Elizabeth Pohler

Desmoglein 1 deficiency results in severe dermatitis, multiple allergies and metabolic wasting

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 have a crucial role in maintaining epidermal integrity and barrier function. Mutations causing SAM syndrome 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. Our deciphering of the pathogenesis of SAM syndrome substantiates the notion that allergy may result from a primary structural epidermal defect.

Intragenic Copy Number Variation within Filaggrin Contributes to the Risk of Atopic Dermatitis with a Dose-Dependent Effect

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 (χ2 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.

The Barrett’s Antigen Anterior Gradient-2 Silences the p53 Transcriptional Response to DNA Damage

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 Ser15 and Ser392 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 Ser15 in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser15 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.

Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. 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 α- and γ-adaptin–binding protein p34, located at a previously linked locus at 15q22. α- and γ-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.

Alteration of glutathione S-transferase levels in Barrett's metaplasia compared to normal oesophageal epithelium

Objective Oesophageal cancer associated with the premalignant condition Barretts 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, Barretts 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 Barretts 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 Barretts tissue compared to normal oesophageal tissue. Nuclear staining featured in some of the normal tissue sections, but not in Barretts tissue. Conclusion The reduction in GST expression suggested in Barretts 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 Barretts mucosa.

Heterozygous Mutations in AAGAB Cause Type 1 Punctate Palmoplantar Keratoderma with Evidence for Increased Growth Factor Signaling

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.

Punctate palmoplantar keratoderma type 1: a novel AAGAB mutation and efficacy of etretinate.

Punctate palmoplantar keratoderma type 1 (PPKP1, OMIM#148600), also known as the Buschke-FischerBraurer type, is a rare form of palmoplantar keratoderma that is autosomal dominantly inherited (1). PPKP1 is clinically characterised by multiple punctate hyperkeratotic papules affecting the palmar and plantar skin, with considerable phenotypic variation among patients (2). These circumscribed papules gradually coalesce and increase in number with age (2). The lesions typically start to appear in early adolescence but sometimes develop later in life. In 2012, linkage analysis and whole-exome sequencing identified heterozygous null mutations within AAGAB as a cause of PPKP1 (2, 3). AAGAB encodes αand γ-adaptin binding protein p34, which is involved in clathrin-mediated vesicle transport (2). Loss-of-function mutations in AAGAB result in haploinsufficiency of p34 (2). To date, 20 AAGAB null variants have been identified in Scottish, Irish, English, German, Tunisian, Chinese Mexican and Japanese populations (2–8). Here we report a Japanese case with PPKP1 carrying a novel AAGAB null mutation.

Mutations in the SASPase Gene (ASPRV1) Are Not Associated with Atopic Eczema or Clinically Dry Skin

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 (<1%) and therefore unlikely to be significant on a population level, although it is still possible that these rare mutations could contribute significantly to individual disease risk. Mutations P206P and L325L result in synonymous changes and are therefore unlikely to be pathogenic. All of the non-synonymous mutations we identified (Supplementary Figure 1) affect amino acid residues outside the active protease site of SASPase (Bernard et al., 2005), however the effect of these mutations on SASPase activity remains to be determined experimentally. Finally, we used custom-designed TaqMan® allelic discrimination assays (Supplementary Table 1) to screen for the V187I and V243A mutations which reduce and abolish SASPase-mediated profilaggrin cleavage respectively (Matsui et al., 2011). However we failed to detect these mutations in any of the discovery cohorts, nor in the Irish atopic eczema cohort, indicating that these mutations are likely to be specific to the Japanese population. Although our results failed to find an association between ASPRV1 gene mutations and atopic eczema or clinically dry skin in the European populations that we studied, they do not exclude the possibility that an association exists in other ethnicities. In the populations that we studied, other factors which modulate SASPase activity could contribute instead, such as the actions of protease inhibitors which provide a powerful counterbalance against excessive protease activities (Hewett et al., 2005). Profilaggrin-filaggrin processing is a tightly regulated process involving not just SASPase but multiple proteases such as elastase 2 (Bonnart et al. 2010) and the serine proteases matriptase/MT-SP1 (List et al., 2003) and prostasin (Leyvraz et al., 2005). A greater understanding of the proteases and inhibitors involved in profilaggrin-filaggrin processing will be required to fully appreciate their contribution to skin barrier dysfunction.

Abstract B182: Molecular basis for clinical development of the novel CDK2/9 inhibitor CYC065 in oncology

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 Cyclacel9s 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.

Abstract 4178: The novel PLK1 inhibitor, CYC140: Identification of pharmacodynamic markers, sensitive target indications and potential combinations

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