Biology subjects

Spisak, S.

Publications and source records attributed to Spisak, S..

15 recordsLinked to original sources

bioRxiv2026

One-carbon metabolism controls colorectal cancer cell state and chemosensitivity through chromatin remodeling

Folate is co-administered with fluoropyrimidines in colorectal cancer to potentiate thymidylate synthase inhibition, yet its therapeutic effects are paradoxically inconsistent across disease and treatment settings. Here we show that one-carbon metabolism remodels epithelial identity before treatment and thereby alters fluoropyrimidine response. Folate restoration shifted colorectal cancer cells along a polarized epithelial axis from a stem-like toward a differentiated state, whereas folate deprivation favored the opposite configuration. These pretreatment states differed markedly in 5-fluorouracil sensitivity. Folate restoration increased sensitivity across colorectal cancer cell lines and patient-derived organoids, whereas folate deprivation or methotrexate increased therapy-induced senescence. State-dependent sensitivity was not explained by proliferation alone, as stem-like cells were least responsive under the same conditions in which their S-phase occupancy was highest. Mechanistically, folate restoration increased methylation potential, supported EZH2-dependent H3K27me3 remodeling, reorganized chromatin accessibility and transcription-factor engagement, and reduced canonical WNT activity. In human colorectal tissue, stem-like program activity was higher and differentiation program activity lower in carcinoma than in normal mucosa, with adenomas intermediate, a pattern opposite to folate-induced remodeling. Folate is therefore not merely a cofactor for thymidylate synthase inhibition but an instructive metabolic regulator of the epithelial state that stratifies fluoropyrimidine response.

molecular biology↗

bioRxiv2024

Chemical perturbations impacting histone acetylation governcolorectal cancer differentiation

Dysregulated epigenetic programs that restrict differentiation, reactivate fetal genes, and confer phenotypic plasticity are critical to colorectal cancer (CRC) development. By screening a small molecule library targeting epigenetic regulators using our dual reporter system, we found that inhibiting histone deacetylase (HDAC) 1/2 promotes CRC differentiation and anti-tumor activity. Comprehensive biochemical, chemical, and genetic experiments revealed that on-target blockade of the HDAC1/2 catalytic domain mediated the differentiated phenotype. Unbiased profiling of histone posttranslational modifications induced by HDAC1/2 inhibition nominated acetylation of specific histone lysine residues as potential regulators of differentiation. Genome-wide assessment of implicated marks indicated that H3K27ac gains at HDAC1/2-bound regions associated with open chromatin and upregulation of differentiation genes upon HDAC1/2 inhibition. Disrupting H3K27ac by degrading acetyltransferase EP300 rescued HDAC1/2 inhibitor-mediated differentiation of a patient-derived CRC model using single cell RNA-sequencing. Genetic screens revealed that DAPK3 contributes to CRC differentiation induced by HDAC1/2 inhibition. These results highlight the importance of specific chemically targetable histone modifications in governing cancer cell states and epigenetic reprogramming as a therapeutic strategy in CRC. BRIEF SUMMARYHDAC1/2 inhibition promotes colorectal cancer differentiation via gains in H3K27ac, which can be reversed by blocking its acetyltransferase EP300.

cancer biology↗

bioRxiv2023

Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer

Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators that regulate these key cellular programs in CRC, we developed an endogenous reporter system by genome-editing human CRC cell lines with knock-in fluorescent reporters at the SOX9 and KRT20 locus to report aberrant stem cell-like activity and differentiation, respectively, and then performed pooled genetic perturbation screens. Constructing a dual reporter system that simultaneously monitored aberrant stem cell-like and differentiation activity in the same CRC cell line improved our signal to noise discrimination. Using a focused-library CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs, we identified factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency in CRC and required for in vivo growth of human CRC models. These studies highlight the utility of a biologically designed endogenous reporter system to uncover novel therapeutic targets for drug development.

cancer biology↗

bioRxiv2023

Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors.

Gastroesophageal adenocarcinoma (GEA) is an aggressive, often lethal, malignancy that displays marked chromosomal instability (CIN). To understand adaptive responses that enable CIN, we analyzed paired normal, premalignant, and malignant gastric lesions from human specimens and a carcinogen-induced mouse model, observing activation of replication stress, DNA damage response (DDR), and cell cycle regulator p21 in neoplastic progression. In GEA cell lines, expression of DDR markers correlated with ploidy abnormalities, including high-level focal amplifications and whole-genome duplication (WGD). Moreover, high expression of DNA damage marker H2AX correlated with CIN, WGD, and inferior patient survival. By developing and implementing a composite diagnostic score that incorporates TP53 mutation status, ploidy abnormalities, and H2AX expression, among other genomic information, we can identify GEA cell lines with enhanced sensitivity to DDR pathway inhibitors targeting Chk1/2 and Wee1. Anti-tumor properties were further augmented in combination with irinotecan (SN38) but not gemcitabine chemotherapy. These results implicate specific DDR biomarkers and ploidy abnormalities as diagnostic proxy that may predict premalignant progression and response to DDR pathway inhibitors.

cancer biology↗

bioRxiv2023

Nucleotide excision repair deficiency is a targetable therapeutic vulnerability in clear cell renal cell carcinoma.

