Abstract
BackgroundSeveral small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy.MethodsIB3–1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[wildtype]CFTR-treated IB3–1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients.ResultsCF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures.ConclusionsAdjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.
Highlights
Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic disease due to mutations in the CFTR gene [1,2,3]
Experimental design In this study, we treated IB3–1 Cystic Fibrosis (CF) cells with various combinations of digitoxin and Vertex drugs (VX-661, VX-770, and VX-809) and interrogated IκBα/NFκB signaling, chemokine and cytokine secretion, and transcriptional responses (Fig. 1a)
Digitoxin dose-dependently suppresses secretion of GM-CSF, IL-6 and IL-8 under baseline and TNFα-activated conditions (Fig. 2c-e). These data are consistent with prior data showing dose-dependent suppressive effects of digitoxin on secretion of IL-8 from CF cells when incubated under baseline conditions only [33]
Summary
Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic disease due to mutations in the CFTR gene [1,2,3]. The common CF mutation [F508del] [4, 5], and the less common [G551D] mutation [6,7,8,9], are both associated with a hyper-proinflammatory phenotype that is due to constitutive activation of the TNFα/NFκB signaling pathway [10,11,12,13,14]. A licensed combination of the corrector drug VX-661 with VX-770 has been shown to be safer and possibly more effective in CF patients homozygous [17] and heterozygous [18] for the [F508del] CFTR mutation. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy
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