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Cytokines dimerize/oligomerize surface receptors to activate signaling. While cytokine receptors preferentially bind only one member of the JAK family, ancestral cytokine receptors, such as Gp130, promiscuously recruit different JAKs to elicit their activities. Here, we have explored how the identity of JAKs in Gp130 signaling complexes can regulate functional outcomes. Using a synthetic biology approach, we show that Gp130 bound to different JAKs propagates distinct STAT activation profiles. While Gp130-JAK1 complexes activated both, STAT1 and STAT3 very potently, Gp130-JAK2 complexes exhibited a clear preference for STAT3 activation. Gp130-TYK2 complexes triggered overall weaker signaling but with diminished STAT specificity. The three JAKs competed for binding to Gp130 and led to differential activation of phospho-Tyr in the Gp130 intracellular domain. JAK1, JAK2 and to a lower extent TYK2 bound with comparable affinities to Gp130, and in response to IL-6 stimulation efficiently drove Gp130 dimerization. However, the three JAKs differentially affected Gp130 surface expression, identifying JAK-dependent receptor trafficking as a critical determinant of signaling plasticity. Our results provide new mechanistic insights into how differential functional coupling in Gp130-JAK complexes translates into unique signaling signatures that likely contribute to its large functional diversity.

Cytokines are soluble factors vital for mammalian physiology. Cytokines elicit highly pleiotropic activities, characterized by their ability to induce a wide spectrum of functional responses in a diverse range of cell subsets, which makes their study very challenging. Cytokines activate signalling via receptor dimerization/oligomerization, triggering activation of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. Given the strong crosstalk and shared usage of key components of cytokine signalling pathways, a long-standing question in the field pertains to how functional diversity is achieved by cytokines. Here, we discuss how biophysical - for example, ligand-receptor binding affinity and topology - and cellular - for example, receptor, JAK and STAT protein levels, endosomal compartment - parameters contribute to the modulation and diversification of cytokine responses. We review how these parameters ultimately converge into a common mechanism to fine-tune cytokine signalling that involves the control of the number of Tyr residues phosphorylated in the receptor intracellular domain upon cytokine stimulation. This results in different kinetics of STAT activation, and induction of specific gene expression programs, ensuring the generation of functional diversity by cytokines using a limited set of signalling intermediaries. We describe how these first principles of cytokine signalling have been exploited using protein engineering to design cytokine variants with more specific and less toxic responses for immunotherapy.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modelling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with Systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Summary Cytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4+ T helper 1 (Th-1) cells to address the molecular bases defining cytokine functional pleiotropy. We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses., Graphical Abstract, Highlights • CDK8 regulates IL-6-mediated STAT3 S727 phosphorylation in primary human T cells • CDK8 controls STAT3 activity by limiting its resident time at gene loci • CDK8 inhibition increases IL-6-mediated Th17 differentiation, How IL-6 elicits its immune pleiotropic activities is not fully understood. Martinez-Fabregas et al. show that CDK8 represses IL-6-mediated transcription by limiting STAT3 resident time at the gene loci. By regulating CDK8 expression levels, immune cells can adapt their responses to STAT3-activating cytokines.

Interleukin-10 (IL-10) is a dimeric cytokine with both immunosuppressive and immunostimulatory activities; however, IL-10-based therapies have shown only marginal clinical benefits. Here, we explored whether the stability of the IL-10-receptor complex contributes to the immunomodulatory potency of IL-10. We generated an IL-10 mutant with enhanced affinity for its IL-10Rβ receptor using yeast surface display. Compared to the wild-type cytokine, the affinity-enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signalling complexes and triggered greater STAT1 and STAT3 activation in human monocytes and CD8(+) T cells. These effects in turn led to more robust induction of IL-10-mediated gene expression programs at low ligand concentrations in both human cell subsets. IL-10-regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and in CD8(+) T cell exhaustion. At non-saturating doses, IL-10 did not induce key components of its gene expression program, which may explain its lack of efficacy in clinical settings. Our engineered IL-10 variant showed a more robust bioactivity profile than that of wild-type IL-10 at low doses in monocytes and CD8(+) T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with wild-type IL-10. Our study provides insights into how IL-10-receptor complex stability fine-tunes IL-10 biology and opens new opportunities to revitalise failed IL-10 therapies.

