Background: Clinical management of allergic diseases has been hampered by the lack of safe and convenient tests to reliably identify culprit allergens and to closely follow changes in disease activity over time. Because allergy diagnosis is a complex and laborious multistep procedure, there is an urgent need for simpler but still functionally accurate ex vivo assays allowing objective diagnosis, substantiating treatment choices, and quantifying therapeutic responses. Objective: In this study, we sought to develop a novel functional cell-based assay that relies on passive sensitization of allergic effector cells with patient serum, circumventing current limitations in allergy diagnosis. Methods: We genetically engineered a conditional homeobox B8 (Hoxb8)-immortalized progenitor line from the bone marrow of mice that are transgenic for the human high-affinity IgE receptor (Fc epsilon RIa). These cells can be reproducibly differentiated into mature Hoxb8 mast cells within 5 days of culture in virtually unlimited numbers. Results: We demonstrate that the established Hoxb8 mast cell assay can be used to accurately measure total IgE levels, identify culprit allergens, longitudinally monitor allergen-specific immunotherapy, and potentially determine the time point of tolerance induction upon allergen-specific immunotherapy in patients with allergy. To facilitate the analysis of large testing volumes, we demonstrate a proof-of-concept for a high-throughput screening application based on fluorescent cell barcoding using the engineered Hoxb8 mast cells. Conclusions: Our results indicate that this novel mast cell assay could represent a valuable tool to support clinicians in the identification of IgE-mediated allergies and in the quantification of treatment efficacy as well as duration of therapeutic response.

Actin-Related Protein-Testis1 (ARP-T1)/ACTRT1 gene mutations cause the Bazex-Dupré-Christol Syndrome (BDCS) characterized by follicular atrophoderma, hypotrichosis, and basal cell cancer. Here, we report an ARP-T1 interactome (PXD016557) that includes proteins involved in ciliogenesis, endosomal recycling, and septin ring formation. In agreement, ARP-T1 localizes to the midbody during cytokinesis and the basal body of primary cilia in interphase. Tissue samples from ARP-T1-associated BDCS patients have reduced ciliary length. The severity of the shortened cilia significantly correlates with the ARP-T1 levels, which was further validated by ACTRT1 knockdown in culture cells. Thus, we propose that ARP-T1 participates in the regulation of cilia length and that ARP-T1-associated BDCS is a case of skin cancer with ciliopathy characteristics.

Acetaminophen (APAP) is one of the most commonly used analgesic and anti-pyretic drugs, and APAP intoxication is one of the main reasons for liver transplantation following liver failure in the Western world. While APAP poisoning ultimately leads to liver necrosis, various programmed cell death modalities have been implicated, including ER stress-triggered apoptosis. The BCL-2 family member BOK (BCL-2-related ovarian killer) has been described to modulate the unfolded protein response and to promote chemical-induced liver injury. We therefore investigated the impact of the loss of BOK following APAP overdosing in mice. Surprisingly, we observed sex-dependent differences in the activation of the unfolded protein response (UPR) in both wildtype (WT) and Bok-/- mice, with increased activation of JNK in females compared with males. Loss of BOK led to a decrease in JNK activation and a reduced percentage of centrilobular necrosis in both sexes after APAP treatment; however, this protection was more pronounced in Bok-/- females. Nevertheless, serum ALT and AST levels of Bok-/- and WT mice were comparable, indicating that there was no major difference in the overall outcome of liver injury. We conclude that after APAP overdosing, loss of BOK affects initiating signaling steps linked to ER stress, but has a more minor impact on the outcome of liver necrosis. Furthermore, we observed sex-dependent differences that might be worthwhile to investigate.

