Advanced Science: Biosecurity Primitive: Polymerase X-based Genetic Physical Unclonable Functions (Pertsemlidis)

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Abstract

A Physical Unclonable Function (PUF) is a security primitive that exploits inherent variations in manufacturing protocols to generate unique, random-like identifiers. These identifiers are used for authentication and encryption purposes in hardware security applications in the semiconductor industry. Inspired by the success of silicon PUFs, herein it leverages Terminal deoxynucleotidyl Transferase (TdT), a template-independent polymerase belonging to the X-family of DNA polymerases, to augment the intrinsic entropy generated during DNA lesion repair and rapidly produce genetic PUFs that satisfy the following properties: robustness (i.e., they repeatedly produce the same output), uniqueness (i.e., they do not coincide with any other identically produced PUF), and unclonability (i.e., they are virtually impossible to replicate). Furthermore, a post-sequencing feature selection methodology based on logistic regression to facilitate PUF classification is developed. This experimental and computational pipeline drastically reduces production time and cost compared to conventional genetic barcoding without compromising the stringent PUF criteria of uniqueness and unclonability. These results provide novel insights into the function of TdT and represent a major step toward utilizing PUFs as a biosecurity primitive for cell line authentication and provenance attestation.\

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Raushan Kurmasheva, PhD, was awarded the Translation to Cure (T2C) Award by the CURE Childhood Cancer foundation

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Cure CC FoundA cancer diagnosis is always devastating and is especially overwhelming when the patient is a child. This is why the CURE Childhood Cancer Foundation is on a mission to advance research that will bring an end to childhood cancers.

The foundation prioritizes research that is on the fast track to discovering new treatments in the next few years and research into hard-to-treat childhood cancers. Over the past 12 years, the foundation has invested more than $45 million in research at leading pediatric cancer research institutions across the United States.

Recently, Raushan Kurmasheva, PhD, Assistant Professor at the Greehey Children’s Cancer Research Institute and the Department of Molecular Medicine at The University of Texas Health Science Center at San Antonio, was awarded a $330,000 Translation to Cure (T2C) award from the CURE foundation.

The two-year grant will fund her research project titled “Advancing Innovative and Effective Therapies for Children with Malignant Rhabdoid Tumors.”

Malignant Rhabdoid Tumors are a rare, fast-growing cancer that is most common in infants and toddlers. This cancer often begins in the kidneys but can also occur in soft tissues and the brain. Five-year survival rates for this type of cancer are 20-25%. Current treatments for this cancer are surgery, aggressive chemotherapy, stem cell transplant, and radiation therapy (if the child is over six months old).

The project will explore a novel combination therapy using next-generation selective poly (ADP-ribose) polymerase 1 (PARP1) inhibitors and DNA-damaging agents to target tumors.

Kurmasheva said there are few treatment options for this type of cancer, and there is an urgent need for more effective therapies. For young infants with MRT, for example, radiotherapy is not an option. She said the new therapy they are developing has strong potential for clinical translational value.  Through this project, her team will establish preclinical data that will support future clinical trials with the ultimate goal of improving outcomes for children with aggressive malignancies.

“Malignant rhabdoid tumors mostly affect babies and toddlers under the age of 3,” said Kurmasheva. “These very young patients have very few treatment options. With support from the CURE Childhood Cancer Foundation, we will develop therapies that not only shrink tumors but also avoid toxic side effects, ensuring treatments are safe and help preserve the children’s long-term health. Our goal is to bring new hope to children and families facing this heartbreaking diagnosis.”

