Biology subjects

Johnson, K. C.

Publications and source records attributed to Johnson, K. C..

13 recordsLinked to original sources

bioRxiv2025

TET CpG sequence context specific DNA demeth-ylation shapes progression of IDH-mutant gliomas

BackgroundTreatment decisions in IDH-mutant oligodendrogliomas are shaped by tumor aggressiveness, underscoring the need for objective grading of these malignant brain tumors. Material and MethodsWe collected 302 primary and recurrent resections from oligodendrogliomas and performed Ki-67 staining, proteomics and DNA methylation profiling. Results & conclusionDuring tumor progression, DNA methylation of oligodendrogliomas changed along a continuum. This continuum is linked to increased epigenetic aging, methylation of transcription factors and Ki-67+ cell density, and to large scale DNA demethylation. Demethylation was correlated with CpGs flanking sequences preferred by TET enzymes. We confirmed these findings in previously profiled astrocytomas, indicating IDH-mutant gliomas progress along a shared epigenetic axis. We developed an objective DNA methylation based prognostic continuous grading coefficient (CGC{psi}) that captured these changes and outperformed WHO grading for oligodendrogliomas. Our findings underscore the potential of DNA methylation-based grading to more accurately reflect tumor biology and inform clinical decision-making in IDH-mutant gliomas.

cancer biology↗

bioRxiv2025

Tumor-initiating genetics and therapy drive divergent molecular evolution in IDH-mutant gliomas

Astrocytomas and oligodendrogliomas are slow-growing and treatment-sensitive IDH-mutant gliomas diagnosed at ages 30-50. Local tumor regrowth and treatment resistance is inevitable resulting in 3-10 year astrocytoma and up to >20 years oligodendroglioma survival. We sought to identify genetic changes associated with tumor evolution in response to therapy through multi-timepoint whole-genome/whole-exome sequencing of 206 IDH-mutant glioma patient samples collected through the Glioma Longitudinal Analysis (GLASS) Consortium. We validated known genomic markers of tumor progression, including hypermutation and CDKN2A homozygous deletion, and discovered novel genetic alterations that distinguish the response to treatment in astrocytomas compared to oligodendrogliomas. Point mutations in PIK3CA, PIK3R1, and NOTCH1 were newly acquired in recurrent oligodendrogliomas and associated with increased mutation rates. Focal oncogene amplifications, together with CDKN2A homozygous deletions, were associated with an increase in recurrence-specific chromosomal imbalances in astrocytomas. Mutational signature analysis revealed additional differences and detected enrichment for the SBS11, and SBS119 mutational signatures after temozolomide treatment in both IDH-glioma subtypes, whereas astrocytomas showed increased ID8 signatures after radiotherapy. These signatures suggest that the genomes of oligodendroglioma and astrocytoma adapt to the selective pressures of tumor progression and treatment in different ways. However, in both IDH-mutant glioma subtypes we observed a convergence of acquired driver gene alterations with genome-wide changes and worse patient outcomes, signaling selection of treatment-refractory clones. By identifying new prognostic markers and delineating the genomic divergence of oligodendrogliomas and astrocytomas after diagnosis, our results suggest that different DNA damage response mechanisms are engaged following chemo- and radiation therapy.

cancer biology↗

bioRxiv2024

Enhancing reproducibility and decentralization in single cell research with biocytometry

Biomedicine today is experiencing a shift towards decentralized data collection, which promises enhanced reproducibility and collaboration across diverse laboratory environments. This inter-laboratory study evaluates the performance of biocytometry, a method utilizing engineered bioparticles for enumerating cells based on their surface antigen patterns. In a decentralized framework, spanning 78 assays conducted by 30 users across 12 distinct laboratories, biocytometry consistently demonstrated significant statistical power in discriminating numbers of target cells at varying concentrations as low as 1 cell per 100,000 background cells. User skill levels varied from expert to beginner capturing a range of proficiencies. Measurement was performed in a decentralized environment without any instrument cross-calibration or advanced user training outside of a basic instruction manual. The results affirm biocytometry to be a viable solution for immunophenotyping applications demanding sensitivity as well as scalability and reproducibility and paves the way for decentralized analysis of rare cells in heterogeneous samples.

cell biology↗

bioRxiv2021

The epigenetic evolution of gliomas is determined by their IDH1 mutation status and treatment regimen

Tumor adaptation or selection is thought to underlie therapy resistance of gliomas. To investigate the longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 143 matched initial and recurrent patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) gliomas. IDHwt gliomas showed a longitudinally stable epigenome with relatively low levels of global methylation, whereas the epigenome of IDHmut gliomas showed initial high levels genome-wide of DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. By integrating DNA methylation and gene expression data, adaptive changes of putative master regulators of the cell cycle and of differentiation were seen in IDHmut recurrent tumors. Furthermore, relapses of IDHmut tumors were accompanied by histological progression which in turn influenced survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment differed between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment for IDHmut gliomas. Our study provides one of the largest cohorts of paired glioma samples profiled with epigenomics, transcriptomics and genomics; and our results demonstrate that the treatment of IDHmut gliomas reshapes the epigenome towards an IDHwt-like phenotype. Accordingly, the prevalent practice of early genotoxic treatment in this patient population may need to be revisited.