Cytokines are highly pleiotropic ligands that critically contribute to a balanced immune response. We have an incomplete understanding of how cytokines elicit their functional pleiotropy, which has limited their therapeutic use. Here, using Interleukin-6 (IL-6) as a model system, we have performed detailed phosphoproteomic and transcriptomic studies in human Th-1 cells to address the molecular bases defining cytokine functional pleiotropy. We have identified CDK8 as a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses. We found that CDK8 is a major regulator of the IL-6 phosphoproteome and interacts with STAT3 in the nucleus upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecules inhibitors, reduced the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 Ser727 phosphorylation. This, in turn, resulted in retention of tyrosine phosphorylated STAT3 in the nucleus, which increased the binding of STAT3 to target DNA sites in the genome with a concomitant increase in STAT3 mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity via modulation of its gene loci resident time, critically contributing to tuning STAT3 mediated responses.

Interleukin-10 is a dimeric cytokine with both immune-suppressive and immune-stimulatory activities. Despite its immuno-modulatory potential, IL-10-based therapies have shown only marginal benefits in the clinic. Here we have explored whether the stability of the IL-10-receptor complex contributes to IL-10 immuno-modulatory potency. For that, we have generated an IL-10 mutant with greatly enhanced affinity for its IL-10Rβ receptor via yeast surface display. The affinity enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signaling complexes than the wild-type cytokine and triggered more potent STAT1 and STAT3 activation in human monocytes and CD8 T cells. This in turn led to more robust induction of IL-10-mediated gene expression programs at a wide range of ligand concentrations in both human cell subsets. IL-10 regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and genes involved in CD8 T cell exhaustion. Interestingly, at non-saturating doses, IL-10 lost key components of its gene-expression program, which may explain its lack of efficacy in clinical settings. Remarkably, our engineered IL-10 variant exhibited a more robust bioactivity profile than IL-10 wt at all the doses tested in monocytes and CD8 T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with IL-10 wt. Our study provides unique insights into how IL-10-receptor complex stability fine-tunes IL-10 biology, and opens new opportunities to revitalize failed IL-10 therapies.

Summary Stable isotope labeling by amino acid-based high-resolution phosphoproteomics is a powerful technique that allows for direct comparison of cells stimulated under different experimental conditions. This feature makes it the ideal methodology to identify cytokine signaling networks. Here, we present an optimized protocol for the isolation and identification of phosphopeptides from IL-6-stimulated primary human Th-1 cells. For complete details on the use and execution of this protocol, please refer to Martinez-Fabregas et al. (2020)., Graphical abstract, Highlights • Phosphoproteomics protocol for studying cytokines signaling in immune cells • Tips for the isolation and expansion of primary human Th-1 cells • Detailed protocol for the efficient SILAC labelling of human Th-1 cells, Stable isotope labeling by amino acid-based high-resolution phosphoproteomics is a powerful technique that allows for direct comparison of cells stimulated under different experimental conditions. This feature makes it the ideal methodology to identify cytokine signaling networks. Here, we present an optimized protocol for the isolation and identification of phosphopeptides from IL-6-stimulated primary human Th-1 cells.

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

Cytokines activate downstream signaling networks via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered variants of IL-6 with different affinities to the gp130 receptor chain to investigate how cytokine receptor binding kinetics influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes, from minimal to full agonist, and induced biased signaling, with changes in receptor binding kinetics affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This, in turn, leads to a graded gene expression response and substantial differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