Nature Communications: Loss of CD98HC phosphorylation by ATM impairs antiporter trafficking and drives glutamate toxicity in Ataxia telangiectasia (Bishop Lab)

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July Carolina Romero, Sonal S. Tonapi,Manish Parihar,Eva Loranc,Henry E. Miller,Liesl A. Lawrence,Nicklas Bassani,Daniel G. Robledo,Lin Cao,Jia Nie,Kairi Kanda,Aiola Stoja,Natalia Garcia,Aparna Gorthi,Brian J. Stoveken,Teresa W-M Fan,Teresa A. Cassel,Shan Zha,James D. Lechleiter,Nicolas Musi,Lily Q. Dong,Andrew N. Lane &Alexander J. R. Bishop

Abstract

Ataxia-telangiectasia is a rare genetic disorder characterized by neurological defects, immunodeficiency, cancer predisposition, radiosensitivity, decreased blood vessel integrity, and diabetes. ATM, the protein mutated in Ataxia-telangiectasia, responds to DNA damage and oxidative stress, but its functional relationship to the progressive clinical manifestation of this disorder is not understood. CD98HC chaperones cystine/glutamate and cationic/neutral amino acid antiporters to the cell membrane, and CD98HC phosphorylation by ATM accelerates membrane localization to acutely increase amino acid transport. Loss of ATM impacts tissues reliant on heterodimeric amino acid transporters relevant to Ataxia-telangiectasia phenotypes, such as endothelial cells (telangiectasia) and pancreatic α-cells (fatty liver and diabetes), with toxic glutamate accumulation. Bypassing the antiporters restores intracellular metabolic balance in ATM-deficient cells and mouse models. These findings provide insight into the long-known benefits of N-acetyl cysteine in Ataxia-telangiectasia cells beyond oxidative stress through removing glutamate excess by producing glutathione.

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BioMolecular NMR Assignments: The 1H, 15N and 13C backbone resonance assignments of the N-terminal (1-149) domain of Serpine mRNA Binding Protein 1 (SERBP1) (Libich Lab)

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Abstract

Serpine mRNA-Binding Protein 1 (SERBP1) is an RNA-binding protein implicated in diverse cellular functions, including translational regulation, tumor progression, and stress response. It interacts with ribosomal subunits, RNA, and proteins involved in stress granules, contributing to processes such as phase separation and epigenetic regulation. Recent studies have shown SERBP1’s role in glioblastoma progression and its involvement in ribosomal regulation. Structurally, SERBP1 contains N- and C-terminal hyaluronan-binding domains, two RG/RGG motifs, and is predicted to be predominantly disordered. Here, we report the backbone resonance assignment and secondary structure propensities of SERBP1’s N-terminal residues (1–149). Using NMR spectroscopy, we identified a stable α-helix (residues 28–40) and transient structural elements. These findings provide insight into the structural features of SERBP1 that may mediate its interactions with ribosomal subunits, RNA, and other binding partners, laying a foundation for future structural studies of its functional mechanisms.

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Science: CTC1-STN1-TEN1 controls DNA break repair pathway choice via DNA end resection blockade (Sung & Libich Labs)

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Cody M. Rogers, Hardeep Kaur, Michelle L. Swift , Vivek B. Raina , Shuo Zhou , Ajinkya S. Kawale  Shahrez Syed , Korilynn G. Kelly , Angela M. Jasper , Sameer Salunkhe, Youngho Kwon , Jeffrey Wang, Aida Badamchi Shabestari, James M. Daley, Adam Sacks , Maria E. Gaczynska , Pawel A. Osmulski , Yashpal Rawal, Nozomi Tomimatsu, Simon A. Gayther, Kate Lawrenson, Sandeep Burma, Elizabeth V. Wasmuth , Shaun K. Olsen , Weixing Zhao , Robert Hromas , David S. Libich , Alexander V. Mazin , Daohong Zhou, Eric C. Greene , Dipanjan Chowdhury , and Patrick Sung

Editor’s summary

DNA breakage can cause chromosome rearrangements, which are a hallmark of cancer. DNA break repair can occur by DNA joining or homologous recombination, with the latter being dependent on the gene BRCA1, mutations of which cause familial breast and ovarian cancers. BRCA1 promotes DNA resection, a process that creates a crucial DNA intermediate needed for successful repair. Rogers et al. uncovered a multilayered mechanism by which the CTC1-STN1-TEN1 (CST) complex, a known DNA-joining factor, negatively regulates DNA resection. Importantly, they showed that BRCA1 alleviates the CST-mediated DNA resection blockade. These findings have implications for understanding drug resistance arising in tumors deficient in BRCA1. —Di Jiang