Hereditary keratodermas and ichthyoses comprise a large collection of genodermatoses for which underlying mutations in more than 100 genes have been identified (Oji et al., 2010Oji V. Tadini G. Akiyama M. Blanchet Bardon C. Bodemer C. Bourrat E. et al.Revised nomenclature and classification of inherited ichthyoses: results of the first Ichthyosis Consensus Conference in Soreze 2009.J Am Acad Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). The implicated genes are involved in a multitude of biological pathways. Ceramides are important for cutaneous barrier function (Borodzicz et al., 2016Borodzicz S. Rudnicka L. Mirowska-Guzel D. Cudnoch-Jedrzejewska A. The role of epidermal sphingolipids in dermatologic diseases.Lipids Health Dis. 2016; 15: 13Crossref PubMed Scopus (47) Google Scholar) and cutaneous proliferation and differentiation (Uchida, 2014Uchida Y. Ceramide signaling in mammalian epidermis.Biochim Biophys Acta. 2014; 1841: 453-462Crossref PubMed Scopus (64) Google Scholar). In most organisms, they are synthesized by three different biochemical pathways, named de novo, sphingomyelinase, and salvage pathways (Hannun and Obeid, 2008Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat Rev Mol Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2205) Google Scholar, Kihara, 2016Kihara A. Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides.Prog Lipid Res. 2016; 63: 50-69Crossref PubMed Scopus (92) Google Scholar, Kitatani et al., 2008Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell Signal. 2008; 20: 1010-1018Crossref PubMed Scopus (354) Google Scholar). The de novo pathway (Linn et al., 2001Linn S.C. Kim H.S. Keane E.M. Andras L.M. Wang E. Merrill Jr., A.H. Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption.Biochem Soc Trans. 2001; 29: 831-835Crossref PubMed Scopus (0) Google Scholar) critically depends on 3-ketodihydrosphingosine reductase (KDSR) catalyzing the reduction of 3-ketodihydrosphingosine to dihydrosphingosine, which serves as a substrate for ceramide synthases 1–6. Recessive mutations in CERS3 cause autosomal recessive congenital ichthyosis (Eckl et al., 2013Eckl K.M. Tidhar R. Thiele H. Oji V. Hausser I. Brodesser S. et al.Impaired epidermal ceramide synthesis causes autosomal recessive congenital ichthyosis and reveals the importance of ceramide acyl chain length.J Invest Dermatol. 2013; 133: 2202-2211Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, Radner et al., 2013Radner F.P. Marrakchi S. Kirchmeier P. Kim G.J. Ribierre F. Kamoun B. et al.Mutations in CERS3 cause autosomal recessive congenital ichthyosis in humans.PLOS Genet. 2013; 9e1003536Crossref PubMed Scopus (101) Google Scholar). Mutations in the ELOVL4 gene, required for the synthesis of ultra-long ceramides, are the underlying cause of a recessive disorder characterized by ichthyosis, intellectual disability, and spastic quadriplegia (Aldahmesh et al., 2011Aldahmesh M.A. Mohamed J.Y. Alkuraya H.S. Verma I.C. Puri R.D. Alaiya A.A. et al.Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.Am J Hum Genet. 2011; 89: 745-750Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). With great interest, we read the recent reports on mutations in the KDSR gene causing either autosomal recessive progressive symmetric erythrokeratoderma (Boyden et al., 2017Boyden L.M. Vincent N.G. Zhou J. Hu R. Craiglow B.G. Bayliss S.J. et al.Mutations in KDSR cause recessive progressive symmetric erythrokeratoderma.Am J Hum Genet. 2017; 100: 978-984Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) or a spectrum of keratinization disorders with associated thrombocytopenia characterized by palmoplantar and anogenital hyperkeratosis or a more generalized phenotype resembling harlequin ichthyosis (Takeichi et al., 2017Takeichi T. Torrelo A. Lee J.Y.W. Ohno Y. Lozano M.L. Kihara A. et al.Biallelic mutations in KDSR disrupt ceramide synthesis and result in a spectrum of keratinization disorders associated with thrombocytopenia.J Invest Dermatol. 2017; 137: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Siblings carrying two KDSR mutations were reported to have thrombocytopenia and moderate anemia, which improved over the first decade of life but which presented only with very minor skin pathology (Bariana et al., 2019Bariana T.K. Labarque V. Heremans J. Thys C. De Reys M. Greene D. et al.Sphingolipid dysregulation due to lack of functional KDSR impairs proplatelet formation causing thrombocytopenia.Haematologica. 