dear editor, Loricrin keratoderma (syn. Camisa syndrome, OMIM 604117) is a rare autosomal dominant genodermatosis characterized by palmoplantar keratoderma and ichthyosis.1, 2 It is caused by mutations in loricrin, a small basic protein synthesized in the upper granular layer, which becomes a major constituent of the cornified cell envelope.3 Seven distinct mutations in loricrin have been reported in 15 unrelated pedigrees to date.4, 5, 6 We report a multi‐generation family with prominent ichthyosis and palmoplantar involvement due to a novel mutation in loricrin. The proband was a 14‐year‐old boy who presented with generalized dryness and scaling affecting his trunk and all four limbs, previously thought to be ichthyosis vulgaris, which was reported from early childhood (Fig. 1a). Clinical examination revealed widespread, prominent ichthyosis and mild diffuse transgredient hyperkeratosis of palms and soles. There was no evidence of atopic dermatitis or keratosis pilaris. His mother and eight other family members were similarly affected (Fig. 1b). Subsequently, a further child was born with a collodion membrane followed by generalized ichthyosis. Figure 1 (a) Pedigree of family with loricrin keratoderma. Filled symbols represent affected individuals. Whole exome sequencing was performed on one affected family member. *marks individuals who were screened for loricrin mutation by Sanger sequencing. (b–d) ... Biopsies of affected skin were processed for light and electron microscopy by standard methods (upper back of the proband's mother) or for electron microscopy only (affected acral skin from the proband's grandfather).7 Following informed consent, genomic DNA was extracted from blood or saliva samples from 10 affected and unaffected family members (Fig. 1a). A whole exome sequencing approach was taken to analyse the proband's DNA (Methods S1; see Supporting Information). Light microscopy of skin from the proband's mother showed mild hyperkeratosis, a normal granular layer and no significant parakeratosis (Fig. 2a). Electron microscopy of (i) affected acral skin demonstrated mild intracellular oedema, abundant keratohyaline granules in upper layers, with desmosomes and keratin filaments appearing intact and of (ii) affected upper back skin, vacuolar changes and disruption of suprabasal keratinocytes (Fig. ​(Fig.3).3). Whole exome sequencing directed at relevant epidermal genes revealed a novel heterozygous duplication mutation in the loricrin gene in exon 2 (designated c.806dupG), with an insertion of a single base pair resulting in a frameshift leading to a delayed termination codon and elongation of the protein by 22 amino acids (Fig. 2d). The mutation was confirmed by Sanger sequencing (Methods S2; see Supporting Information) and was present in affected individuals but was not in unaffected family members (Figs 1a, ​a,2b,c).2b,c). This mutation is not on the dbSNP database or NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS/). Figure 2 (a) A biopsy from the proband's mother's skin showing mild hyperkeratosis, a normal granular layer and no significant parakeratosis (original magnification × 20). (b–d) Mutation analysis. (b) Normal loricrin sequence in exon 2, showing ... Figure 3 Semithin sections and ultrastructural images. In semithin sections (a) there was epidermal acanthosis and compact hyperkeratosis with a pattern of diffuse vacuolar change. These vacuoles (small arrows) were predominantly observed in the suprabasal keratinocyte ... Six different heterozygous insertion mutations in loricrin in 14 unrelated pedigrees have previously been reported and one heterozygous deletion in a further pedigree2, 4, 5, 6, 8, 9, 11, 12, 13 (see Supporting Information; Table S1). All six insertion mutations are single base‐pair insertions leading to delayed termination codons with the most frequent mutation 730insG being present in eight of the 14 published families.2, 4, 8, 9, 11, 12, 13 This region of the loricrin gene is thought to be a mutation hotspot because of the presence of six consecutive guanine nucleotides.12All single‐base‐pair insertion and deletion mutations lead to a frameshift and delayed termination, thus elongating the protein by 22 amino acids and changing the Gly‐Lys‐rich domain into an Arg‐Leu‐rich terminal domain,2 except for two pedigrees of Brazilian origin, where the new protein is 25 amino acids longer than wild‐type protein.5 The mechanism of action of these mutations is thought to relate to preferential localization of mutant loricrin in the nucleus due to the formation of nuclear localization sequences within the arginine‐rich mutant loricrin.14 It has been suggested that the abnormal nuclear protein may disrupt the apoptotic process in terminal differentiation of keratinocytes in mouse models, thus supporting the hypothesis that the phenotype of loricrin keratoderma is caused by the synthesis of mutant loricrin rather than by the lack of wild‐type loricrin.14, 15 No clear genotype‐phenotype associations for pedigrees with specific mutations have currently been identified.4, 5, 13 The phenotype of loricrin keratoderma is heterogeneous. The common clinical features in both the 15 previously reported pedigrees and our new pedigree are palmoplantar keratoderma, usually of a honeycomb pattern, and generalized ichthyosis. Other features, including knuckle pads, pseudoainhum/hyperconstricting bands with autoamputation of digits and collodion babies, are variably reported. Previously, Gedicke et al.13 have suggested that the term ‘mutilating keratoderma with ichthyosis’ is not entirely suitable, due to the variation in phenotypes previously reported. Similarly, given the prominence of generalized ichthyosis with lesser palmoplantar involvement in this pedigree, and the presence of generalized ichthyosis in all previously reported pedigrees, we suggest that the condition could be described as loricrin ichthyosis rather than loricrin keratoderma.

