Hofman DA, Ruiz-Orera J, Yannuzzi I, Murugesan R, Brown A, Clauser KR, Condurat AL, van Dinter JT, Engels SA, Goodale A, et al. Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma. bioRxiv. 2023. doi:10.1101/2023.05.04.539399

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets. 

Bae JH, Liu R, Roberts E, Nguyen E, Tabrizi S, Rhoades J, Blewett T, Xiong K, Gydush G, Shea D, et al. Single duplex DNA sequencing with CODEC detects mutations with high sensitivity. Nat Genet. 2023.

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Dreishpoon MB, Bick NR, Petrova B, Warui DM, Cameron A, Booker SJ, Kanarek N, Golub TR, Tsvetkov P. FDX1 regulates cellular protein lipoylation through direct binding to LIAS. bioRxiv. 2023. doi:10.1101/2023.02.03.526472

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.

Martin-Alonso C, Tabrizi S, Xiong K, Blewett T, Patel S, An Z, Sridhar S, Bekdemir A, Shea D, Amini AP, et al. A nanoparticle priming agent reduces cellular uptake of cell-free DNA and enhances the sensitivity of liquid biopsies. bioRxiv. 2023. doi:10.1101/2023.01.13.524003

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.

Tabrizi S, Martin-Alonso C, Xiong K, Blewett T, Sridhar S, An Z, Patel S, Rodriguez-Aponte S, Naranjo CA, Wang S-T, et al. An intravenous DNA-binding priming agent protects cell-free DNA and improves the sensitivity of liquid biopsies. 2023. doi:10.1101/2023.01.13.523947

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.


Panditharatna E, Marques JG, Wang T, Trissal MC, Liu I, Jiang L, Beck A, Groves A, Dharia NV, Li D, et al. BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma. 2022. doi:10.1158/2159-8290.CD-21-1491

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.


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

Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, Rossen J, Joesch-Cohen L, Humeidi R, Spangler RD, et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. 2022;375(6586):1254–1261. doi:10.1126/science.abf0529

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.

Bondeson DP, Mullin-Bernstein Z, Oliver S, Skipper TA, Atack TC, Bick N, Ching M, Guirguis AA, Kwon J, Langan C, et al. Systematic profiling of conditional degron tag technologies for target validation studies. 2022;13(1):5495. doi:10.1038/s41467-022-33246-4

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.