Biology subjects

Karlan, B. Y.

Publications and source records attributed to Karlan, B. Y..

8 recordsLinked to original sources

bioRxiv2026

Deformability screening identifies NUDT5 as a mediator of cellular mechanobiology

How cells deform, sense, and respond to mechanical cues drives physiological and disease processes ranging from development to cancer metastasis; however, unbiased approaches to identify mechanical mediators are lacking. We screened 1280 compounds to identify modulators of cancer cell deformability using a cellular filtration assay and identified 92 compounds that significantly reduced deformability of ovarian cancer cells; top hits also reduced migration and invasion. Connectivity mapping of the top 21 compounds identified NUDT5 (Nudix hydrolase 5) as a predicted mechanical mediator; transcriptomic analyses implicated NUDT5 in mechanobiology and metabolic processes. We confirmed that NUDT5 mediates intracellular ATP and cellular mechanical behaviors, including morphology and deformability. In ovarian cancer, increased NUDT5 levels were associated with higher tumor stage and worse patient survival; NUDT5 inhibition reduced migration and colony formation in vitro and peritoneal tumor burden in mice. These findings establish deformability-based screening as a platform for discovering mechanical mediators and identify NUDT5 as a therapeutic target in ovarian cancer. TeaserScreening cells based on deformability provides an unbiased approach to identify NUDT5 as a mediator of cell mechanics

cell biology↗

bioRxiv2026

Genome-wide classification of tumor-derived reads from bulk long-read sequencing

DNA extracted from tissue samples typically derives from a complex mixture of cell types. Without single cell analysis, it has been generally impossible to determine the cell type of origin for most molecules. One clear example of this is in the complex milieu of a human neoplasm. Here, we develop ROCIT (https://github.com/tobybaker/rocit), a transformer-based model to classify the tumor or non-tumor origin of individual reads from bulk tumor samples sequenced with long-read whole genome sequencing. Using somatic mutations to derive training data, ROCIT uses read-level methylation patterns to accurately classify reads from any-where in the genome without requiring the adjacent normal tissue or the explicit identification of tumor differentially methylated regions. We apply ROCIT to a cohort of prostate and ovarian tumors and demonstrate high classification accuracy across the entire genome. We then demonstrate the potential of ROCIT predictions to improve somatic variant calling. ROCIT represents a major step forward in the analysis of bulk tumors with long-reads, enabling the accurate and sensitive identification of reads with specific cell types of origin genome-wide.

bioinformatics↗

bioRxiv2025

The mutational landscape defines the proteome and spatial organization of tumor, stroma and immune cells in ovarian cancer

High-grade serous ovarian cancer (HGSOC) is a highly aggressive and lethal form of ovarian cancer. Challenges to diagnosis and treatment include a lack of effective screening methods for early detection, the absence of cancer-specific symptoms, and the development of chemoresistance. The genomic instability of HGSOC, further complicated by homologous recombination deficiency (HRD), leads to heterogeneity in HGSOC tumors and patient response to treatment. This makes it challenging to develop a single, effective diagnostic and treatment approach for this disease. Proteogenomic studies have provided some insight into HGSOC biology, but a deeper understanding of the tumor proteome through chemotherapy and disease recurrence is needed. Here we have profiled the proteome of tumors from 29 HGSOC patients before and after multiple rounds of chemotherapy. The proteome of HGSOC tumors remained unchanged for individual patients even after numerous rounds of chemotherapy. Differential expression analysis revealed known and novel proteins associated with chemoresistance and HRD status, further supported by similar changes at the genetic and epigenetic levels. We found that HRD affected proteins related to immune pathways. HRD was also associated with more shared T cell receptor (TCR) CDR3 repertoires of tumor-infiltrating T cells and increased neoantigen counts. ERAP1, a protein involved in peptide trimming before antigen presentation to immune cells via MHC-class I, was overexpressed in homologous recombination proficient (HRP) tumors and negatively correlated to neoantigen count. Its potential role in immune suppression in HRP tumors makes it an attractive therapeutic target that may be effective in combination with immunotherapy, particularly for HRP tumors. 26-plex immunostaining of HGSOC whole tissues further revealed significant differences in the spatial proximities of immune cells to each other and to tumor cells based on HRD status. Through proteomic and imaging analysis, this work has shown that the immune landscape of HGSOC tumors is influenced by homologous recombination status and identified candidate drivers of HGSOC biology.

cancer biology↗

bioRxiv2025

The mutational and clonality profile of HGSOC is established early in tumor development and conserved throughout therapy resistance