Abstract

Antagonistic activities of the 53BP1 axis and the tumor suppressor BRCA1-BARD1 determine whether DNA double-strand breaks (DSBs) are repaired by end joining or homologous recombination. We show that the CTC1-STN1-TEN1 (CST) complex, a central 53BP1 axis component, suppresses DNA end resection by EXO1 and the BLM-DNA2 helicase-nuclease complex but acts by distinct mechanisms in restricting these entities. Whereas BRCA1-BARD1 alleviates the CST-imposed EXO1 blockade, it has little effect on BLM-DNA2 restriction. CST mutants impaired for DNA binding or BLM–EXO1 interaction exhibit a hyper-resection phenotype and render BRCA1-deficient cells resistant to poly(ADP–ribose) polymerase (PARP) inhibitors. Our findings mechanistically define the crucial role of CST in DNA DSB repair pathway choice and have implications for understanding cancer therapy resistance stemming from dysfunction of the 53BP1 axis.

PNAS: Phase separation of the oncogenic fusion protein EWS::FLI1 is modulated by its DNA-binding domain (Libich & Bishop Labs)

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Emily E. Selig  Erich J. Sohn  Aiola Stoja, Alma K. Moreno-Romero, Shivani Akula, Xiaoping Xu, Alexander J. R. Bishop, and David S. Libich

Significance

The oncogenic fusion protein, EWS::FLI1, responsible for more than 85% of Ewing sarcoma tumors, combines the transactivation domain from EWS and the DNA-binding domain (DBD) from Friend leukemia integration 1 (FLI1). The fusion impacts the function of wild-type EWS and drives oncogenesis via aberrant transcriptional and splicing changes as well as defects in the DNA-damage response. Both components of the fusion are required for oncogenesis, suggesting a synergistic function between the domains. Here, the authors describe the structural underpinnings of EWS::FLI1’s effect on EWS, mediated by the FLI1 DBD, that enhances the phase separation propensity of EWS and drives aberrant changes to the physical properties of condensates.

Abstract

Ewing sarcoma (EwS) is an aggressive cancer of bone and soft tissue that predominantly affects children and young adults. A chromosomal translocation joins the low-complexity domain (LCD) of the RNA-binding protein EWS (EWSLCD) with the DNA-binding domain of Friend leukemia integration 1 (FLI1DBD), creating EWS::FLI1, a potent fusion oncoprotein essential for EwS development and responsible for over 85% of EwS tumors. EWS::FLI1 forms biomolecular condensates in vivo and promotes tumorigenesis through the mediation of aberrant transcriptional changes and by interfering with the normal functions of nucleic acid-binding proteins like EWS through a dominant-negative mechanism. In particular, the expression of EWS::FLI1 in EwS directly interferes with the biological functions of EWS leading to alternate splicing events and defects in DNA-damage repair pathways. Though the EWSLCD is capable of phase separation, here we report a direct interaction between FLI1DBD and EWSLCD that enhances condensate formation and alters the physical properties of the condensate. This effect was conserved for three related E-twenty-six transformation-specific (ETS) DNA-binding domains (DBDs) while DNA binding blocked the interaction with EWSLCD and inhibited EWS::FLI1 condensate formation. NMR spectroscopy and mutagenesis studies confirmed that ETS DBDs transiently interact with EWSLCD via the ETS DBDs “wings.” Together these results revealed that ETS DBDs, particularly FLI1DBD, enhance EWSLCD condensate formation and rigidity, supporting a model in which electrostatic and structural interactions drive condensate dynamics with implications for EWS::FLI1-mediated transcriptional regulation in EwS.

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Cell Reports: Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation (Sung, Libich Labs)

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Highlights

BRCA2 OB-fold DNA-binding domain has specificity for ssDNA
BRCA2 C-terminal DNA-binding domain has specificity for dsDNA
OB-fold DNA-binding domain is needed for DNA repair and replication fork protection
C-terminal DNA-binding domain functions primarily in replication fork protection

Summary

Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.

