Abstract Background: Gene fusions, resulting from chromosomal rearrangements, play a critical role in the oncogenesis of various cancers, often acting as diagnostic markers and therapeutic targets. Their detection is important for understanding tumor biology and guiding personalized treatment strategies. With the evolution of economical sequencing technologies, hypothesis-free gene fusion detection in cancer genomics is becoming more accessible. This study demonstrates the use of the cost-effective Ultima Genomics UG100 platform for a comprehensive analysis of gene fusions, uniquely integrating Whole Genome Sequencing (WGS) and Whole Transcriptome Sequencing (WTS). This integration capitalizes on the strengths of both methods: WGS, which enables a broad analysis of structural variations including extragenic events, and WTS for more functional characterization of expressed gene fusions and abnormal isoforms, offering a synergistic approach that enhances the detection and characterization of genomic rearrangements. Methods: Five cancer cell lines (K562, THP-1, NCI-H660, Kasumi-1, MV4-11) with 29 previously detected gene fusions were sequenced, using both WTS and WGS, in addition to a standard fusion reference sample (Seraseq Fusion RNA Mix, with 16 RNA fusions). The libraries were sequenced on a UG100 sequencer using single ended reads of length ~250bp and analyzed with publicly available software: STAR-Fusion, combined with FusionInspector were used to detect RNA fusions; Manta and GridSS were used to detect DNA structural variations, including translocations. Results: 93% of the documented fusion events were detected in DNA and in RNA, in addition to the 16 evaluated just in RNA (100% detected). Sequence data from both WTS and WGS on the UG100 platform successfully identified a spectrum of gene fusions in both DNA and RNA, thereby confirming the platform's detection capabilities in both types of assays. The structural variation events detected in the WGS were comprised mainly of translocations, but also deletions and duplications. Conclusions: The Ultima Genomics UG100 sequencer has proven to be a cost-effective and efficient tool for the genome-wide discovery and validation of gene fusions, which can be impactful in clinical oncology settings. This study underscores the potential of using an integrated WTS and WGS approach on a single platform to advance our understanding of the genomic complexity of cancer, paving the way for more effective diagnostic and therapeutic strategies. Citation Format: Gila Lithwick-Yanai, Sarah Pollock, Danit Lebanony, Keren Ben Simhon, Sima Benjamin, David Bogumil, Nika Iremadze, Matt Sexton, Peleg Winer, Maya Levy, Shlomit Gilad, Josh Haimes, Giulia Corbet, Jennifer Pavlica, Doron Lipson. Cost-effective, comprehensive detection of gene fusions from DNA and RNA using the UG100 sequencer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 315.
Abstract Myelodysplastic Syndromes (MDS) are a group of blood malignancies characterized by aberrant differentiation of hematopoietic stem and progenitor cells (HSPC) in the bone marrow that results in inefficient hematopoiesis and high risk of transformation to acute myeloid leukemia. In order to detect aberrant HSC differentiation in blood samples sensitively and accurately, we explored the use of scRNA-seq of CD34-enriched cells from peripheral blood in identifying differentiation trajectories that are abnormal in patient samples versus expected normal development. A total of 685,000 CD34-enriched PBMCs from 93 samples were analyzed using 10X 3’ v3 scRNA-seq library preparation. For increased efficiency, the samples were multiplexed in groups of 5 individuals and were later identified based on individual natural genetic variation using a custom genotyping assay or low-pass WGS. We then sequenced these 24 libraries on a UG100 sequencer to yield an average of 20,000 reads per cell. Six of these libraries were also sequenced on a NovaSeq. Comparison between scRNA profiles generated on the two different sequencers yielded highly similar results, including a similar ability to demultiplex the patient samples using genetic markers, accurately quantify gene modules and determine cell populations.To analyze the scRNA data, we used a novel computational framework, Metacell, to reconstruct metacell modules for both healthy and disease samples allowing us to find variability in the differentiation process between the disease samples and the normal baseline state. Two patients with MDS which had transformed to acute myelogenous leukemia (AML) demonstrated unique and distinct metacell clusters that were significantly separate from normal HSPC differentiation patterns, demonstrating distinct clusters with elevated levels of BCL2 which is an existing therapy target in secondary AML. Our results demonstrate that scRNA-seq analysis of peripheral blood HSPCs samples can be used to detect aberrations in HSC development in MDS patients and serve as a prognostic tool for stratification of patients with aggressive disease and drug response. Given the cost-efficiency of the entire process, we believe this is one of the first examples of the potential practical utility of single-cell analysis in a clinical setting. Citation Format: Eti Meiri, Nili Saar-Furer, Nimrod Rappoport, Sarah Pollock, Gila Lithwick-Yanai, Zohar Shipony, Nika Iremadze, Doron Lipson, Amos Tanay, Liran Shlush. Single-cell analysis of CD34-enriched blood cells reveals early prognostic markers of myelodysplastic syndromes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5467.