genomics↗

bioRxiv2021

Longitudinal analysis of diffuse glioma reveals cell state dynamics at recurrence associated with changes in genetics and the microenvironment

To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA and/or DNA sequencing data from temporally separated tumor pairs of 292 adult patients with IDH-wild-type or IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their malignant cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells. Collectively, our results uncover recurrence-associated changes that could be targetable to shape disease progression following initial diagnosis.

cancer biology↗

bioRxiv2020

Live-cell imaging shows uneven segregation of extrachromosomal DNA elements and transcriptionally active extrachromosomal DNA clusters in cancer

Oncogenic extrachromosomal DNA elements (ecDNAs) promote intratumoral heterogeneity, creating a barrier for successful cancer treatments. The underlying mechanisms are poorly understood and studies are hampered in part by a lack of adequate tools enabling studies of ecDNA behavior. Here, we show that single-cell ecDNA copy numbers follow a Gaussian distribution across tumor cells in vitro and in patient glioblastoma specimens, suggesting uneven ecDNA segregation during mitosis. We established a CRISPR-based approach which leverages unique ecDNA breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying this method during mitosis revealed disjointed ecDNA inheritance patterns, providing an explanation for rapid ecDNA accumulation in cancer. Post-mitosis, ecDNAs tended to cluster and clustered ecDNAs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new lights of mechanisms through which ecDNAs contribute to oncogenesis.

cancer biology↗

bioRxiv2020

Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to universal therapeutic resistance. Here, we integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes, and bulk multi-omic profiles across 11 adult IDH-mutant or IDH-wild-type gliomas to delineate sources of intratumoral heterogeneity. We found that local DNA methylation instability, or epimutation burden, was elevated in more aggressive tumors, reflected intratumoral variability, linked with transcriptional disruption, and associated with environmental stress response. We show that the activation of cell-state specific transcription factors is impacted by epimutations and that loosened epigenetic control may facilitate cellular plasticity. Our analyses support that somatic copy number alterations (SCNAs) promote epigenetic instability and that SCNAs largely precede epigenetic and transcriptomic diversification during glioma evolution. We confirmed the link between genetic and epigenetic instability by analyzing larger cohorts of bulk longitudinally collected and spatially separated DNA methylation data. Increased DNA methylation instability was associated with accelerated disease progression, and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work provides an integrative framework to better understand glioma evolution and highlights the importance of epigenetic heterogeneity in shaping therapeutic response.

cancer biology↗

bioRxiv2020

DNA methylation classification in diffuse glioma shows little spatial heterogeneity after adjusting for tumor purity

Intratumoral heterogeneity is a hallmark of diffuse gliomas. We used neuronavigation to acquire 133 image-guided and spatially-separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma, which we characterized using DNA methylation arrays. Samples were obtained from regions with and without imaging abnormalities. Methylation profiles were analyzed to devise a three-dimensional reconstruction of genetic and epigenetic heterogeneity. Molecular aberrations indicated that tumor was found outside imaging abnormalities, underlining the infiltrative nature of this tumor and the limitations of current routine imaging modalities. We demonstrate that tumor purity is highly variable between samples and largely explains apparent epigenetic spatial heterogeneity. Indeed, we observed that DNA methylation subtypes are highly conserved in space after adjusting for tumor purity. Genome-wide heterogeneity analysis showed equal or increased heterogeneity among normal tissue when compared to tumor. These findings were validated in a separate cohort of 61 multi-sector tumor and 64 normal samples. Our findings underscore the infiltrative nature of diffuse gliomas and suggest that heterogeneity in DNA methylation is innate to somatic cells and not a characteristic feature of this tumor type.

cancer biology↗

bioRxiv2019

Comparative molecular life history of spontaneous canine and human gliomas

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole genome-, exome-, transcriptome-and methylation-sequencing of 81 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, p53 and cell cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.\n\nSignificanceDiffuse gliomas are the most common malignant brain tumors, with high-grade tumors carrying a dismal prognosis. Preclinical models have proven themselves as poor predictors of clinical efficacy. Spontaneous glioma in dogs provides an attractive alternative model, because of their comparable tumor microenvironment and tumor life history. We determined the similarities and differences between human and canine gliomas through genomic profiling, and leveraged our datasets to identify conserved somatic drivers, mutational processes and temporal ordering of somatic glioma events across species. We show that canine gliomas resemble human gliomas at (epi-)genetic levels and are more reminiscent of pediatric than adult disease, thus rationalizing sporadic canine glioma as a preclinical model tailored to measuring treatment efficacies in patients with canine or human glioma.