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Trends in Molecular Medicine: The RB protein: more than a sentry of cell cycle entry (Sung Lab)

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Highlights

The retinoblastoma protein (RB) signal transduction pathway suppresses tumor development and is impaired in most cancers.
RB pathway disruption in tumors promotes a broad range of malignant properties, such as cancer-associated blood vessel formation, metastasis, inflammation, immune evasion, cell survival, and metabolic reprogramming, as well as genomic instability.
Impaired RB pathway signaling in cancers promotes various types of genomic instability due to disrupted cell biological processes, including cell cycle progression, DNA replication, DNA repair, centrosome duplication, chromosome segregation, and chromatin organization.
While RB pathway alterations promote cancer development and treatment resistance, they also offer therapeutic opportunities, including some based on new concepts, such as synthetic lethality and oncolytic viruses.

Abstract

Genomic instability is a hallmark of cancer. It fuels cancer progression and therapy resistance. As ‘the guardian of the genome’, the tumor suppressor protein p53 protects against genomic damage. Canonically, the retinoblastoma protein (RB) is ‘the sentry of cell cycle entry’, as it dictates whether a cell enters the cell cycle to divide. However, the RB pathway also controls myriad non-canonical cellular processes, including metabolism, stemness, angiogenesis, apoptosis, and immune surveillance. We discuss how frequent RB pathway inactivation and underlying mechanisms in cancers affect these processes. We focus on RB’s – rather than p53’s – ‘guardian of the genome’ functions in DNA replication, DNA repair, centrosome duplication, chromosome segregation, and chromatin organization. Finally, we review therapeutic strategies, challenges, and opportunities for targeting RB pathway alterations in cancer.
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Nature Communications: Epigenetic silencing of DNA sensing pathway by FOXM1 blocks stress ligand-dependent antitumor immunity and immune memory (Rao, Chen, Zheng, Sung Labs et al)

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Abstract

The interplay between tumor cells and the microenvironment significantly influences cancer progression. Here, we report a significant role of the transcription factor FOXM1 in shaping the tumor immune landscape. Single-cell sequencing reveals that tumor-intrinsic FOXM1 creates an immune-suppressive tumor microenvironment by inhibiting expression of stress ligands (including ULBP1) on cancer cells, thereby blocking NKG2D-NKG2DL interactions critical for priming natural killer- and T cell-mediated cytotoxicity of cancer cells. FOXM1 suppresses ULBP1 expression by epigenetically silencing the DNA-sensing protein STING using a DNMT1-UHRF1 complex, which in turn inhibits the unfolded protein response protein CHOP from activating ULBP1. Importantly, cancer patients with higher levels of FOXM1 and DNMT1, and lower levels of STING and ULBP1, have worse survival and are less responsive to immunotherapy. Collectively, our findings provide key insight into how a tumor-intrinsic transcription factor epigenetically shapes the tumor immune microenvironment, with strong implications for refining existing and designing new cancer immunotherapies.

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NPJ – Precision Oncology: Benchmarking mouse contamination removing protocols in patient-derived xenografts genomic profiling (Zheng, Wang, Kurmasheva, Houghton, Lai, & Chen Labs)

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Abstract

Patient-derived xenograft (PDX) models are widely used in cancer research. Genomic and transcriptomic profiling of PDXs are inevitably contaminated by sequencing reads originated from mouse cells. Here, we examine the impact of mouse read contamination on RNA sequencing (RNAseq), Whole Exome Sequencing (WES), and Whole Genome Sequencing (WGS) data of 21 PDXs. We also systematically benchmark the performance of 12 computational protocols for removing mouse reads from PDXs. We find that mouse read contamination increases expression of immune and stromal-related genes, and inflates the number of somatic mutations. However, detection of gene fusions and copy number alterations is minimally affected by mouse read contamination. Using gold standard datasets, we find that pseudo-alignment protocols often demonstrate better prediction performance and computing efficiency. The best performing tool is a relatively new tool Xengsort. Our results emphasize the importance of removing mouse reads from PDXs and the need to adopt new tools in PDX genomic studies.

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