Human Artificial Chromosomes for Cancer Research and Functional Genomics

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Over the past years, we intensified our analysis of tandemly repeated ribosomal DNA (rDNA) sequences, particularly focusing on their structure and variation within the human genome. These rDNA arrays play essential roles in nucleolus formation and critical cellular processes such as ribosome biogenesis, cell cycle regulation, and stress response. The extent of divergence among human rDNA units remains incompletely characterized. To address this gap, we applied Transformation-Associated Recombination (TAR) cloning technology to isolate individual rDNA units. This approach revealed an unexpectedly high level of heterogeneity in mature 18S/28S rRNA sequences, suggesting that some of these variants might affect ribosome dynamics. For functional analysis, we utilized a yeast rDNA assay where the chromosomal rDNA repeats are completely deleted and cell growth depends on a single rDNA repeat unit expressed from a 2-micron plasmid. By plasmid shuffling, this system enables examination of mutational effects on translational accuracy and rRNA structures involved in ribosome assembly and function. Currently, we are studying sequence variants identified in 24 conserved regions of 28S rRNA shared between humans and yeast. So far, among 12 variants analyzed, two proved lethal in yeast, and three increased sensitivities to aminoglycoside antibiotics. We continue to investigate the mechanisms underlying these defects. In addition to human rDNA studies, we identified a novel gene of unknown function within the human rDNA intergenic spacer region (IGS), which we named ORF3. This gene encodes a previously uncharacterized 190-amino acid, 22-kDa protein. In human cells, ORF3 localizes to both the nucleus and cytoplasm and is associated with RNA complexes. Furthermore, ORF3 expression is upregulated under stress conditions such as viral transformation and senescence induced by etoposide, ionizing radiation, or oxidative stress, suggesting a role in chromatin dynamics or the cellular stress response. This project is carried out in collaboration with Dr. Nagaraja (NIA)). We also continued to advance our work with synthetic tetO-HAC vector to investigate human kinetochore assembly and maintenance - processes critical for accurate chromosome segregation during cell division. Beyond fundamental research, this platform has implications in gene therapy and synthetic biology, where precise chromosome manipulation is essential. To expand tetO-HAC's utility, we developed two novel assays specifically designed to measure chromosome instability (CIN), a hallmark of many cancers. These assays enable (i) quantitative evaluation of how various anticancer drugs and cytotoxic plant extracts affect chromosome transmission fidelity, allowing us to rank compounds by their ability to induce or suppress CIN, and (ii) functional genomic screens to identify novel CIN-related genes, potentially unveiling new cancer therapeutic targets.Using this platform, we performed a genome-wide RNA interference (RNAi) screen targeting ~19,000 genes to systematically assess their roles in chromosome stability. This large-scale screen identified 834 candidate CIN-associated genes. Many of these genes function in critical mitotic pathways, including chromosome segregation and cell cycle checkpoint regulation, underscoring their central role in maintaining chromosomal integrity during mitosis. Notably, we identified SENP6 as a novel CIN gene; while previously implicated in kinetochore function, its direct role in chromosome instability was unrecognized. Future studies will utilize advanced computational tools to map protein interactions and molecular networks involving these novel CIN factors. Chromosome instability is present in approximately 80-90% of solid tumors, emphasizing the clinical relevance of the CIN genes we discovered. Expression analyses revealed that several top candidate genes - AMY2B, ALAD, PDGFRA, PPIE, VEZ1, and TTC19 - correlate significantly with poor survival outcomes in multiple cancers, including lung, adrenal cortical, ovarian, and breast cancers. Additionally, downregulation of CIN-related genes associates with worse prognosis in genomically unstable tumors such as small cell lung cancer (SCLC), adrenal cortical carcinoma (ACC), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), ovarian, and breast cancers, highlighting their potential as prognostic biomarkers. Integrating synthetic lethality data revealed therapeutic strategies targeting CIN genes alongside their synthetic lethal partners to enhance cancer treatment efficacy. A notable translational insight was the identification of fostamatinib, an FDA-approved thrombocytopenia drug, as a CIN-inducing compound - suggesting opportunities for drug repurposing in oncology. Collectively, our tetO-HAC system not only facilitates novel CIN gene discovery and therapeutic target identification but also offers a powerful platform for personalized cancer diagnostics and tailored treatment strategies, marking a significant step forward in precision oncology. This work has recently accepted for publication in PNAS, in collaboration with Dr. Earnshaw (Wellcome Trust Center. Another project employs linear and circular HAC assay systems to investigate the effects of telomere-targeting compounds that inhibit Heat Shock Protein 90 (Hsp90), a molecular chaperone stabilizing key oncogenic proteins, including hTERT and shelterin complex components. Hsp90 is critical for hTERT folding, stabilization, and nuclear localization, thereby regulating telomerase activity. Hsp90 inhibitors such as 17-AAG and TAS-116 disrupt this function, leading to hTERT degradation, telomerase inhibition, and induction of telomere dysfunction-induced foci (TIFs) - hallmarks of telomere uncapping and DNA damage signaling that contribute to chromosomal instability (CIN). To evaluate these effects, we employed a HAC assay using two distinct HAC cell lines: one harboring a linear EGFP-expressing HAC (with telomeres) and the other a circular EGFP-expressing HAC (lacking telomeres). Compounds targeting telomeres cause selective destabilization and loss of the linear HAC, observed by loss of EGFP signal, while the circular HAC remains stable unless off-target effects occur. We tested five Hsp90 inhibitors - 17-AAG, TAS-116, XL-888, SNX-2112, and STA-9090 - for their capacity to induce telomere dysfunction. All five selectively induced linear HAC loss, indicating telomere-specific effects. TAS-116 showed the strongest inhibition of telomere replication, also causing telomere shortening, telomere-associated DNA damage, and increased micronuclei formation - indicators of CIN. Importantly, TAS-116 is in clinical trials and has demonstrated promising efficacy and low toxicity as monotherapy and combined with immune checkpoint inhibitors. Together, these findings support Hsp90 inhibitors as effective anti-cancer agents targeting telomere maintenance pathways and selectively inducing CIN in cancer cells. Further work is ongoing to refine the HAC-based screening platform, test additional compounds, and investigate downstream CIN effects in cancer models. In addition, we are developing a novel system in which both linear and circular HACs are introduced into a single, isogenic cell line. This unified dual-HAC cell model will allow us to more precisely assess whether a given compound or gene specifically affects telomere maintenance or overall chromosome stability, without the confounding influence of different cellular backgrounds. This new approach will enable more accurate, side-by-side comparisons in a single experimental context. The work is currently in progress.