PurposeDue to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. Experimental DesignWe used functional assays that detect UV-induced 6-4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. ResultsFunctional assays showed NER deficiency in ccRCC cells. Irofulven sensitivity increased in some cell lines. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. ConclusionsccRCC cell line based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells. Translational relevanceDNA repair deficiencies can be therapeutically targeted by synthetic lethal-based strategies in cancer. However, clear cell renal cell carcinoma (ccRCC) has not benefitted from this therapeutic approach due to a lack of evidence for the presence of specific DNA repair pathway deficiencies. Here, we demonstrate that ccRCC harbors a therapeutically targetable DNA repair pathway aberration, nucleotide excision repair (NER) deficiency. ccRCC cell lines displayed robust signs of NER deficiency as determined by functional assays and some of these cell lines also displayed NER deficiency induced mutational signatures. These cell lines are also sensitive to irofulven, an abandoned anticancer agent that creates DNA lesions which can only be repaired by the NER pathway. We estimate that up to 10% of ccRCC cases may respond to NER-directed therapy with irofulven based on NER deficiency associated mutational signatures and PTGR1 expression levels, which is an enzyme required to activate irofulven.

cancer biology↗

bioRxiv2023

Epigenetic Regulation Explains The Functionality Behind Colon Cancer Specific Biomarker Septin9

Despite advancements in early cancer detection and prevention methods, colorectal cancer (CRC) remains a significant global health problem. It is the third most common cancer and the second leading cause of cancer-related deaths worldwide. Additionally, there has been a marked increase of incidence in young adults, and the reasons for this tendency are not fully understood. Therefore, the need for more effective diagnostic methods of assessing disease risk at early stage is crucial. One of the newly developed blood-based circulating biomarkers with promising potential is the short hypermethylated region located at the Septin9 intronic region. Several clinical studies have proven its performance and applicability. However, the molecular mechanism behind this consistent and recurrent feature present in most of the CRC and related precancerous stages and why it is specific and advantageous for CRC development are poorly understood. Here, we used comprehensive epigenetic and gene expression profile analyses from different sources of human clinical samples and cell line data to link specific hypermethylation events at the Septin9 intronic loci, which initiate alternative transcription of the Septin9 gene. Through our investigation of TCGA-COAD RNA-seq samples (n=287), we found that there was no significant difference in global Septin9 levels between normal and tumor samples. However, we did observe a significant alteration in the transcript variant ratio between v1 and v2, suggesting the use of an alternative promoter. Our findings were further supported by our analysis of ATAC-seq data, which revealed that the v2 promoter conferred higher chromatin accessibility, which correlated with the expression of the v2 isoform. However, this was not supported by promoter or enhancer activity as measured by H3K27ac signals. Hypermethylation at the v2 promoter was confirmed in tumor samples, providing a possible explanation for the switch in variants. Protein sequence analysis confirmed small differences between Septin9 variant A(v1) and B(v2). However, AlphaFold2 indicates a substantial difference at the N terminus, which could impact protein phosphorylation. We hypothesize, that variant A (v1) and variant B (v2) are required for normal cell functions but shifting the balance towards v1 is more favourable for the tumor. Although very little is known about Septin9 and its function in CRC biology, we are confident that our study will help to emphasize the importance of understanding regulatory mechanisms behind tumor-specific biomarkers and helps to improve the application.

cancer biology↗

bioRxiv2022

Porcine cytomegalovirus detection by nanopore-based metagenomic sequencing in a Hungarian pig farm

The rapid diagnosis of infectious diseases has an essential impact on their control, treatment and recovery. Oxford Nanopore Technologies (ONT) sequencing opens up a new dimension in applying clinical metagenomics. In a large-scale pig farm in Hungary, four fattening and one piglet nasal swab pooled samples were sequenced using ONT for metagenomic analysis. Long reads covering 53.69% of the porcine cytomegalovirus genome were obtained in the piglet sample. The 650 bp long read matching the glycoprotein B gene of the virus is sequentially most similar to Japanese, Chinese and Spanish isolates.

genomics↗

bioRxiv2022

Diagnostic tool to identify and treat DNA repair deficient gastroesophageal adenocarcinomas