TO THE EDITOR Punctate palmoplantar keratoderma (punctate PPK or PPKP) is a rare autosomal dominant disorder of keratinization. Three variants of this disease have been described; PPKP1 (OMIM ♯148600, Buschke–Fischer–Brauer type) is characterized by the progressive development of discrete areas of hyperkeratosis on the palms and soles, followed by more extensive diffuse hyperkeratosis on the pressure-bearing areas of plantar skin. Linkage analyses of families affected by PPKP1 have previously identified a locus within 15q22–q24 (Martinez-Mir et al., 2003; Gao et al., 2005), but two Chinese pedigrees with a PPKP1 phenotype demonstrated linkage on chromosome 8q24.13–q24.21 (Zhang et al., 2004). Recently, nonsense mutations in AAGAB, the gene encoding alpha- and gamma-adaptin-binding protein p34, were reported in three PPKP1 families (Giehl et al., 2012). Simultaneously, we applied whole-exome sequencing and reported heterozygous loss-of-function mutations in AAGAB in 18 PPKP1 kindreds (Pohler et al., 2012). AAGAB is located on chromosome 15q22, within one of the previously reported linkage regions (Martinez-Mir et al., 2003; Pohler et al., 2012). We now report the AAGAB genotype in a further 12 PPKP1 patients from 6 independent kindreds of Scottish, English, and Mexican ancestry. This study was carried out in accordance with the Declaration of Helsinki Principles, and all subjects gave written informed consent.

The relative contribution of immunological dysregulation and impaired epithelial barrier function to allergic diseases is still a matter of debate. Here we describe a new syndrome featuring severe dermatitis, multiple allergies and metabolic wasting (SAM syndrome) caused by homozygous mutations in DSG1. DSG1 encodes desmoglein 1, a major constituent of desmosomes, which connect the cell surface to the keratin cytoskeleton and play a crucial role in maintaining epidermal integrity and barrier function. SAM syndrome-causing mutations resulted in lack of membrane expression of DSG1, leading to loss of cell-cell adhesion. In addition, DSG1 deficiency was associated with increased expression of a number of genes encoding allergy-related cytokines. The deciphering of the pathogenesis of SAM syndrome substantiates the notion that allergy may result from a primary structural epidermal defect.

