Kelsey Keith

Pyruvate dehydrogenase complex (PDHc) deficiency (PDCD) is a primary mitochondrial disorder characterized by neurodevelopmental disability, altered intermediary metabolism, and early mortality. Dichloroacetate (DCA), a pyruvate analog, activates PDHc and remains under clinical investigation for treatment of PDCD. Here, we studied the in vivo efficacy of a five‐point log concentration range of DCA on animal health and metabolism in C. elegans with feeding RNA interference (RNAi) expression knockdown of either PDHA‐1 or DLD‐1 homologs at graded degrees to model variable disease severity. These worm models recapitulate phenotypic features of PDCD observed in human patients, including reduced survival, delayed growth, locomotor impairment, and elevated lactate and/or pyruvate tissue levels. DCA treatment appeared well‐tolerated, with no gross morphologic toxicity seen at doses up to 25 mM. Significantly improved health, survival, tissue lactate levels, and mitochondrial physiology were observed at 25 mM in pdha‐1(RNAi) knockdown animals. DCA treatment in dld‐1(RNAi) C. elegans models (undiluted, 1:20 dilution, and 1:100 dilution) significantly improved survival, neuromuscular function, and metabolic phenotypes primarily in the moderate (1:20) and/or mild (1:100) dld‐1(RNAi) deficiency strains, but not in full‐dose dld‐1(RNAi) . Importantly, linear growth, neuromuscular activity, and mitochondrial physiology were significantly improved with DCA treatment even in the most severe dld‐1(RNAi) undiluted model. Overall, preclinical modeling provides objective evidence of DCA therapeutic efficacy in C. elegans knockdown strains for two well‐conserved homologs of PDHA1 and DLD representing distinct genetic etiologies of PDCD, with demonstrated beneficial effects on survival, healthspan, tissue lactate, and mitochondrial physiology. These data further confirm that DCA's therapeutic effect correlates with PDHc disease phenotype severity in dld‐1(RNAi) animals.

Accurate variant detection using nanopore long-read transcriptome data remains challenging. Here we present NanoTS—a deep learning-based tool for single nucleotide polymorphism detection from diverse types of nanopore transcriptome sequencing data. NanoTS outperforms existing methods, achieving F₁ scores above 0.980 and 0.966 on nanopore direct RNA and cDNA sequencing data, respectively, for single nucleotide polymorphisms with at least five supporting reads. Notably, NanoTS shows strong improvements over existing methods for allelically imbalanced variants. We also demonstrate that NanoTS enables accurate detection and genotype calling of pathogenic variants underlying Mendelian disorders, highlighting its potential clinical utility.

Background: While somatic mitochondrial dysfunction occurs in diverse cancers, the association between oncogenesis and germline mitochondrial gene pathogenic variants remains unclear. Further, few clinical observations have been reported of cancer occurring in primary mitochondrial disease (PMD) patients. Objectives: To improve understanding of the potential modulating role for PMD gene disorders in cancer prevalence. Design: 727 individuals, including 100 with PMD, from 97 unrelated families were retrospectively surveyed to assess their history of individual cancer occurrence. Methods: We evaluated survey responses by characterizing the cancer prevalence among the study cohort and comparing to the general U.S. population via the National Cancer Institute (NCI) Surveillance, Epidemiology, and End Results (SEER) database. Odds ratio calculation was performed to determine the association of survey responses and cancer prevalence. Results: Although overall cancer prevalence in PMD probands and their families was elevated compared to the NCI SEER rate (8800 vs 5600 cases per 100,000), odds ratio calculation determined that PMD did not significantly increase the likelihood of developing cancer, with a non-significant trend observed toward less cancer occuring in PMD that needs to be explored in further studies. Cancer prevalence was significantly correlated with advanced age. Significantly reduced prevalence of prostate cancer was seen across the entire cohort. Surprisingly, while low absolute prevalence ( n = 3), a 9-fold increased odds ratio of cancer was seen in POLG patients relative to those with other causes of PMD. Conclusion: No evidence of increased cancer odds was identified in a cohort of PMD patients and their close relatives. Interestingly, a possible inverse association, which did not reach statistical significance, was suggested between mitochondrial disease status and cancer odds. Future prospective investigations in larger PMD kindreds are warranted to validate and evaluate potential mechanistic relations between cancer prevalence and PMD.

