Glutamine metabolism and vesicant pathogenesis

The National Institutes of Health Office of the Director awarded The Johns Hopkins University $247,337 on August 18, 2026, under the Trans-NIH Research Support program (CFDA 93.310) to investigate glutamine metabolism and vesicant pathogenesis in response to NOFO-PA-25-093.

The project will characterize the metabolic consequences of nitrogen mustard and phenylarsine oxide exposure in keratinocytes using in vivo mouse models of skin exposure, performing untargeted imaging mass spectrometry for spatial metabolomics and single-cell RNA sequencing to elucidate shared transcriptional changes in metabolic pathways. The work will assess how vesicants disrupt glutamine metabolism, impairing de novo nucleotide synthesis and glutathione production pathways and thereby increasing oxidative stress and cytotoxicity. A second aim will elucidate the protective roles of glutamine metabolism in DNA repair and glutathione-mediated defense mechanisms upon vesicant exposure, using pharmacological inhibitors of glutamine metabolism and evaluating the therapeutic potential of glutamine supplementation in murine models.

Performance of work occurs in Baltimore, Maryland. The award carries a period of performance through July 31, 2029. This is a project grant, a form of assistance funding under the Trans-NIH Research Support program, which covers multiple offices in the NIH Office of the Director and evaluates applications for scientific and technical merit by peer review and program relevance by national advisory council.

Abstract

Chemical warfare vesicants, such as nitrogen mustard (NM) and phenylarsine oxide (PAO), pose significant threats to human health through their capacity to induce oxidative stress and DNA damage, leading to substantial cytotoxicity and tissue injury. However, the consequences of vesicants on cellular metabolism that lead to pathogenesis remain insufficiently explored. In response to nofo-PA-25-093, this proposal will investigate the shared metabolic consequences of NM and PAO exposure, aiming to identify novel therapeutic strategies against chemical warfare vesicants. Glutamine is the most abundant amino acid in the body, playing vital roles in supporting cellular metabolism, redox balance, and signaling, especially in keratinocytes, the principal cells of the epidermis. Our central hypothesis posits that vesicants disrupt glutamine metabolism in keratinocytes, impairing DE novo nucleotide synthesis and glutathione production pathways, thereby leading to augmented oxidative stress and cytotoxicity. In aim 1, we will characterize the metabolic effects of NM and PAO on keratinocytes using in vivo mouse models of skin exposure, performing untargeted imaging mass spectrometry for spatial metabolomics alongside single-cell RNA sequencing (SCRNASEQ) to elucidate shared transcriptional changes in metabolic pathways at single-cell resolution. This data will provide insights into the metabolic shifts triggered by NM and PAO, focusing on alterations in glutamine-related metabolites and associated pathways. Aim 2 will elucidate the protective roles of glutamine metabolism in DNA repair and glutathione-mediated defense mechanisms upon vesicant exposure. We will utilize pharmacological inhibitors of glutamine metabolism to assess how suppression impacts DNA repair and oxidative stress responses in murine models. We will also evaluate the therapeutic potential of glutamine supplementation and mechanistic target of rapamycin (MTOR) activation, positing that enhanced glutamine metabolism may alleviate vesicant-induced pathogenesis. In aim 3, to confirm the relevance of our findings in human contexts, we will investigate the effects of vesicants on glutamine metabolism in human organotypic skin cultures through metabolomics and RNA sequencing, examining how NM and PAO exposure alters glutamine and its downstream metabolites while assessing their roles in cellular injury and repair processes. This research will significantly advance our understanding of how chemical warfare vesicants disrupt metabolic pathways in keratinocytes and elucidate the protective role of glutamine during vesicant exposure. By targeting glutamine metabolism as a novel countermeasure strategy, we may provide a straightforward and effective intervention for mitigating the effects of chemical warfare agents. Ultimately, the findings from this work are anticipated to have broad implications for the treatment of chemical agent-induced injuries and could pave the way for targeted therapies that enhance keratinocyte survival and function under stress conditions.