To the Editor A key event during epidermal differentiation is the proteolytic breakdown of profilaggrin into “free” filaggrin monomers. A recent study has shown that the skin specific retroviral-like aspartic protease (SASPase) plays a key role in profilaggrin-filaggrin processing (Matsui et al., 2011). SASPase cleaves the linker peptide between the individual filaggrin monomers of profilaggrin and on a hairless mouse background, loss of SASPase leads to dry, scaly skin with reduced stratum corneum hydration accompanied by accumulations of profilaggrin-filaggrin intermediates but an absence of filaggrin monomers (Matsui et al., 2011). In this same study several missense mutations in the SASPase gene in atopic eczema patients and controls were identified, some of which were shown to have a detrimental effect on the ability of SASPase to cleave the profilaggrin linker peptide. Given the important role of filaggrin in skin barrier function and maintaining stratum corneum hydration (O’Regan and Irvine, 2010), these results prompted us to question whether aberrant profilaggrin-filaggrin processing due to altered SASPase activity could provide an alternative pathogenic mechanism for atopic eczema or clinically dry skin. To answer this question, the entire coding region of the SASPase gene, ASPRV1, was amplified by PCR in a single fragment and fully sequenced. To maximize our chances of finding mutations that might be associated with atopic eczema or clinically dry skin we sequenced ASPRV1 from three discovery cohorts; 96 paediatric atopic eczema cases from Ireland, 96 atopic eczema cases from the Cape Town region of South Africa (Xhosa people) and 99 cases of clinically dry skin from patients referred to dermatology clinics in Glasgow, Scotland. Atopic eczema in the Irish paediatric cases was diagnosed using the UK Diagnostic criteria (Williams et al., 1994); atopic eczema in the Xhosa people was diagnosed by experienced dermatologists. Clinically dry skin was defined using a previously published scoring system (Sergeant et al., 2009). Demographic and clinical data relating to the discovery cohorts are shown in Table 1. Table 1 Demographic and clinical data relating to eczema and dry skin cases and population controls Sequencing of the ASPRV1 gene in the discovery cohorts identified a total of 5 non-synonymous mutations and 2 synonymous mutations (Table 2). None of the ASPRV1 mutations identified in a previous Japanese study (Matsui et al., 2011) were detected in our discovery cohorts. We then investigated some of these mutations further by screening an additional 259 Irish atopic eczema cases and 167 Scottish dry skin cases (which included the original 93 cases from the discovery cohort) using custom-designed TaqMan® allelic discrimination assays for the mutations V74I, G87R and S333F (Supplementary Table 1). The G87R mutation was identified only in a single case of atopic eczema and the V74I and S333F mutations were not detected in any of these additional cases. In the 167 cases of dry skin, the G87R and S333F mutations were found only in single cases (ie. in the original discovery cohort) and the V74I mutation was not found in any of the cases. We then carried out two independent case-control studies to investigate any association between the T49A mutation and atopic eczema and clinically dry skin (Supplementary Table 2). 442 Irish atopic eczema cases (which included the original 92 cases from the discovery cohort) and 458 Irish population controls were screened using a TaqMan® allelic discrimination assay. There was no association between the T49A mutation and atopic eczema in the Irish study: chi-square p=0.415, odds ratio 0.98 (95% confidence interval 0.81–1.18). Similarly, screening of 167 clinically dry skin cases and 100 Scottish population controls failed to reveal any association between the T49A mutation and dry skin: p=0.479, odds ratio 0.90 (0.63–1.28). Power calculations showed that the eczema case-control study had >80% power to detect an odds ratio of 1.5 or above and the dry skin case-control study study had >70% power to detect an odds ratio of 2.0 (Quanto 1.2.4, University of Southern California, http://hydra.usc.edu/gxe/). Since FLG null mutations are known to have such a strong effect on eczema risk, it is possible that the effect of ASPRV1 mutations may only be apparent in FLG wild-type individuals. Therefore the four most prevalent FLG null mutations (R501X, 2282del4, R2447X and S3247X) were screened in each of the cases and controls using methods described previously (Kezic et al., 2011; Sandilands et al., 2007). The statistical analyses for each study were repeated after excluding individuals carrying FLG null mutations, but there was still no evidence of association between ASPRV1 mutation T49A and eczema or clinically dry skin (Supplementary Table 3). Table 2 dbSNP minor allele frequencies of ASPRV1 polymorphisms identified in the discovery cohorts With the exception of T49A and to a lesser extent L325L, the remaining ASPRV1 mutations that we identified were rare (

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics(1-3). Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding alpha- and gamma-adaptin binding protein p34, located at a previously linked locus at 15q22. alpha- and gamma-adaptin binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53-mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium. Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.

Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.