Background and aimsDNA repair deficiency is a common feature of cancer. Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in solid tumors. HR deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of DNA repair pathway deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Furthermore, whether DNA repair deficient GEA have enhanced responsiveness to platinum chemotherapy and sensitivity to PARP inhibitors is not well characterized. MethodsUsing whole exome and genome sequencing data, we measured various HR deficiency-associated mutational signatures in patient specimen of gastric, esophageal and colorectal cancer specimens and gastric cancer cell lines. Gold-standard immunofluorescence assays were used to confirm HR and NER deficiency in cancer cell lines. The relationship between PARP inhibitor treatment and tumor response was evaluated in patients with gastric cancer. Drug sensitivity was determined using standard in vitro cell culture assays. Single-cell RNA-sequencing was performed to evaluate gastric cancer response to commonly used chemotherapeutics. ResultsWe found that a significant subset of GEA, but very few colorectal tumors, show evidence of HR deficiency by mutational signature analysis (HRD score). Gastric cancer cell lines with high HRD mutational signature scores demonstrated functional HR deficiency by RAD51 assay and increased sensitivity to platinum and PARP inhibitors. There was a positive association between HRD scores and tumor response in patients with gastric cancer treated with a PARP inhibitor on a clinical trial. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by DDB2 proteo-probe assay. Single-cell RNA-sequencing revealed that, in addition to inducing general apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, which may explain the outlier sensitivity. ConclusionA subset of upper gastrointestinal tumors have genomic features of HR and NER deficiency and therefore may be more likely to benefit from platinum chemotherapy and PARP inhibition.

cancer biology↗

bioRxiv2022

A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and strongly hormone receptor positive. Yet, they are relatively resistant to tamoxifen and are associated with inferior long-term outcomes compared to invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. Through a comprehensive analysis of the epigenome of ILC in pre-clinical models and clinical samples we found that compared to IDC, ILC has a distinct chromatin state that is linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This results in an ILC-unique FOXA1-estrogen receptor (ER) axis that promotes the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1-ER axis leads to retained ER chromatin binding after tamoxifen treatment thereby facilitating tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the auto-induction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, we show that ILC is characterized by a unique cell state and FOXA1-ER axis that dictate tumor progression and offer a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC to optimize endocrine treatments in this breast cancer subtype.

cancer biology↗

bioRxiv2021

Mobile antimicrobial resistance genes in probiotics

Even though people around the world tend to consume probiotic products for their beneficial health effects on a daily basis, recently, concerns were outlined regarding the uptake and potential intestinal colonisation of the bacteria that they transfer. These bacteria are capable of executing horizontal gene transfer (HGT) which facilitates the movement of various genes, including antimicrobial resistance genes (ARGs), among the donor and recipient bacterial populations. Within our study, 47 shotgun sequencing datasets deriving from various probiotic samples (isolated strains and metagenomes) were bioinformatically analysed. We detected more than 70 ARGs, out of which rpoB mutants conferring resistance to rifampicin, tet(W/N/W) and potentially extended-spectrum beta-lactamase (ESBL) coding TEM-116 were the most common. Numerous ARGs were associated with integrated mobile genetic elements, plasmids or phages promoting the HGT. Our findings raise clinical and public health concerns as the consumption of probiotic products may lead to the transfer of ARGs to human gut bacteria.

genomics↗

bioRxiv2020

H3k27ac-HiChIP in prostate cell lines identifies risk genes for prostate cancer susceptibility

Genome-wide association studies (GWAS) have identified more than 140 prostate cancer (PrCa) risk regions which provide potential insights into causal mechanisms. Multiple lines of evidence show that a significant proportion of PrCa risk can be explained by germline causal variants that dysregulate nearby target genes in prostate-relevant tissues thus altering disease risk. The traditional approach to explore this hypothesis has been correlating GWAS variants with steady-state transcript levels, referred to as expression quantitative trait loci (eQTLs). In this work, we assess the utility of chromosome conformation capture (3C) coupled with immunoprecipitation (HiChIP) to identify target genes for PrCa GWAS risk loci. We find that interactome data confirms previously reported PrCa target genes identified through GWAS/eQTL overlap (e.g., MLPH). Interestingly, HiChIP identified links between PrCa GWAS variants and genes well-known to play a role in prostate cancer biology (e.g., AR) that are not detected by eQTL-based methods. We validate these findings through CRISPR interference (CRISPRi) perturbation of the variant-containing regulatory elements for NKX3-1 and AR in the LNCaP cell line. Our results demonstrate that looping data harbor additional information beyond eQTLs and expand the number of PrCa GWAS loci that can be linked to candidate susceptibility genes.

genomics↗

bioRxiv2019

Genomic aberration based molecular signatures efficiently characterize homologous recombination deficiency in prostate cancer