cancer biology↗

bioRxiv2019

Longitudinal Study of Leukocyte DNA Methylation and Biomarkers for Cancer Risk in Older Adults

BackgroundChanges in DNA methylation over the course of life may provide an indicator of risk for cancer. We explored longitudinal changes in CpG methylation from blood leukocytes, and likelihood of a future cancer diagnosis.\n\nMethodsPeripheral blood samples were obtained at baseline and at follow-up visit from 20 participants in the Health, Aging and Body Composition prospective cohort study. Genome-wide CpG methylation was assayed using the Illumina Infinium Human MethylationEPIC (HM850K) microarray.\n\nResultsGlobal patterns in DNA methylation from CpG-based analyses showed extensive changes in cell composition over time in participants who developed cancer. By visit year 6, the proportion of CD8+ T-cells decreased (p-value = 0.02), while granulocytes cell levels increased (p-value = 0.04) among participants diagnosed with cancer compared to those who remained cancer-free (cancer-free vs. cancer-present: 0.03 {+/-} 0.02 vs. 0.003 {+/-} 0.005 for CD8+ T-cells; 0.52 {+/-} 0.14 vs. 0.66 {+/-} 0.09 for granulocytes). Epigenome-wide analysis identified three CpGs with suggestive p-values [≤] 10-5 for differential methylation between cancer-free and cancer-present groups, including a CpG located in MTA3, a gene linked with metastasis. At a lenient statistical threshold (p-value [≤] 3 x 10-5), the top 10 cancer-associated CpGs included a site near RPTOR that is involved in the mTOR pathway, and the candidate tumor suppressor genes REC8, KCNQ1, and ZSWIM5. However, only the CpG in RPTOR (cg08129331) was replicated in an independent data set. Analysis of within-individual change from baseline to Year 6 found significant correlations between the rates of change in methylation in RPTOR, REC8 and ZSWIM5, and time to cancer diagnosis.\n\nConclusionThe results show that changes in cellular composition explains much of the cross-sectional and longitudinal variation in CpG methylation. Additionally, differential methylation and longitudinal dynamics at specific CpGs could provide powerful indicators of cancer development and/or progression. In particular, we highlight CpG methylation in the RPTOR gene as a potential biomarker of cancer that awaits further validation.

cancer biology↗

bioRxiv2018

Genome-wide abundance of 5-hydroxymethylcytosine in breast tissue reveals unique function in dynamic gene regulation and carcinogenesis

5-hydroxymethylcytosine (5hmC) is generated by oxidation of 5-methylcytosine (5mC), however little is understood regarding the distribution and functions of 5hmC in mammalian cells. We determined the genome-wide distribution of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC is differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. In independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Our findings provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue and demonstrate that 5hmC is positioned to contribute to gene regulatory functions which protect against carcinogenesis.

genomics↗

bioRxiv2017

Reference-free deconvolution of DNA methylation signatures identifies common differentially methylated gene regions on 1p36 across breast cancer subtypes

Breast cancer is a complex disease and studying DNA methylation (DNAm) in tumors is complicated by disease heterogeneity. We compared DNAm in breast tumors with normal-adjacent breast samples from The Cancer Genome Atlas (TCGA). We constructed models stratified by tumor stage and PAM50 molecular subtype and performed cell-type reference-free deconvolution on each model. We identified nineteen differentially methylated gene regions (DMGRs) in early stage tumors across eleven genes (AGRN, C1orf170, FAM41C, FLJ39609, HES4, ISG15, KLHL17, NOC2L, PLEKHN1, SAMD11, WASH5P). These regions were consistently differentially methylated in every subtype and all implicated genes are localized on chromosome 1p36.3. We also validated seventeen DMGRs in an independent data set. Identification and validation of shared DNAm alterations across tumor subtypes in early stage tumors advances our understanding of common biology underlying breast carcinogenesis and may contribute to biomarker development. We also provide evidence on the importance and potential function of 1p36 in cancer.

genomics↗

bioRxiv2017

DNA methylation differences at regulatory elements are associated with the cancer risk factor age in normal breast tissue

BackgroundThe underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies.\n\nResultsHere we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors: age, body mass index, parity, and family history of disease with DNA methylation adjusting for potential variation in cell-type proportions. We identified 787 CpG sites that demonstrated significant associations (Q-value < 0.01) with subject age. Notably, DNA methylation was not strongly associated with the other evaluated breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations of normal breast tissue (n = 18) and normal-adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in cancer in both pre-invasive (n = 40, P=3.0E-03) and invasive breast tumors (n = 731, P=1.1E-13).\n\nConclusionsDNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

epidemiology↗