Introduction: CYC140 is a selective and potent ATP-competitive inhibitor of PLK1, which has completed IND-enabling studies. The aim of this translational project was to inform the clinical development path of CYC140. Esophageal cancer was investigated as a potential target indication based on unmet medical need and the observation that PLK1 is frequently overexpressed in esophageal tumors and carries a poor prognosis. Experimental procedures: The anticancer activity of CYC140 was examined across multiple tumor types to identify sensitive target indications or tumor subsets. 6 h pulse exposure to CYC140 was used to determine sensitivity in a panel of over 250 cancer cell lines, including 15 esophageal cancer cell lines, using CellTiter Glo and resazurin-based assays. Candidate pharmacodynamic markers were examined in malignant and non-malignant cells. Drug combination testing was undertaken in several esophageal cell lines using approved and targeted agents. Solid tumor and leukemia xenograft models were performed to assess CYC140 dosing schedules and efficacy. Summary: Inhibition of PLK1 by CYC140 perturbs the entry into and exit from mitosis. In malignant cells, CYC140 treatment leads to the appearance of mitotic cells with monopolar spindles and a persistent increase in the proportion of cells in G2 and M phase, resulting in complete growth inhibition and induction of cell death. In non-malignant cells, the growth arrest is transient, and cells resume cycling once compound is removed. Short (6 h) pulse treatments of CYC140 maximise the difference in cellular response between malignant esophageal cell lines and cells of a non-malignant origin. In the esophageal cell line panel, CYC140 cellular IC50 correlates with induction of apoptosis. The effect of CYC140 on pharmacodynamic markers of PLK-1 activity such as phospho-nucleophosmin and phospho-histone H3 was characterized in malignant and non-malignant cells. Several promising combinations of CYC140 with targeted agents were identified, including EGFR inhibitors, HDAC inhibitors and PI3K pathway inhibitors, and CYC140 can also be combined with cytotoxic agents approved for use in esophageal cancer, such as cisplatin or irinotecan. CYC140 anti-tumor efficacy was demonstrated in solid tumor and leukemic xenograft models with responses including tumor regression and tumor-free cures. Conclusions: CYC140 is a promising anti-cancer agent with potent anti-proliferative activity and therapeutic potential in a variety of cancers, including esophageal cancer and acute leukemia. The mode of action of CYC140 is consistent with PLK1 inhibition and cell death is preferentially triggered in sensitive malignant cells. Suitable pharmacodynamic markers and several promising combinations have been identified that could assist clinical development of CYC140. Citation Format: Sylvie Moureau, Craig MacKay, Chiara Saladino, Elizabeth Pohler, Karin Kroboth, Jonathan Hollick, Daniella Zheleva, Sheelagh Frame, David Blake. The novel PLK1 inhibitor, CYC140: Identification of pharmacodynamic markers, sensitive target indications and potential combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4178. doi:10.1158/1538-7445.AM2017-4178

