Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 regulates protein lipoylation by directly binding to the lipoyl synthase (LIAS) enzyme and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss-of-function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling of cells growing in either normal or low glucose conditions established that FDX1 loss-of-function results in the induction of both compensatory metabolism related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-functions is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.

Liquid biopsies are enabling minimally invasive monitoring and molecular profiling of diseases across medicine, but their sensitivity remains limited by the scarcity of cell-free DNA (cfDNA) in blood. Here, we report an intravenous priming agent that is given prior to a blood draw to increase the abundance of cfDNA in circulation. Our priming agent consists of nanoparticles that act on the cells responsible for cfDNA clearance to slow down cfDNA uptake. In tumor-bearing mice, this agent increases the recovery of circulating tumor DNA (ctDNA) by up to 60-fold and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. We envision that this priming approach will significantly improve the performance of liquid biopsies across a wide range of clinical applications in oncology and beyond.

Blood-based, or "liquid," biopsies enable minimally invasive diagnostics but have limits on sensitivity due to scarce cell-free DNA (cfDNA). Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo . Here, we sought to augment the level of circulating tumor DNA (ctDNA) detected in a blood draw by attenuating the clearance of cfDNA in vivo . We report a first-in-class intravenous DNA-binding priming agent given 2 hours prior to a blood draw to recover more cfDNA. The DNA-binding antibody minimizes nuclease digestion and organ uptake of cfDNA, decreasing its clearance at 1 hour by over 150-fold. To improve plasma persistence and limit potential immune interactions, we abrogated its Fc-effector function. We found that it protects GC-rich sequences and DNase-hypersensitive sites, which are ordinarily underrepresented in cfDNA. In tumor-bearing mice, priming improved tumor DNA recovery by 19-fold and sensitivity for detecting cancer from 6% to 84%. These results suggest a novel method to enhance the sensitivity of existing DNA-based cancer testing using blood biopsies.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.

Significance:

Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma

Copper is an essential co-factor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copperdependent, regulated cell death is distinct from known death mechanisms, and is dependent on mitochondrial respiration. We show that copper-dependent death occurs via direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss leading to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. Wedemonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies morebroadly, wehavemadethesereagents,andadetailedprotocol,available as a community resource.

In the original version of this article, the given and family names of Uri Ben-David were incorrectly structured. The original article has been corrected.

Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR-Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2-encoding a phosphate importer-is correlated with sensitivity to loss of the phosphate exporter XPR1, both in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2, XPR1 copy number amplifications and XPR1 messenger RNA overexpression. Mechanistically, in SLC34A2-high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic "vacuolar" structures preceding cell death. These data point to the XPR1-KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.

Genomic evolution of patient-derived xenografts (PDXs) may lead to their gradual divergence away of their tumors of origin. We previously reported the genomic evolution of the copy number (CN) landscapes of PDXs during their engraftment and passaging¹. However, whether PDX models are highly stable throughout passaging², or can evolve CNAs rapidly^1,3, remains controversial. Here, we reassess the genomic evolution of PDXs using DNA-based CN profiles. We find strong evidence for genomic evolution in the DNA-based PDX data: a median of ~10% of the genome is differentially altered between matched primary tumors (PTs) and PDXs across cohorts (range, 0% to 73% across all models). In 24% of the matched PT-PDX samples, over a quarter of the genome is differentially affected by CN alterations. Moreover, in matched analyses of PTs and their derived PDXs at multiple passages, later-passage PDXs are significantly less similar to their parental PTs than earlier-passage PDXs, indicative of genomic divergence. We conclude that PDX models indeed evolve throughout their derivation and propagation, and that the phenotypic consequences of this evolution ought to be assessed in order to determine its relevance to the proper application of these valuable cancer models.