Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility.

The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.5786delC (p.Pro1929Leufs(∗)8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected person's skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and ß4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins.

Kindlin-1 Regulates Epidermal Growth Factor Receptor Signaling.

Kindler syndrome is an autosomal recessive genodermatosis that results from mutations in the FERMT1 gene encoding t kindlin-1. Kindlin-1 localizes to focal adhesion and is known to contribute to the activation of integrin receptors. Most cases of Kindler syndrome show a reduction or complete absence of kindlin-1 in keratinocytes, resulting in defective integrin activation, cell adhesion, and migration. However, roles for kindlin-1 beyond integrin activation remain poorly defined. In this study we show that skin and keratinocytes from Kindler syndrome patients have significantly reduced expression levels of the EGFR, resulting in defective EGF-dependent signaling and cell migration. Mechanistically, we show that kindlin-1 can associate directly with EGFR in vitro and in keratinocytes in an EGF-dependent, integrin-independent manner and that formation of this complex is required for EGF-dependent migration. We further show that kindlin-1 acts to protect EGFR from lysosomal-mediated degradation. This shows a new role for kindlin-1 that has implications for understanding Kindler syndrome disease pathology.

Drug-Repositioning Screens Identify Triamterene as a Selective Drug for the Treatment of DNA Mismatch Repair Deficient Cells.

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial.Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivoResults: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks.Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880-90. ©2016 AACR.

Targeting Mismatch Repair defects: A novel strategy for personalized cancer treatment.

The DNA Mismatch Repair (MMR) pathway is a fundamental cellular process required to repair mispaired bases introduced routinely during DNA replication. Given this critical role in the maintenance of genome stability, it is not surprising that underlying defects in the MMR pathway occur in both hereditary and sporadic cancers. Furthermore, the MMR status greatly influences the sensitivity of cells to many common chemotherapeutic agents. Therefore, novel strategies are being investigated to exploit the loss of MMR in these cancers and to identify personalized therapeutic strategies to target MMR deficient tumours. In this review, we describe recent advances in strategies to target MMR deficient tumours using a synthetic lethal approach. We discuss new ways to target mutations secondary to MMR deficiency and suggest potential new therapies to optimise treatment outcome. We highlight ongoing clinical studies focussing on novel ways of preventing and treating MMR deficient cancers.

Kindlin-1 Regulates Keratinocyte Electrotaxis.

Kindler syndrome (KS) is an autosomal recessive blistering skin disease resulting from pathogenic mutations in FERMT1. This gene encodes kindlin-1, a focal adhesion protein involved in activation of the integrin family of extracellular matrix receptors. Most cases of KS show a marked reduction or complete absence of the kindlin-1 protein in keratinocytes, resulting in defective cell adhesion and migration. Electric fields also act as intrinsic regulators of adhesion and migration in the skin, but the molecular mechanisms by which this occurs are poorly understood. Here we show that keratinocytes derived from KS patients are unable to undergo electrotaxis, and this defect is restored by overexpression of wild-type kindlin-1 but not a W612A mutation that prevents kindlin-integrin binding. Moreover, deletion of the pleckstrin homology domain of kindlin-1 also failed to rescue electrotaxis in KS cells, indicating that both integrin and lipid binding are required for this function. Kindlin-1 was also required for the maintenance of lamellipodial protrusions during electrotaxis via electric field-activated ß1 integrin. Indeed, inhibition of ß1 integrins also leads to loss of electrotaxis in keratinocytes. Our data suggest that loss of kindlin-1 function may therefore result in epithelial insensitivity to electric fields and contribute to KS disease pathology.

BPAG1-e restricts keratinocyte migration through control of adhesion stability.

Bullous pemphigoid antigen 1 (BPAG1-e, also known as BP230) is a member of the plakin family of hemidesmosome cytoskeletal linker proteins that is encoded by an isoform of the dystonin (DST) gene. Recently, we reported two unrelated families with homozygous nonsense mutations in this DST isoform that led to ultrastructural loss of hemidesmosomal inner plaques and clinical features of trauma-induced skin fragility. We now demonstrate that keratinocytes isolated from these individuals have significant defects in adhesion, as well as increased cell spreading and migration. These mutant keratinocytes also display reduced levels of ß4 integrins at the cell surface but increased total protein levels of keratin-14 and ß1 integrins. These alterations in cell behavior and protein expression were not seen in control keratinocytes in which BPAG1-e expression had been silenced by stable expression of short hairpin RNA to target DST. The failure of knockdown approaches to recapitulate the changes in morphology, adhesion, and migration seen in patient cells therefore suggests such approaches are not appropriate to study loss of this protein in vivo. The contrasting findings in keratinocytes harboring naturally occurring mutations, however, demonstrate a previously unappreciated key role for BPAG1-e in regulating keratinocyte adhesion and migration and suggest a requirement for this protein in controlling functional switching between integrin types in epithelial cells.