Introduction: Double-hit lymphomas (DHL), defined by concurrent MYC and BCL2 rearrangements, have poor prognosis compared to standard-risk diffuse large B cell lymphomas (DLBCL). Current treatment regimens involve multiple chemotherapies, but do not specifically exploit these molecular features of the disease. Several studies have established that: (i) DLBCL show frequent overexpression of Mcl-1, an anti-apoptotic member of the Bcl-2 family (30% and 50% in the ABC and GCB subtypes, respectively), (ii) MYC-driven lymphomas are highly sensitive to depletion of Mcl-1, (iii) MYC overexpression and inhibition of CDK activity are synthetically lethal and (iv) resistance to BH3 mimetics targeting Bcl-2 can be conferred by upregulation of Mcl-1. CYC065 is a specific and potent CDK2/9 inhibitor, currently in a Phase 1 trial in patients with advanced cancer (NCT02552953). The mechanism of action of CYC065 involves rapid reduction of the levels of both Mcl-1 and MYC, suggesting a therapeutic rationale for investigating this agent in DLBCL. This preclinical study explored the single agent activity of CYC065 in B cell lymphoma, and its potential to combine with other molecularly targeted agents of interest, including venetoclax (ABT-199, a Bcl-2 inhibitor), and BET inhibitors. Methods: Single agent activity of CYC065 was explored using short pulse treatments (6-8 h) in B cell lymphoma cell lines. Viability and total cell number were assessed 24, 48 or 72 h following treatment. Levels of MYC, Bcl-2 and Mcl-1 were determined by Western blotting at baseline and following treatment with CYC065. Cell fate was examined by flow cytometry. CYC065 was combined with venetoclax or BET inhibitors ((+)-JQ-1, GSK525762 and OTX-015), which were administered concomitantly for a 6 h pulse or up to 72 h. Combination data were analyzed by the Chou & Talalay method. Results: The median IC50 for CYC065 in 13 B cell lymphoma cell lines was 0.43 μM. No obvious difference was observed between ABC and GCB subtypes of DLBCL, and DHL DLBCL lines had similar IC50 values to non-DHL DLBCL lines (median 0.47 μM vs 0.29 μM; p = 0.95). As expected from the target inhibitory profile, CYC065 caused a rapid decrease in the phosphorylation of S2 of the CTD of RNA polymerase II followed by downregulation of Mcl-1 and MYC, and rapid induction of apoptosis in sensitive cell lines. CYC065 had no impact on Bcl-2 levels. Combining CYC065 with venetoclax was highly synergistic in DHL lines (median CI values range from 0.1-0.8), and resulted in >90% cell death. The combination of CYC065 and BET inhibitors was also highly synergistic. Conclusions: CYC065 targets key oncogenic and survival pathways in DLBCL. CYC065 is a potent and effective inducer of cell death and combines synergistically with Bcl-2 or BET inhibitors in B cell lymphoma cell lines, including DHLs, which represent an unmet clinical need. Citation Format: Sheelagh M. Frame, Elizabeth Pohler, Craig MacKay, Daniella Zheleva, David Blake. CYC065, a novel CDK2/9 inhibitor, is an effective inducer of cell death and synergizes with BCL2 and BET inhibitors in B-cell lymphoma, including double-hit lymphomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1309.

Loss-of-function variants within the filaggrin gene (FLG) increase the risk of atopic dermatitis. FLG also demonstrates intragenic copy number variation (CNV), with alleles encoding 10, 11, or 12 filaggrin monomers; hence, CNV may affect the amount of filaggrin expressed in the epidermis. A total of 876 Irish pediatric atopic dermatitis cases were compared with 928 population controls to test the hypothesis that CNV within FLG affects the risk of atopic dermatitis independently of FLG-null mutations. Cases and controls were screened for CNV and common FLG-null mutations. In this population the 11-repeat allele was most prevalent (allele frequency 51.5%); the 10-repeat allele frequency was 33.9% and the 12-repeat allele frequency was 14.6%. Having excluded FLG mutation carriers, the control group had a significantly higher number of repeats than cases (chi(2) P = 0.043), and the odds ratio of disease was reduced by a factor of 0.88 (95% confidence interval 0.78-0.98, P-0.025) for each additional unit of copy number. Breakdown products of filaggrin were quantified in tape-stripped stratum corneum from 31 atopic dermatitis patients and urocanic acid showed a positive correlation with total copy number. CNV within FLG makes a significant, dose-dependent contribution to atopic dermatitis risk, and therefore treatments to increase filaggrin expression may have therapeutic utility