Primary mitochondrial diseases (PMD) have limited disease-modifying therapies, currently applicable to only 3 of over 400 discrete gene disorders. Cycloheximide (CHX) is a global cytosolic translation inhibitor we previously reported to rescue PMD preclinical models, although its toxicity precluded clinical development. To identify specific mediators underlying CHX treatment benefit in PMD, SOMAscan-based proteomics was performed in complex I deficient and genetic disease fibroblast cell line models grown in galactose. Thrombopoietin (THPO) and insulin-like growth factor binding protein 5 (IGFBP5) were the only two differentially regulated proteins, together with ERK/MAPK pathway dysregulation, identified upon CHX treatment in PMD versus healthy control cells. THPO inhibition by siRNA or pharmacologic approaches rescued stress-induced viability loss in patient fibroblasts having diverse PMD gene etiologies, and significantly improved mitochondrial stress, linear growth, and neuromuscular function in a classical ndufs2 ^-/- C. elegans model. IGFBP5 overexpression by lentiviral or mRNA approaches rescued cell viability across distinct PMD gene etiologies, as did IGF1 pharmacologic inhibition across both PMD mutant and C. elegans models. MAPK pharmacologic inhibition rescued multiple distinct complex I disease cells’ survival, as well as mitochondrial stress in SLC25A46 ^-/- C. elegans . Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models. Additionally, single or combined pharmacologic inhibition of THPO or IGF1 significantly enhanced primary and metastatic osteosarcoma cell death. Collectively, targeted small molecule and genetic modulation of THPO, IGF1, or MAPK recapitulated the significant therapeutic benefit of CHX in PMD, while avoiding global translation inhibition. These novel PMD therapies likely confer benefit by attenuating MAPK-driven autophagy and potentially promoting noncanonical glucose uptake, improving cellular energy balance. Overall, these glucose signaling cellular pathway targets hold broad therapeutic promise for PMD patients, warranting further clinical research development.

Diagnosing rare genetic diseases remains a major challenge despite widespread clinical testing. Long-read RNA sequencing (RNA-seq) offers a powerful approach to capturing the effects of genetic variants on the transcriptome, yet challenges with sequencing coverage, cost, tissue selection, and scalability have limited its clinical adoption. To address this, we developed STRIPE (Sequencing Targeted RNAs Identifies Pathogenic Events), a targeted long-read RNA-seq–based strategy for rare disease diagnosis and variant interpretation. STRIPE enables deep sequencing of full-length transcripts for any customized disease-specific gene panel such that a wide range of clinically informative readouts, including transcript aberrations and sequence variants, can be detected at haplotype-level resolution. Applying STRIPE to 88 individuals spanning two major rare disease groups, we accurately reidentified known pathogenic variants and revealed their transcript consequences, including many unexpected ones. For 8 of 15 splice site region variants, we observed more complex RNA processing defects beyond single exon skipping or cryptic splice site activation. Notably, we find that donor splice site variants frequently activate cryptic intronic polyadenylation sites, leading to premature transcript termination. Leveraging unique strengths of long-read RNA-seq, STRIPE also resolved variants of uncertain significance and uncovered disease-causing variants in five previously undiagnosed individuals. Overall, STRIPE is a powerful, adaptable, and scalable strategy with broad potential to improve clinical variant interpretation and advance genetic diagnosis of rare diseases.