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States alone. Nearly 80% of patients with HGSOC tumors will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Intratumor heterogeneity is believed to be a key feature of recurrence and resistance in HGSOC tumors, with early studies reporting diverse and complex mechanisms of the seeding of metastatic sites, including metastasis reseeding the primary tumor. Few studies have investigated temporal changes to clonality and structural variants through disease recurrence and the development of chemoresistance as surgical debulking is not frequently performed at this later stage. We performed multi-omic profiling in paired chemo-naive and chemoresistant tumors from 32 HGSOC patients to investigate the mutational and genomic landscape of disease progression. Somatic mutation profiles were largely conserved through disease progression. Mutational burdens did not significantly differ across recurrence but were driven by homologous recombination repair deficiency status. Clonal composition and dynamics were measured through variant allele frequency alterations as tumors progressed from primary chemo-naive to recurrent chemo-resistant tumors. A novel candidate driver gene, MDC1, from the homologous recombination repair pathway, was significantly mutated and over-represented in patients with homologous recombination proficient tumors, with somatic mutations clustered in a single exon. Tumor evolution and phylogeny revealed that few changes in clonal abundance and complexity occur across the disease course in these patients. Taking structural variants into account, homologous recombination repair proficient (HRP) tumors tend to be polyclonal while homologous recombination repair deficient (HRD) tumors tend to be monoclonal, accompanied by a longer progression-free survival than the HRP patients. Three distinct classes of tumors were identified by structural variant signature analysis: tumors defined by DNA losses, tumors defined by DNA gains, and tumors defined by copy number neutral changes, which were largely defined by HRD status. Each class displayed distinct regions of the chromosome that were frequently affected by large scale SV events (>5Mb). Although no regions were frequently altered in recurrent tumors, GO analysis revealed that recurrent tumors have a significantly reduced immune response, which was not seen in the primary tumors. Ultra long read sequencing validated a majority of the SVs identified in short read sequencing and identified additional SVs undetected by short reads. These analyses identify that the phenotype of high grade serous ovarian tumors as defined by mutation and clonality profiles is established early in disease development and remain largely unchanged through chemotherapy and recurrence. This, when considered with the significant inter-patient heterogeneity identified in HGSOC, demonstrates the need for personalized therapies based on tumor profiling.

genomics↗

bioRxiv2024

Replication stress marker phospho-RPA2 predicts response to platinum and PARP inhibitors in homologous recombination-proficient ovarian cancer

BackgroundOvarian cancer treatment includes cytoreductive surgery, platinum-based chemotherapy, and often poly (ADP-ribose) polymerase (PARP) inhibitors. Homologous recombination (HR)-deficiency is a well-established predictor of therapy sensitivity. However, over 50% of HR-proficient tumors also exhibit sensitivity to standard-of-care treatments. Currently, there are no biomarkers to identify which HR-proficient tumors will be sensitive to standard-of-care therapy. Replication stress may serve as a key determinant of response. MethodsWe evaluated phospho-RPA2-T21 (pRPA2) foci via immunofluorescence as a potential biomarker of replication stress in formalin-fixed, paraffin-embedded tumor samples collected at diagnosis from patients treated with platinum chemotherapy (discovery cohort: n = 31, validation cohort: n = 244) or PARP inhibitors (n = 87). Recurrent tumors (n = 37) were also analyzed. pRPA2 scores were calculated using automated imaging analysis. Samples were defined as pRPA2-High if > 16% of cells had [≥] 2 pRPA2 foci. ResultsIn the discovery cohort, HR-proficient, pRPA2-High tumors demonstrated significantly higher rates of pathologic complete response to platinum chemotherapy than HR-proficient, pRPA2-Low tumors. In the validation cohort, patients with HR-proficient, pRPA2-High tumors had significantly longer survival after platinum treatment than those with HR-proficient, pRPA2-Low tumors. Additionally, the pRPA2 assay effectively predicted survival outcomes in patients treated with PARP inhibitors and in recurrent tumor samples. ConclusionOur study underscores the importance of considering replication stress markers alongside HR status in therapeutic planning. Our work suggest that this assay could be used throughout a patients treatment course to expand the number of patients receiving effective therapy while reducing unnecessary toxicity.

cancer biology↗

bioRxiv2020

DNA methylation landscapes of matched primary and recurrent high grade serous ovarian cancers are preserved throughout disease progression and chemoresistance.

