Transcriptional Dynamics of NRF2 Overexpression and KEAP1-NRF2 Inhibitors in Human Cell Line and Primary Lung Cells.

Oxidative stress in the human lung is caused by both internal (e.g., inflammation) and external stressors (smoking, pollution, and infection) to drive pathology in a number of lung diseases. Cellular damage caused by oxidative damage is reversed by several pathways, one of which is the antioxidant response. This response is regulated by the transcriptional factor NRF2, which has the ability to regulate the transcription of more than 250 genes. In disease, this balance is overwhelmed, and the cells are unable to return to homeostasis. Several pharmacological approaches aim to improve the antioxidant capacity by inhibiting the interaction of NRF2 with its key cytosolic inhibitor, KEAP1. Here, we evaluate an alternative approach by overexpressing NRF2 from chemically modified RNAs (cmRNAs). Our results demonstrate successful expression of functional NRF2 protein in human cell lines and primary cells. We establish a kinetic transcriptomic profile to compare antioxidant response gene expression after treatment of primary human bronchial epithelial cells with either KEAP1 inhibitors or cmRNAs. The key gene signature is then applied to primary human lung fibroblasts and alveolar macrophages to uncover transcriptional preferences in each cell system. This study provides a foundation for the understanding of NRF2 dynamics in the human lung and provides initial evidence of alternative ways for pharmacological interference.

Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy.

Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.

Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA.

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models.

Vacuolar-ATPase Inhibition Blocks Iron Metabolism to Mediate Therapeutic Effects in Breast Cancer.

Generalized strategies to improve breast cancer treatment remain of interest to develop. In this study, we offer preclinical evidence of an important metabolic mechanism underlying the antitumor activity of inhibitors of the vacuolar-type ATPase (V-ATPase), a heteromultimeric proton pump. Specifically, our investigations in the 4T1 model of metastatic breast cancer of the V-ATPase inhibitor archazolid suggested that its ability to trigger metabolic stress and apoptosis associated with tumor growth inhibition related to an interference with hypoxia-inducible factor-1α signaling pathways and iron metabolism. As a consequence of disturbed iron metabolism, archazolid caused S-phase arrest, double-stranded DNA breaks, and p53 stabilization, leading to apoptosis. Our findings link V-ATPase to cell-cycle progression and DNA synthesis in cancer cells, and highlight the basis for the clinical exploration of V-ATPase as a potentially generalizable therapy for breast cancer.