to nematode infection. In addition, mice lacking PPAR-γ in T cells had greatly reduced frequencies of TH2 cells in visceral adipose tissue. Mechanistically, PPAR-γ appeared to promote the expression of the IL-33 receptor on the surface of TH2 cells. These results pinpoint PPAR-γ as a factor that drives type 2 responses in allergy, worm infection, and visceral adipose tissue