Biology subjects

Anderson, K. S.

Publications and source records attributed to Anderson, K. S..

6 recordsLinked to original sources

bioRxiv2025

RNA Transcripts Serve as a Template for Double-Strand Break Repair in Human Cells

Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Pol{zeta}) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.

molecular biology↗

bioRxiv2023

Testing Adaptive Therapy Protocols using Gemcitabine and Capecitabine on a Mouse Model of Endocrine-Resistant Breast Cancer

Highly effective cancer therapies often face limitations due to acquired resistance and toxicity. Adaptive therapy, an ecologically inspired approach, seeks to control therapeutic resistance and minimize toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life over maximum cell kill. In preparation for a clinical trial in breast cancer, we used large populations of MCF7 cells to rapidly generate endocrine-resistance breast cancer cell line. We then mimicked second line therapy in ER+ breast cancers by treating the endocrine-resistant MCF7 cells in a mouse xenograft model to test adaptive therapy with capecitabine, gemcitabine, or the combination of those two drugs. Dose-modulation adaptive therapy with capecitabine alone increased survival time relative to MTD, but not statistically significant (HR: 0.22, 95% CI 0.043-1.1 P = 0.065). However, when we alternated the drugs in both dose modulation (HR = 0.11, 95% CI: 0.024 - 0.55, P = 0.007) and intermittent adaptive therapies significantly increased survival time compared to high dose combination therapy (HR = 0.07, 95% CI: 0.013 - 0.42; P = 0.003). Overall, survival time increased with reduced dose for both single drugs (P < 0.01) and combined drugs (P < 0.001). Adaptive therapy protocols resulted in tumors with lower proportions of proliferating cells (P = 0.0026) and more apoptotic cells (P = 0.045). The results show that Adaptive therapy outperforms high-dose therapy in controlling endocrine-resistant breast cancer, favoring slower-growing tumors, and showing promise in two-drug alternating regimens.

cancer biology↗

bioRxiv2020

Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2

A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with Mpro, 17 were chosen for evaluation in a kinetic assay for Mpro inhibition. Remarkably 14 of the compounds at 100-M concentration were found to reduce the enzymatic activity and 5 provided IC50 values below 40 M: manidipine (4.8 M), boceprevir (5.4 M), lercanidipine (16.2 M), bedaquiline (18.7 M), and efonidipine (38.5 M). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1, and P2 pockets of Mpro. Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.

pharmacology and toxicology↗

bioRxiv2018

Detection of residual disease after neoadjuvant therapy in breast cancer using personalized circulating tumor DNA analysis

Accurate detection of minimal residual disease (MRD) can guide individualized management of early stage cancer patients, but current diagnostic approaches lack adequate sensitivity. Circulating tumor DNA (ctDNA) analysis has shown promise for recurrence monitoring but MRD detection immediately after neoadjuvant therapy or surgical resection has remained challenging. We have developed TARgeted DIgital Sequencing (TARDIS) to simultaneously analyze multiple patient-specific cancer mutations in plasma and improve sensitivity for minute quantities of residual tumor DNA. In 77 reference samples at 0.03%-1% mutant allele fraction (AF), we observed 93.5% sensitivity. Using TARDIS, we analyzed ctDNA in 34 samples from 13 patients with stage II/III breast cancer treated with neoadjuvant therapy. Prior to treatment, we detected ctDNA in 12/12 patients at 0.002%-1.04% AF (0.040% median). After completion of neoadjuvant therapy, we detected ctDNA in 7/8 patients with residual disease observed at surgery and in 1/5 patients with pathological complete response (odds ratio, 18.5, Fishers exact p=0.032). These results demonstrate high accuracy for a personalized blood test to detect residual disease after neoadjuvant therapy. With additional clinical validation, TARDIS could identify patients with molecular complete response after neoadjuvant therapy who may be candidates for nonoperative management.\n\nOne Sentence SummaryA personalized ctDNA test achieves high accuracy for residual disease.

genomics↗

bioRxiv2018

Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes

The application of Cas9 for genetic and epigenetic therapies in humans raises concerns over immunogenicity of this foreign protein. We report pre-existing human CD8+ T cell immunity to Streptococcus pyogenes Cas9 in the majority of healthy individuals screened. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity.

immunology↗

bioRxiv2017

Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies.

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of cytidine to uridine in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis - for example, A3G deaminates the third cytidine in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy, which we have used to capture an A3G-ssDNA interaction. We characterized an A3G-DNA binding pocket that is important for the enzyme to scan the DNA for its hotspot. The results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.

biophysics↗