Osteosarcoma is the most common pediatric bone tumor yet has limited treatment options, especially for metastatic cases with a 20% adjusted 5-year survival rate. Current therapies are non-specific, involving primary tumor resection with DNA-damaging chemotherapies like methotrexate, doxorubicin, and cisplatin. Few effective treatment options exist for metastases. Targeting metabolism involving cancer’s reduced mitochondrial functionality remains underexplored in osteosarcoma. We investigated the therapeutic potential in human osteosarcoma primary and metastatic cell lines of metabolic modulating drugs including metformin, cycloheximide, mitochondrial ETC inhibitors (antimycin A, metformin), dichloroacetate, and imipridones (ONC201, ONC206) on mitochondrial function and cell viability, individually and combined under various nutrient conditions across our lines. Results confirmed osteosarcoma cells are more dependent on glucose than osteoblasts but also require mitochondrial function for survival, highlighting the therapeutic potential of metabolic pathways. Osteosarcoma cell viability was reduced when any metabolic drug treatment was combined with conditions forcing reliance on mitochondrial OXPHOS capacity. Combination metabolic therapies, particularly ONC201/ONC206/metformin in 143B cells, and to a lesser extent DCA and ONC201 with either ONC206 or antimycin A, showed enhanced cytotoxicity compared to single agents, with a good therapeutic index based on minimal toxicity to normal osteoblast cells. The degree of effectiveness varied across cell lines, underscoring the importance of personalized treatment strategies. RNA-Seq transcriptome analysis revealed that effective nutrient and metabolic drug treatments triggered widespread regulatory changes in osteosarcoma cells involving increased translation/splicing with decreased mitochondrial processes such as cholesterol biosynthesis. These results demonstrate the utility of developing combined metabolic and chemotherapeutic treatments for osteosarcoma.

Mitochondrial complex I (CI) deficiency represents a common biochemical pathophysiology underlying Leigh syndrome spectrum (LSS), manifesting with progressive multi-system dysfunction, lactic acidemia, and early mortality. To facilitate mechanistic studies and rigorous screening of therapeutic candidates for CI deficient LSS, we used CRISPR/Cas9 to generate an ndufs2 ^-/- 16 bp deletion zebrafish strain . ndufs2 ^-/- larvae exhibit markedly reduced survival, severe neuromuscular dysfunction including impaired swimming capacity, multiple morphologic malformations, reduced growth, hepatomegaly, uninflated swim bladder, yolk retention, small intestines, and small eyes and pupils with abnormal retinal ganglion cell layer. Transcriptome profiling of ndufs2 ^-/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Similar transcriptomic profiles were observed in ndufs2 ^-/- missense mutant C. elegans ( gas-1(fc21) ) and two human CI-disease fibroblast cell lines stressed in galactose media. ndufs2 ^-/- zebrafish had 80% reduced CI enzyme activity. Unbiased metabolomic profiling showed increased lactate, TCA cycle intermediates, and acyl-carnitine species. One-carbon metabolism associated pathway alterations appear to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in ndufs2 ^-/- larvae. Overall, ndufs2 ^-/- zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.

Background: In 2019, the Open Pediatric Brain Tumor Atlas (OpenPBTA) was created as a global, collaborative open-science initiative to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Here, we present an extension of the OpenPBTA called the Open Pediatric Cancer (OpenPedCan) Project, a harmonized open-source multiomic dataset from 6,112 pediatric cancer patients with 7,096 tumor events across more than 100 histologies. Combined with RNA sequencing (RNA-seq) from the Genotype-Tissue Expression and The Cancer Genome Atlas projects, OpenPedCan contains nearly 48,000 total biospecimens (24,002 tumor and 23,893 normal specimens). Findings: We utilized Gabriella Miller Kids First workflows to harmonize whole-genome sequencing (WGS), whole exome sequencing (WXS), RNA-seq, and Targeted Sequencing datasets to include somatic SNVs, indels, copy number variants, structural variants, RNA expression, fusions, and splice variants. We integrated summarized Clinical Proteomic Tumor Analysis Consortium whole-cell proteomics and phospho-proteomics data and miRNA sequencing data, as well as developed a methylation array harmonization workflow to include m-values, beta-values, and copy number calls. OpenPedCan contains reproducible, dockerized workflows in GitHub, CAVATICA, and Amazon Web Services (AWS) to deliver harmonized and processed data from over 60 scalable modules, which can be leveraged both locally and on AWS. The processed data are released in a versioned manner and accessible through CAVATICA or AWS S3 download (from GitHub) and queryable through PedcBioPortal and the National Cancer Institute’s pediatric Molecular Targets Platform. Notably, we have expanded Pediatric Brain Tumor Atlas molecular subtyping to include methylation information to align with the World Health Organization 2021 Central Nervous System Tumor classifications, allowing us to create research-grade integrated diagnoses for these tumors. Conclusions: OpenPedCan data and its reproducible analysis module framework are openly available and can be utilized and/or adapted by researchers to accelerate discovery, validation, and clinical translation.

