An enhanced Eco1 retron editor enables precision genome engineering in human cells from a single-copy integrated lentivirus.

Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast; however, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity as major contributors to poor retron editor efficiency. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to liberate the sgRNA and ncRNA. These modifications yielded a ncRNA with 5'- and 3'-end protection and an sgRNA with minimal 5' extension. This strategy increased steady-state ncRNA levels and rescued Cas9 activity leading to enhanced efficiency of the Eco1 retron editor in human cells. The enhanced Eco1 retron editor enabled the insertion of missense mutations in human cells from a single integrated lentivirus, thereby ensuring genotype-phenotype linkage over multiple cell divisions. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells. Here we present an enhanced Eco1 retron editor that enables efficient introduction of missense mutations in human cells from a single heritable genome copy.

Unraveling cysteine deficiency-associated rapid weight loss.

Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.

Cellular adaptation to cancer therapy along a resistance continuum.

Advancements in precision oncology over the past decades have led to new therapeutic interventions, but the efficacy of such treatments is generally limited by an adaptive process that fosters drug resistance1. In addition to genetic mutations2, recent research has identified a role for non-genetic plasticity in transient drug tolerance3 and the acquisition of stable resistance4,5. However, the dynamics of cell-state transitions that occur in the adaptation to cancer therapies remain unknown and require a systems-level longitudinal framework. Here we demonstrate that resistance develops through trajectories of cell-state transitions accompanied by a progressive increase in cell fitness, which we denote as the 'resistance continuum'. This cellular adaptation involves a stepwise assembly of gene expression programmes and epigenetically reinforced cell states underpinned by phenotypic plasticity, adaptation to stress and metabolic reprogramming. Our results support the notion that epithelial-to-mesenchymal transition or stemness programmes-often considered a proxy for phenotypic plasticity-enable adaptation, rather than a full resistance mechanism. Through systematic genetic perturbations, we identify the acquisition of metabolic dependencies, exposing vulnerabilities that can potentially be exploited therapeutically. The concept of the resistance continuum highlights the dynamic nature of cellular adaptation and calls for complementary therapies directed at the mechanisms underlying adaptive cell-state transitions.

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases Tregs, and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

The pleiotropic functions of reactive oxygen species in cancer.

Cellular redox homeostasis is an essential, dynamic process that ensures the balance between reducing and oxidizing reactions within cells and thus has implications across all areas of biology. Changes in levels of reactive oxygen species can disrupt redox homeostasis, leading to oxidative or reductive stress that contributes to the pathogenesis of many malignancies, including cancer. From transformation and tumor initiation to metastatic dissemination, increasing reactive oxygen species in cancer cells can paradoxically promote or suppress the tumorigenic process, depending on the extent of redox stress, its spatiotemporal characteristics and the tumor microenvironment. Here we review how redox regulation influences tumorigenesis, highlighting therapeutic opportunities enabled by redox-related alterations in cancer cells.

Metabolic reprogramming by histone deacetylase inhibition preferentially targets NRF2-activated tumors.

The interplay between metabolism and chromatin signaling is implicated in cancer progression. However, whether and how metabolic reprogramming in tumors generates chromatin vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway, which drives aggressive and chemo-resistant disease. Using a chromatin-focused CRISPR screen, we report that NRF2 activation sensitizes LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDACs). This association is observed across cultured cells, mouse models, and patient-derived xenografts. Integrative epigenomic, transcriptomic, and metabolomic analysis demonstrates that HDAC inhibition causes widespread redistribution of H4ac and its reader protein, which transcriptionally downregulates metabolic enzymes. This results in reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest NRF2 activation as a potential biomarker for effective repurposing of HDAC inhibitors to treat solid tumors.