CYC065 is a novel CDK2/9 inhibitor. Following completion of IND-enabling studies CYC065 has been cleared by the FDA for first-in-human clinical trials. This study aims to establish the mechanistic rationale and dosing schedule for targeting tumors that are either dependent on sustained expression of certain CDK9 transcriptional targets including Mcl-1 and MYC, or on activation of CDK2, e.g. by overexpression of cyclin E. CYC065 is efficacious in ALL and AML preclinical models, including MLL rearranged (MLLr) leukemia1. Mcl-1 is critical for survival of AML2, and MLLr AML are dependent on CDK9-driven expression of MLL target genes3. CYC065 is effective in cyclin E-overexpressing tumor models, such as uterine serous carcinoma4 and trastuzumab-resistant Her2+ breast cancer5. Pharmacologic CDK inhibition or CDK knockdown is synthetically lethal with overexpressed MYC in triple negative breast cancer (TNBC)6. A common feature of the basal-like subtype of TNBC is amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor. The efficacy and mechanism of action of CYC065 was analyzed in AML and basal-like TNBC cell panels by measuring the effects on cell proliferation, levels of key CDK9-dependent proteins, cell cycle distribution and apoptosis induction. We show that submicromolar CYC065 inhibits CDK9-dependent RNA Polymerase II phosphorylation, resulting in downregulation of target proteins and rapid induction of apoptosis. Sensitive tumor subtypes undergo rapid apoptosis after short pulse treatments at doses and durations predicted to be clinically achievable and well tolerated. However, significant apoptosis induction was not observed in non-malignant cells despite showing similar upstream effects. The data predicts a therapeutic window between cancer and normal proliferating cells and indicates the potential for stratification markers to enrich for sensitive patients. Combinability of anti-cancer agents is an important consideration for clinical development. In preclinical models Cyclacel's first generation CDK inhibitor seliciclib combines effectively with the novel nucleoside analogue sapacitabine (or its active metabolite CNDAC). This combination is currently being evaluated in a phase 1 clinical trial (NCT00999401)7. We show that CYC065 combines effectively with CNDAC in a TNBC panel. Increased expression and activity of other anti-apoptotic Bcl-2-family members can counteract the pro-apoptotic effects of Mcl-1 downregulation by CYC065. This can be overcome by combined treatment with CYC065 and Bcl-2 inhibitors such as ABT-199, resulting in a highly synergistic combination. CYC065 has the potential to be efficacious in cancers that require sustained expression of CDK9-dependent transcripts or activation of CDK2. These include Mcl-1-dependent and MLLr leukemia, MYC-driven lymphoma, and MYC- or cyclin E-dependent breast or gynecological cancers. CYC065 can act synergistically with selected DNA damaging or targeted agents. Together this data suggests that CYC065 may be a promising novel anti-cancer agent. 1) Saladino et al. AACR 2015, Abs 1650 2) Glaser et al. Genes Dev. 2012, 26, 120-5 3) Dou & Hess. Int J Haematol. 2008, 87, 10-8 4) Cocco et al. AACR, 2015, Abs 3103 5) Scaltriti et al. Proc Natl Acad Sci USA. 201, 108, 3761-6 6) Horiuchi et al. J Exp Med. 2012, 209, 679-96 7) Shapiro et al. AACR, 2013, Abs LB-202 Citation Format: Craig MacKay, Sheelagh Frame, Chiara Saladino, Elizabeth Pohler, Daniella Zheleva, David G. Blake. Molecular basis for clinical development of the novel CDK2/9 inhibitor CYC065 in oncology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B182.

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53-mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium. Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.

In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human ρ0 teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.

The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser(15) and Ser(392) and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser(15) in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser(15) kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.

Objective Oesophageal cancer associated with the premalignant condition Barrett's oesophagus has increased in incidence over the last few years. Phase II detoxifying enzymes, including glutathione S-transferases (GSTs) protect the mucosa from carcinogens, which can cause oxidative damage to cells. Therefore, a reduction in these anti-oxidant enzymes can increase the risk of carcinogenesis. The aim of this study was to compare the extent of GST expression in normal oesophageal tissue, Barrett's oesophagus and oesophageal adenocarcinoma. Design Antibodies raised against GST alpha, GST mu, GST pi and microsomal GST were used to identify expression of these proteins in tissue sections. Method Paraffin-embedded sections were stained using standard immunohistochemical techniques to demonstrate the pattern of expression of GST proteins in biopsy specimens. Twelve sections of Barrett's metaplasia and an equal number of specimens from normal oesophageal tissue were examined, together with sections from adenocarcinoma and normal gastric mucosa. Results Expression of the GST enzymes appeared to be reduced in Barrett's tissue compared to normal oesophageal tissue. Nuclear staining featured in some of the normal tissue sections, but not in Barrett's tissue. Conclusion The reduction in GST expression suggested in Barrett's tissue is an interesting finding, as it is possible that reduced expression of these detoxifying enzymes may contribute to the risk of development of adenocarcinoma in Barrett's mucosa.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8 + T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8 + T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.