2019; 104: 1036-1045Crossref PubMed Scopus (10) Google Scholar). Here, we report an allelic phenotype in a female patient born to healthy unrelated parents with generalized harlequin ichthyosis (Supplementary Figure S1 a–c after 1 day, Supplementary Figure S1d after 1 week), which improved spontaneously within weeks into palmoplantar keratoderma and leukokeratosis anogenitalis without thrombocytopenia, suggesting that disorders caused by KDSR mutations indeed represent a phenotypic spectrum. Institutional ethical approval was obtained for our experiments, and samples were collected after obtaining written, informed patient consent. Clinical examination of the patient showed diffuse noninflammatory palmar and plantar keratoderma with a well-defined border, erythemato–squamous plaques with excoriations involving the dorsum of the hands and feet, hyperkeratosis on the elbows, and generalized xerosis (Figure 1a–d). Nails were overcurved and slightly thickened (Figure 1b and c). Whitish hyperkeratosis of the vulva extending to the perianal region was present (Figure 1e). Symmetrical brachytelephalangy without underlying bone lesion on X-ray was also observed (Figure 1f). No involvement of the face or oral mucosa was found. Hair and teeth were normal. The patient had a normal platelet count. Her younger brother showed no abnormal skin. Histology of a hyperkeratotic plaque showed marked orthokeratosis and parakeratosis and the condensation of an eosinophilic material around the nucleus in a few cells of the spinal layer (Figure 1g and h). Ultrastructurally, the intermediate filaments in the granular layer were highly aggregated and the cytoplasm of the spinous layer was enriched in mitochondria and ribosomes. The basal layer showed no particular abnormalities and the number of mitotic cells was normal. Based on the clinical manifestations in our patient, which corresponded partially with the clinical description reported in the literature, we hypothesized that mutations in the KSDR gene might be the underlying cause. Therefore, we have sequenced all 10 exons of the KDSR gene from the index patient. The analysis revealed that the patient's DNA harbors the compound heterozygous mutations LRG_1196t1:c.592G>A and LRG_1196t1:c.865G>A in exons 6 and 9, respectively (Figure 2a and b). Compound heterozygosity was only found in the patient, whereas her unaffected parents and brother carried only one of the two mutations (Figure 2b). Both gene mutations were not found in the 1,000 Genomes Project (1000 Genomes Project Consortium et al., 2015Auton A. Brooks L.D. Durbin R.M. Garrison E.P. Kang H.M. et al.1000 Genomes Project ConsortiumA global reference for human genetic variation.Nature. 2015; 526: 68-74Crossref PubMed Scopus (6224) Google Scholar). The exon 9 mutation was found in 3 out of 251,184 alleles in the Genome Aggregation database (https://gnomad.broadinstitute.org), whereas the exon 6 mutation was not present in the Exome Aggregation Consortium (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (5668) Google Scholar) or Genome Aggregation databases. The first mutation, denoted as LRG_1196p1:p.(Glu198Lys), concerns a highly conserved amino acid residue lying 12 amino acids toward the C-terminal end of the sequence YxxxK, which is an essential part of the catalytic triad in the nicotinamide adenine dinucleotide–binding Rossman fold of KDSR (Gupta et al., 2009Gupta S.D. Gable K. Han G. Borovitskaya A. Selby L. Dunn T.M. et al.Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals.J Lipid Res. 2009; 50: 1630-1640Crossref PubMed Scopus (14) Google Scholar). The second mutation, LRG_1196p1:p.(Glu289Lys), is situated in the second predicted transmembrane region, which might be important for the correct spatial localization of KDSR into the endoplasmic reticulum membrane (Gupta et al., 2009Gupta S.D. Gable K. Han G. Borovitskaya A. Selby L. Dunn T.M. et al.Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals.J Lipid Res. 2009; 50: 1630-1640Crossref PubMed Scopus (14) Google Scholar, Kihara and Igarashi, 2004Kihara A. Igarashi Y. FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytosolic side of the endoplasmic reticulum membrane.J Biol Chem. 2004; 279: 49243-49250Crossref PubMed Scopus (61) Google Scholar). A large deletion from amino acids 260 to 293 has been reported in patients suffering from progressive symmetric erythrokeratoderma (Boyden et al., 2017Boyden L.M. Vincent N.G. Zhou J. Hu R. Craiglow B.G. Bayliss S.J. et al.Mutations in KDSR cause recessive progressive symmetric erythrokeratoderma.Am J Hum Genet. 2017; 100: 978-984Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Analysis of the functional impact of both mutations on the enzymatic function of KDSR classified them as probably (Glu198Lys, POLYPhen-2 score 1.0) or possibly damaging (Glu289Lys, POLYPhen-2 score 0.932) using POLYPhen-2 (Adzhubei et al., 2010Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. et al.A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (8193) Google Scholar) and PANTHER (Mi et al., 2013Mi H. Muruganujan A. Thomas P.D. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees.Nucleic Acids Res. 2013; 41: D377-D386Crossref PubMed Scopus (1089) Google Scholar) or as deleterious using SIFT (Sim et al., 2012Sim N.L. Kumar P. Hu J. Henikoff S. Schneider G. Ng P.C. SIFT web server: predicting effects of amino acid substitutions on proteins.Nucleic Acids Res. 2012; 40: W452-W457Crossref PubMed Scopus (983) Google Scholar). Furthermore, the web-based software MutationsTaster2 (Schwarz et al., 2014Schwarz J.M. Cooper D.N. Schuelke M. Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age.Nat Methods. 2014; 11: 361-362Crossref PubMed Scopus (1966) Google Scholar) predicted that both mutations would be disease-causing. Immunofluorescence analysis showed normal KDSR expression in patient skin, similar to what has been reported before (Boyden et al., 2017Boyden L.M. Vincent N.G. Zhou J. Hu R. Craiglow B.G. Bayliss S.J. et al.Mutations in KDSR cause recessive progressive symmetric erythrokeratoderma.Am J Hum Genet. 2017; 100: 978-984Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, Takeichi et al., 2017Takeichi T. Torrelo A. Lee J.Y.W. Ohno Y. Lozano M.L. Kihara A. et al.Biallelic mutations in KDSR disrupt ceramide synthesis and result in a spectrum of keratinization disorders associated with thrombocytopenia.J Invest Dermatol. 2017; 137: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The previously reported KDSR mutations and those described herein are found in exons 2 to 6, comprising the active site and the nicotinamide adenine dinucleotide–binding region, and in exon 9, encoding the region around the second transmembrane region, underlining their importance for KDSR function (Figure 2c). However, the number of known mutations are far too low to make any statement regarding genotype and phenotype correlation. The described biallelic KDSR mutations, most likely perturbing the de novo synthesis of sphingolipids, have not been reported previously, to our knowledge, and allow classifying the disorder within a spectrum of KDSR associated phenotypes. Palmoplantar keratoderma with leukokeratosis anogenitalis and transient harlequin fetus is a unique extremely rare clinical phenotype—only one identical case has been published (Itin and Rufli, 1994Itin P.H. Rufli T. Collodion baby with evolution to palmoplantar keratoderma and leukokeratosis anogenitalis. A new disease?.Eur J Dermatol. 1994; 4: 589-592Google Scholar)—but despite our efforts, this patient could not be tracked. However, two cases suffering from palmoplantar keratoderma, leukokeratosis anogenitalis, and thrombocytopenia; a patient with harlequin ichthyosis and thrombocytopenia; and an individual presenting with harlequin ichthyosis, which resolved into generalized erythroderma with fine scaling, and thrombocytopenia were reported in this journal previously (Takeichi et al., 2017Takeichi T. Torrelo A. Lee J.Y.W. Ohno Y. Lozano M.L. Kihara A. et al.Biallelic mutations in KDSR disrupt ceramide synthesis and result in a spectrum of keratinization disorders associated with thrombocytopenia.J Invest Dermatol. 2017; 137: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The particular phenotype in the described patient herein may be linked to the specific and, to our knowledge, previously unreported mutations. Our patient never received retinoids and never asked for treatment. She is now of child-bearing age and has family plans. As for potential mechanism-based therapy, our repeated attempts to culture primary keratinocytes did fail, such that we could not perform experimental preclinical studies in vitro. Nevertheless, topical or systemic treatment with ceramides or induction of alternative ceramide salvage pathways may be of benefit for patients suffering from KDSR mutations, particularly the ones with more severe phenotypes. Data are available upon request. Marcel Huber: https://orcid.org/0000-0003-3821-2378 Elena Chiticariu: https://orcid.org/0000-0002-9921-8020 Daniel Bachmann: https://orcid.org/0000-0003-2757-4218 Lukas Flatz: https://orcid.org/0000-0001-7491-2385 Daniel Hohl: https://orcid.org/0000-0001-6337-7110 The authors state no conflict of interest. The authors thank the family members for their participation. The study was supported by grants from the Swiss National Science Foundation , Switzerland ( 310030_173102/1 ) and the Dind Cottier Foundation , Switzerland. Conceptualization: EC, MH, DH; Data Curation: MH, DB, EC, LF, DH; Formal Analysis: EC, MH, DB, DH; Funding Acquisition: DH; Investigation: MH, EC, DB; Methodology: MH, EC, DH; Project Administration: MH, DH; Resources: MH, EC, DH; Supervision: MH, DH; Validation: MH, DH; Visualization: EC, MH, DH; Writing - Original Draft Preparation: MH, EC; Writing - Review and Editing: MH, DH

BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.

ABSTRACT Actin-Related Protein-Testis1 (ARP-T1)/ ACTRT1 gene mutations cause the Bazex-Dupré-Christol Syndrome (BDCS) characterized by follicular atrophoderma, hypotrichosis and basal cell cancer. Here, we report an ARP-T1 interactome (PXD016557) involved in ciliogenesis, endosomal recycling and septin ring formation. Consequently, ARP-T1 localizes to the midbody during cytokinesis and the basal body of primary cilia in G 0 . Tissue samples from BDCS patients show reduced ciliary length with significant correlations of ARP-T1 expression levels, confirmed by ACTRT1 knock down. We report that BDCS is a novel ciliopathy and the first case of a skin cancer ciliopathy, where ARP-T1 plays a critical role to prevent pathogenesis.

ARP-T1 (Actin-Related Protein-Testis 1) is encoded by the ACTRT1 gene, and mutations in this gene cause Bazex-Dupré-Christol Syndrome (BDCS) associated with Basal Cell Cancer (BCC). Unlike most cases of BCC, BDCS is a dominantly X-linked inherited syndromic form of BCC. The insertion mutation ARP-T1 547_548InsA creating a shift of the reading frame resulting in a truncated protein of 198 amino acids segregates with the disease, and germline mutations of non-coding sequences surrounding ACTRT1 were identified and postulated to belong to enhancers transcribed as non-coding RNAs (eRNAs). ARP-T1 plays a role as a tumor suppressor in BDCS ( Bal et al., 2017 ). We employed mass spectrometry analysis to find ARP-T1 interacting proteins in HeLa cells, normal human epidermal keratinocytes and ciliated differentiated serum starved hTERT-RPE1 cells and found several proteins known to be involved in ciliogenesis - Acetylated Tubulin, TCP8, Hsc70, BAG2, EHD4, EHD2, Rab8A, Septin2, Septin9, Gamma Tubulin. We found that ARP-T1 is expressed not only in testis but also in human hair follicles and in ciliated differentiated cells, and confirmed that ARP-T1 localizes at the basal body of primary cilia. ARP-T1 co-localizes with Rootletin, a major basal body protein, and forms a complex with Gamma Tubulin, which localizes in vicinity of the basal body. Tissue samples from BDCS patients show reduced ciliary length and ARP-T1 expression when compared to normal hair follicles with significant correlations between ARP-T1 expression levels and ciliary length. ACTRT1 knock down confirmed that ARP-T1 was a direct cause to reduced ciliary length. We report that BDCS is a novel ciliopathy and the first case of a skin cancer ciliopathy, where ARP-T1 plays a critical role to prevent pathogenesis. How ARP-T1 interacts with proteins in ciliary machinery to regulate ciliogenesis linked to the actin network will be discussed.

BCL-2-related ovarian killer (BOK) is a pro-apoptotic BAX-like member of the BCL-2 family with suggested tumor suppressor activity. The molecular mechanisms regulating BOK expression are poorly understood and fail to explain a frequent lack of concordance between protein and transcript levels. Here, we describe a potent post-transcriptional mechanism that negatively regulates BOK expression mediated by conserved (AU/U)-rich elements within its 3' UTR. Using proteomics approaches we identified TRIM28 as a key component associating with U-rich elements in the human BOK 3' UTR, resulting in a dramatic reduction of BOK expression. TRIM28 is overexpressed in several cancers, correlating with poor patient outcome, whereas the BOK locus is frequently deleted or its expression downregulated in human cancers. Data mining indicated that, for certain cancers, high TRIM28 and low BOK expression are significantly correlated in the stratum of patients with the worst survival, suggesting that this mechanism might be of potential therapeutic value.

As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p<0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p=0.004, n=35) and three (p=0.031, n=39) as well as in tumors with metastases to hilar (pN1) (p=0.047, n=31) and mediastinal/subcarinal lymph nodes (pN2) (p=0.021, n=18) as opposed to grade one tumors (p=0.688, n=7) and tumors without lymph node metastases (p=0.112, n=51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p=0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGF-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.