Background In 2019, the Open Pediatric Brain Tumor Atlas (OpenPBTA) was created as a global, collaborative open-science initiative to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Here, we extend the OpenPBTA to create the Open Pediatric Cancer (OpenPedCan) Project, a harmonized open-source multi-omic dataset from 6,112 pediatric cancer patients with 7,096 tumor events across more than 100 histologies. Combined with RNA-Seq from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA), OpenPedCan contains nearly 48,000 total biospecimens (24,002 tumor and 23,893 normal specimens). Findings We utilized Gabriella Miller Kids First (GMKF) workflows to harmonize WGS, WXS, RNA-seq, and Targeted Sequencing datasets to include somatic SNVs, InDels, CNVs, SVs, RNA expression, fusions, and splice variants. We integrated summarized CPTAC whole cell proteomics and phospho-proteomics data, miRNA-Seq data, and have developed a methylation array harmonization workflow to include m-values, beta-vales, and copy number calls. OpenPedCan contains reproducible, dockerized workflows in GitHub, CAVATICA, and Amazon Web Services (AWS) to deliver harmonized and processed data from over 60 scalable modules which can be leveraged both locally and on AWS. The processed data are released in a versioned manner and accessible through CAVATICA or AWS S3 download (from GitHub), and queryable through PedcBioPortal and the NCI’s pediatric Molecular Targets Platform. Notably, we have expanded PBTA molecular subtyping to include methylation information to align with the WHO 2021 Central Nervous System Tumor classifications, allowing us to create research-grade integrated diagnoses for these tumors. Conclusions OpenPedCan data and its reproducible analysis module framework are openly available and can be utilized and/or adapted by researchers to accelerate discovery, validation, and clinical translation.

The intestinal microbiota is an important environmental factor implicated in CRC development. Intriguingly, modulation of DNA methylation by gut microbiota has been reported in preclinical models, although the relationship between tumor-infiltrating bacteria and CIMP status is currently unexplored. In this study, we investigated tumor-associated bacteria in 203 CRC tumor cases and validated the findings using The Cancer Genome Atlas datasets. We assessed the abundance of Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae through qPCR analysis and observed enrichment of all four bacterial species in CRC samples. Notably, except for E. coli, all exhibited significant enrichment in cases of CIMP. This enrichment was primarily driven by a subset of cases distinguished by high levels of these bacteria, which we labeled as "Superhigh". The bacterial Superhigh status showed a significant association with CIMP (odds ratio 3.1, p-value = 0.013) and with MLH1 methylation (odds ratio 4.2, p-value = 0.0025). In TCGA CRC cases (393 tumor and 45 adj. normal), bacterial taxa information was extracted from non-human whole exome sequencing reads, and the bacterial Superhigh status was similarly associated with CIMP (odds ratio 2.9, p < 0.001) and MLH1 methylation (odds ratio 3.5, p < 0.001). Finally, 16S ribosomal RNA gene sequencing revealed high enrichment of Bergeyella spp. C. concisus, and F. canifelinum in CIMP-Positive tumor cases. Our findings highlight that specific bacterial taxa may influence DNA methylation, particularly in CpG islands, and contribute to the development and progression of CIMP in colorectal cancer.