Abstract 1558: Therapeutic targeting of cell surface plectin induces anti-cancer immune response and pancreatic cancer regression
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Background: Pancreatic ductal adenocarcinoma (PDA) is the 3rd deadliest cancer, diagnosed typically in advanced stages, with only an 8% 5-year survival rate, thus demonstrating the need for novel therapeutic approaches that significantly enhance chemo- and/or immune-therapy. Our team previously identified a promising functional target for cancer therapy in PDA, cell surface plectin 1 (CSP1) that is aberrantly expressed on PDA cells and thus a cell surface-associated biomarker of cancer. CSP1 expression first becomes apparent in high grade dysplasias, remaining high in early and advanced cancers and in metastases. Our first-in-human imaging trial in PDA patients using a CSP1-targeted imaging agent revealed that CSP is an available target and accessible for binding, a potentially a target for cancer therapy. We hypothesized that a monoclonal antibody (mAb) against CSP1 could lead to novel pancreatic cancer treatment options, thus, we developed a therapeutic mAb, e.g., ZB131, representing a first-in-class antibody selectively targeting CSP1.Methods: ZB131 is a humanized mAb targeted against human plectin 1 (rhSec8) that also binds murine CSP1. ZB131 affinity and its effect on cancer cells including proliferation, cytotoxicity, and migration were tested in vitro using saturation binding, SRB-based survival assays, flow cytometry, and migration assays on various pancreatic cell types and homeostatic "normal" controls. In vivo validation was performed using two nu/nu mouse models bearing subcutaneous MiaPACA2 or Yapc PDA cells, and a syngeneic KPC-derived tumor model to also evaluate immune responses to tumors treated with ZB131 or IgG control.Results: ZB131 exhibits high specificity and high affinity (0.4±0.1nM) to CSP1, and functionally induces G0 growth arrest followed by necrotic cell death of PDA cells in culture, and is synergistic with gemcitabine resulting in a 50-fold decrease in IC50. In vivo, in subcutaneous xenograft models, ZB131 monotherapy decreased PDA tumor volume 5-fold as compared to controls, and in combination with cisplatin resulted in sustained tumor reduction with greater than 85% tumor necrosis. In subcutaneous syngeneic PDA models, ZB131 induced complete tumor regression within 35 days mediated by an anti-tumor immune response as upon tumor rechallenge, full tumor regression was again achieved without additional ZB131 therapy. Leukocyte complexity analysis of regressing PDA tumors versus controls revealed an ~3-fold increase in effector and central memory T cells.Conclusion: CSP1 is a first in class anti-cancer target expressed on the cell surface of PDA, as well as other cancers including ovarian, esophageal and head neck. ZB131, an anti-CSP1 mAb, induces tumor cell intrinsic cell death, as well as a robust anti-tumor T cell response leading to complete tumor regression indicating the potential therapeutic efficacy of ZB131 in late-stage cancers.Citation Format: Julien Dimastromatteo, Amanda Poisonnier, Samantha Perez, Lisa Coussens, Kimberly Kelly. Therapeutic targeting of cell surface plectin induces anti-cancer immune response and pancreatic cancer regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1558.Keywords:
Plectin
Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin’s function in a living organism, we have disrupted its gene in mice. Plectin (−/−) mice died 2–3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (−/−) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (−/−) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation.
Plectin
Epidermolysis bullosa simplex
Hemidesmosome
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Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.
Plectin
Epidermolysis bullosa simplex
Desmin
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The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause skin blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.
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Plectin
Epidermolysis bullosa simplex
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Plectin, a high-molecular-mass cytolinker, is abundantly expressed in the central nervous system (CNS). Currently, a limited amount of data about plectin in the CNS prevents us from seeing the complete picture of how plectin affects the functioning of the CNS as a whole. Yet, by analogy to its role in other tissues, it is anticipated that, in the CNS, plectin also functions as the key cytoskeleton interlinking molecule. Thus, it is likely involved in signalling processes, thereby affecting numerous fundamental functions in the brain and spinal cord. Versatile direct and indirect interactions of plectin with cytoskeletal filaments and enzymes in the cells of the CNS in normal physiological and in pathologic conditions remain to be fully addressed. Several pathologies of the CNS related to plectin have been discovered in patients with plectinopathies. However, in view of plectin as an integrator of a cohesive mesh of cellular proteins, it is important that the role of plectin is also considered in other CNS pathologies. This review summarizes the current knowledge of plectin in the CNS, focusing on plectin isoforms that have been detected in the CNS, along with its expression profile and distribution alongside diverse cytoskeleton filaments in CNS cell types. Considering that the bidirectional communication between neurons and glial cells, especially astrocytes, is crucial for proper functioning of the CNS, we place particular emphasis on the known roles of plectin in neurons, and we propose possible roles of plectin in astrocytes.
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Plectin is a multi-faceted, 500 kDa-large protein, which due to its expression in different isoforms and distinct organs acts diversely as a cytoskeletal crosslinker and signaling scaffold. It functions as a mediator of keratinocyte mechanical stability in the skin, primarily through linking intermediate filaments to hemidesmosomes. Skin fragility may occur through the presence of mutations in the gene encoding for plectin, PLEC, or through the presence of autoantibodies against the molecule. Below, we review the cutaneous manifestations of plectinopathies as well as their systemic involvement in specific disease subtypes. We summarize the known roles of plectin in keratinocytes and fibroblasts and provide an outlook on future perspectives for plectin-associated skin disorders.
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Hemidesmosome
Epidermolysis bullosa simplex
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Epidermolysis bullosa simplex associated with late-onset muscular dystrophy (EBS-MD) is an autosomal recessive disorder resulting from mutations in the plectin gene. The majority of these mutations occur within the large exon 31 encoding the central rod domain and leave the production of a low-level rodless plectin splice variant unaffected. To investigate the function of the rod domain, we generated rodless plectin mice through conditional deletion of exon 31. Rodless plectin mice develop normally without signs of skin blistering or muscular dystrophy. Plectin localization and hemidesmosome organization are unaffected in rodless plectin mice. However, superresolution microscopy revealed a closer juxtaposition of the C-terminus of plectin to the integrin β4 subunit in rodless plectin keratinocytes. Wound healing occurred slightly faster in rodless plectin mice than in wild-type mice, and keratinocytes migration was increased in the absence of the rod domain. The faster migration of rodless plectin keratinocytes is not due to altered biochemical properties because, like full-length plectin, rodless plectin is a dimeric protein. Our data demonstrate that rodless plectin can functionally compensate for the loss of full-length plectin in mice. Thus the low expression level of plectin rather than the absence of the rod domain dictates the development of EBS-MD.
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Epidermolysis bullosa simplex
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Plectin is a cytoskeletal linker protein that has a dumbbell-like structure with a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS-MD), and EB simplex with pyloric atresia (EBS-PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full-length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS-MD and three EBS-PA patients. In EBS-PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS-MD, the expression of the N- and C-terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full-length plectin isoform with residual expression of the rodless plectin isoform leads to EBS-MD, and that complete loss or marked attenuation of full-length and rodless plectin expression underlies the more severe EBS-PA phenotype. These results also clearly account for the majority of EBS-MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS-PA PLEC1 mutations are generally outside exon 31.
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Epidermolysis bullosa simplex
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