BCL-2-related ovarian killer (BOK) is a conserved and widely expressed BCL-2 family member with sequence homology to pro-apoptotic BAX and BAK, but with poorly understood pathophysiological function. Since several members of the BCL-2 family are critically involved in the regulation of hepatocellular apoptosis and carcinogenesis we aimed to establish whether loss of BOK affects diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Short-term exposure to DEN lead to upregulation of BOK mRNA and protein in the liver. Of note, induction of CHOP and the pro-apoptotic BH3-only proteins PUMA and BIM by DEN was strongly reduced in the absence of BOK. Accordingly, Bok-/- mice were significantly protected from DEN-induced acute hepatocellular apoptosis and associated inflammation. As a consequence, Bok-/- animals were partially protected against chemical-induced hepatocarcinogenesis showing fewer and, surprisingly, also smaller tumors than WT controls. Gene expression profiling revealed that downregulation of BOK results in upregulation of genes involved in cell cycle arrest. Bok-/- hepatocellular carcinoma (HCC) displayed higher expression levels of the cyclin kinase inhibitors p19INK4d and p21cip1. Accordingly, hepatocellular carcinoma in Bok-/- animals, BOK-deficient human HCC cell lines, as well as non-transformed cells, showed significantly less proliferation than BOK-proficient controls. We conclude that BOK is induced by DEN, contributes to DEN-induced hepatocellular apoptosis and resulting hepatocarcinogenesis. In line with its previously reported predominant localization at the endoplasmic reticulum, our findings support a role of BOK that links the cell cycle and cell death machineries upstream of mitochondrial damage.

The B-cell lymphoma 2 (BCL-2)-related ovarian killer (BOK) shares sequence homology with the proapoptotic BCL-2 family members BAX and BAK. However, Bok −/− cells are not protected from classic apoptotic triggers and evidence for a proapoptotic role of BOK is derived mostly from overexpression studies (1). BOK localizes preferentially to the endoplasmic reticulum (ER) membrane, where it interacts with IP3-receptors (2, 3). Using cells from their newly generated Bok −/− mouse strain, Carpio et al. propose that BOK is a critical inducer of BAX/BAK-dependent apoptosis in response to ER stress (4). This proposal is in contrast to our earlier report, in which we showed that loss of BOK did not confer resistance toward ER stress in several cell types (2). Underlying reasons for this discrepancy may lie in the initial Sv129:C57BL/6 mixed genetic background of the strain used by Carpio et al. (4) [which may influence the phenotype despite backcrossing (5)] and their targeting strategy of the Bok locus. By targeting exons 2 (containing the START codon) and 3, alternative splicing of exon 1–4 is enabled and is indeed readily detectable based on the RT-PCR analysis in figure 1C of Carpio et al. (4). A resulting ∼1-kb transcript (exon1/4/5), which is not occurring naturally, contains several predicted ORFs and may influence the phenotype of these mice. In contrast, our Bok −/− strain was generated in a pure C57BL/6 genetic background, with no detectable transcript because of targeting of the exon 1 splice donor site along with exon 2 (1).

Neutrophils are essential players in the first-line defense against invading bacteria and fungi. Besides its antiapoptotic role, the inhibitor of apoptosis protein (IAP) family member X-linked IAP (XIAP) has been shown to regulate innate immune signaling. Whereas the role of XIAP in innate signaling pathways is derived mostly from work in macrophages and dendritic cells, it is not known if and how XIAP contributes to these pathways in neutrophils. Here we show that in response to bacterial lipopolysaccharides (LPS), mouse neutrophils secreted considerable amounts of tumor necrosis factor- α (TNF α ) and interleukin-1 β (IL-1 β ) and, in accordance with earlier reports, XIAP prevented LPS-induced hypersecretion of IL-1 β also in neutrophils. Interestingly, and in contrast to macrophages or dendritic cells, Xiap -deficient neutrophils were insensitive to LPS-induced cell death. However, combined loss of function of XIAP and cIAP1/-2 resulted in rapid neutrophil cell death in response to LPS. This cell death occurred by classical apoptosis initiated by a TNF α - and RIPK1-dependent, but RIPK3- and MLKL-independent, pathway. Inhibition of caspases under the same experimental conditions caused a shift to RIPK3-dependent cell death. Accordingly, we demonstrate that treatment of neutrophils with high concentrations of TNF α induced apoptotic cell death, which was fully blockable by pancaspase inhibition in wild-type neutrophils. However, in the absence of XIAP, caspase inhibition resulted in a shift from apoptosis to RIPK3- and MLKL-dependent necroptosis. Loss of XIAP further sensitized granulocyte–macrophage colony-stimulating factor (GM-CSF)-primed neutrophils to TNF α -induced killing. These data suggest that XIAP antagonizes the switch from TNF α -induced apoptosis to necroptosis in mouse neutrophils. Moreover, our data may implicate an important role of neutrophils in the development of hyperinflammation and disease progression of patients diagnosed with X-linked lymphoproliferative syndrome type 2, which are deficient in XIAP.

Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

Background Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods A method is described to massively expand bone marrowderived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.