Healthy mitochondria use an electrochemical gradient across the inner mitochondrial membrane (IMM) to generate energy in the form of ATP. A variety of endogenous and exogenous factors can lead to transient or sustained depolarization of the IMM, including mitochondrial fission events, expression of uncoupling proteins, electron transport chain (ETC) inhibitors, or chemical uncouplers. This depolarization in turn leads to a variety of physiological responses, ranging from selective mitochondrial clearance (mitophagy) to cell death. How cells recognize and ultimately respond to depolarized mitochondria remains incompletely understood. Here we show that the small GTPases RalA and RalB both relocalize to mitochondria following depolarization in a process dependent on clathrin-mediated endocytosis (CME). Furthermore, both genetic and pharmacologic inhibition of RalA and RalB leads to an increase in the activity of the atypical IκB kinase TBK1 both basally and in response to mitochondrial depolarization. This phenotype was also observed following inhibition of Ral relocalization. Collectively, these data suggest a model in which RalA and RalB inhibit TBK1 and that relocalization of Ral to depolarized mitochondria facilitates TBK1 activation through release of this inhibition.
Abstract Nucleotide binding and oligomerization domain containing protein 2 (NOD2) is an intracellular protein that is involved in the recognition of bacterial cell wall derived muramyl-dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders including Crohn’s disease and Blau syndrome. NOD2 is a member of the Nucelotide-binding domain and leucine-rich repeat- containing protein gene family (NLR). Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remains largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl-dipeptide in cellular assays. Using purified recombinant protein, we demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found the purified recombinant protein is able to bind directly to muramyl-dipeptide and can associate with known NOD2 interacting proteins in vitro. Binding of NOD2 to muramyl-dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model that involves binding of nucleotide, muramyl-dipeptide, and oligomerization into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl-dipeptide and assembly of NOD2-containing signaling complexes.
The melon ( Cucumis melo L.) fruit is an important crop and model system for the genomic study of both fleshy fruit development and the Cucurbitaceae family. To obtain an accurate representation of the melon fruit transcriptome based on expressed sequence tag (EST) abundance in 454‐pyrosequencing data, we prepared double‐stranded complementary DNA (cDNA) of melon without the usual amplification and normalization steps. A purification step was also included to eliminate small fragments. Complementary DNAs were obtained from 14 individual fruit libraries derived from two genotypes, separated into flesh and peel tissues, and sampled throughout fruit development. Pyrosequencing was performed using Genome Sequencer FLX (GS FLX) technology, resulting in 1,215,359 reads, with mean length of >200 nucleotides. The global digital expression data was validated by comparative reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR) of 40 selected genes and expression patterns were similar for the two methods. The results indicate that high‐quality, nonbiased cDNA for next‐generation sequencing can be prepared from mature, fleshy fruit, which are notorious for difficulties in ribonucleic acid (RNA) preparation.