Little is known about the role of global DNA methylation in recurrence and chemoresistance of high grade serous ovarian cancer (HGSOC). We performed whole genome bisulfite sequencing (WGBS) and whole transcriptome sequencing (RNA-seq) to establish methylation and gene expression signatures in 62 primary and recurrent tumors from 28 patients diagnosed with stage III/IV HGSOC. Eleven of these patients carried pathogenic germline BRCA1/BRCA2 mutations. Genome-wide methylation and transcriptomic features identified in primary tumors were largely preserved in matched recurrent tumors from the same patient (P-value = 7.16 x 10-7 and 1.41 x 10-3 in BRCA1/2 and non-BRCA1/2 cases respectively). Tumors from BRCA1/2 carriers displayed high levels of heterogeneity, with significantly more shared methylation changes identified between primary and recurrent tumors from non-BRCA1/2 patients, which may be related to the poorer survival we observe in HGSOCs from non-BRCA1/2 carriers (P-value = 0.0056). Partially methylated domains (PMDs) dominated the epigenetic variation across all tumors, and were more hypomethylated in BRCA1/2 than non-BRCA1/2 cases. Differential gene expression analysis identified upregulation of genes from immune pathways including antigen processing and presentation in tumors from BRCA1/2 carriers, implicating increased immune response in the improved survival observed in these patients. In summary, this study shows a previously unreported conservation of methylation and gene expression in recurrent HGSOCs. These data have implications for the possible effectiveness of epigenetic based therapies to treat both primary and recurrent ovarian cancers.

genomics↗

bioRxiv2019

Predicting master transcription factors from pan-cancer expression data

The function of critical developmental regulators can be subverted by cancer cells to control expression of oncogenic transcriptional programs. These "master transcription factors" (MTFs) are often essential for cancer cell survival and represent vulnerabilities that can be exploited therapeutically. The current approaches to identify candidate MTFs examine super-enhancer associated transcription factor-encoding genes with high connectivity in network models. This relies on chromatin immunoprecipitation-sequencing (ChIP-seq) data, which is technically challenging to obtain from primary tumors, and is currently unavailable for many cancer types and clinically relevant subtypes. In contrast, gene expression data are more widely available, especially for rare tumors and subtypes where MTFs have yet to be discovered. We have developed a predictive algorithm called CaCTS (Cancer Core Transcription factor Specificity) to identify candidate MTFs using pan-cancer RNA-sequencing data from The Cancer Genome Atlas. The algorithm identified 273 candidate MTFs across 34 tumor types and recovered known tumor MTFs. We also made novel predictions, including for cancer types and subtypes for which MTFs have not yet been characterized. Clustering based on MTF predictions reproduced anatomic groupings of tumors that share 1-2 lineage-specific candidates, but also dictated functional groupings, such as a squamous group that comprised five tumor subtypes sharing 3 common MTFs. PAX8, SOX17, and MECOM were candidate factors in high-grade serous ovarian cancer (HGSOC), an aggressive tumor type where the core regulatory circuit is currently uncharacterized. PAX8, SOX17, and MECOM are required for cell viability and lie proximal to super-enhancers in HGSOC cells. ChIP-seq revealed that these factors co-occupy HGSOC regulatory elements globally and co-bind at critical gene loci including MUC16 (CA-125). Addiction to these factors was confirmed in studies using THZ1 to inhibit transcription in HGSOC cells, suggesting early down-regulation of these genes may be responsible for cytotoxic effects of THZ1 on HGSOC models. Identification of MTFs across 34 tumor types and 140 subtypes, especially for those with limited understanding of transcriptional drivers paves the way to therapeutic targeting of MTFs in a broad spectrum of cancers.

cancer biology↗

bioRxiv2019

Non-coding Somatic Mutations Converge on the PAX8 Pathway in Epithelial Ovarian Cancer

Transcriptional regulation is highly disease and cell-type specific. We performed H3K27ac chromatin immunoprecipitation and transcriptomic sequencing in primary tumors for the four different subtypes of invasive epithelial ovarian cancer (OC). Histotype-specific regulatory elements (REs) were enriched in enhancers (P<0.001). In silico prediction of putative target genes for histotype-specific REs identified genes (WFDC2, P=5.5x10-5) and pathways (PI3K-Akt signaling, P<0.002) known to be involved in OC development. Some genes (e.g. PAX8 and CA125) are associated with super-enhancers (SEs) in all OCs, while others are histotype-specific, including PPP1R3B which is associated with SEs specific to clear cell OC. Integrated analysis of active chromatin landscapes with somatic single nucleotide variants (SNVs) from whole genome sequencing (WGS) of 232 primary OCs identified frequently mutated REs, including the KLF6 promoter (P=8.2x10-8) and a putative enhancer at chromosome 6p22.1 (P<0.05). In high-grade serous OCs, somatic SNVs clustered in binding sites for the PAX8 binding partner TEAD4 (P=6x10-11), while the collection of cis regulatory elements associated with PAX8 was the most frequently mutated set of enhancers in OC (P=0.003). Functional analyses supported our findings: Knockdown of PPP1R3B in clear cell OC cells significantly reduced intracellular glycogen content, a signature feature of this histotype; and stable knockout of a 635 bp region in the 6p22.1 enhancer induced downregulation of two predicted target genes, ZSCAN16 and ZSCAN12 (P=6.6 x 10-4 and P=0.02). In summary, we have characterized histotype-specific epigenomic and transcriptomic landscapes in OC and defined likely functional REs based on somatic mutation analysis of ovarian tumors.

cancer biology↗