BackgroundProstate cancers with mutations in genes involved in homologous recombination (HR), most commonly BRCA2, respond favorably to PARP inhibition and platinum-based chemotherapy. It is not clear, however, whether other prostate tumors that do not harbor deleterious mutations in these particular genes can similarly be deficient in HR, rendering them sensitive to HR-directed therapies.\n\nTo identify a more comprehensive set of prostate cancer cases with homologous recombination deficiency (HRD) including those cases that do not harbor mutations in known HR genes.\n\nHRD levels can be estimated using various mutational signatures derived from next-generation sequencing data. We used this approach to determine whether prostate cancer cases display clear signs of HRD in somatic tumor biopsies. Whole genome (n=311) and whole exome sequencing data (n=498) of both primary and metastatic prostate adenocarcinomas (PRAD) were analyzed.\n\nResultsKnown BRCA-deficient samples showed robust signs of HR-deficiency associated mutational signatures. HRD-patterns were also detected in a subset of patients who did not harbor germline or somatic mutations in BRCA1/2 or other HR related genes. Patients with HRD signatures had a significantly worse prognosis than patients without signs of HRD.\n\nConclusionsThese findings may expand the number of cases likely to respond to PARP-inhibitor treatment. Based on the HRD associated mutational signatures, 5-8 % of prostate cancer cases may be good candidates for PARP-inhibitor treatment (including those with BRCA1/2 mutations).

cancer biology↗

bioRxiv2019

Allelic imbalance reveals widespread germline-somatic regulatory differences and prioritizes risk loci in Renal Cell Carcinoma

Determining the function of non-coding regulatory variants in cancer is a key challenge transcriptional biology. We investigated genetic (germline and somatic) determinants of regulatory mechanisms in renal cell carcinoma (RCC) using H3K27ac ChIP-seq data in 10 matched tumor/normal samples and RNA-seq data from 496/66 tumor/normal samples from The Cancer Genome Atlas (TCGA). Unsupervised clustering of H3K27ac activity cleanly separated tumor from normal individuals, highlighting extensive epigenetic reprogramming during transformation. We developed a novel method to test each chromatin feature for evidence of an allele-specific quantitative trait locus (asQTL) and evaluate tumor/normal differences in allele-specificity (d-asQTLs) while modelling local structural variation and read overdispersion. At an FDR of 5%, we identified 1,356 unique asQTL chromatin peaks in normal tissues; 2,868 in tumors; and 1,054 d-asQTLs (primarily imbalanced in tumor). The d-asQTL peaks were significantly enriched for RCC genome-wide association study (GWAS) heritability (32x, P=1.8x10-3), more so than any other functional feature including all H3K27ac peaks (12x), super-enhancers (5x), and asQTL genes (4x). Intersection of asQTLs with RCC GWAS loci identified putative functional features for 6/17 known loci including tumor-specific activity at SCARB1, a cholesterol metabolism mediator, which has recently been implicated in RCC progression. We validated the asQTL variant through CRISPR interference (CRISPRi) and demonstrated a concomitant allelic effect on the overlapping enhancer and on downstream SCARB1 expression. Knockdowns of master transcription factors (TFs) involved in the hypoxia pathway altered the expression of SCARB1 in a kidney cancer cell line, consistent with a variant-TF interaction. Genome-wide, d-asQTLs were significantly enriched for tumor-specific binding of hypoxic transcription factors, implicating a more general mechanism for polygenic germline-somatic interaction.

genetics↗

bioRxiv2019

Family aggregation analysis reveals a heritable background of equine grass sickness (dysautonomia)

Equine grass sickness (also known as dysautonomia) is a life-threatening polyneuropathic disease affecting horses with approx. 80% mortality. Since its first description over a hundred years ago, several factors including phenotypic, environmental, management, climate, and intestinal microbiome) have been associated with increased risk of dysautonomia. But despite the extensive research on dysautonomia, its causative factors have yet been identified. A retrospective pedigree and phenotype based genetic epidemiological study was performed to analyze the associations of disease occurrence and the kinship in a Hungarian large scale stud. The pedigree data set containing 1233 horses with 49 affected animals was used in the analysis. The first finding was that among the descendants of some stallions the proportion of affected animals are unexpectedly high, with a maximum of 25% of a stallions descendants affected. Animals with affected siblings have higher odds to be a case (OR: 1.27, 95% CI: 1.01-1.57, p=0.033). Among males in the affected population the odds of dysautonomia is higher than in females (OR: 1.76, 95% CI: 0.95-3.29, p=0.057). Significant familial clustering was observed among the affected animals (GIF p=0.001). Further subgroups were identified with significant (p<0.001) aggregation among close relatives using kinship-based methods. Our analysis of the data and the observed higher disease frequency in males suggests that dysautonomia may have X-linked recessive inheritance as a causal factor. This is the first study providing ancestry data and suggesting a genetic contribution to the likely multifactorial causes of the disease.

genetics↗

bioRxiv2019

A subset of lung cancer cases shows robust signs of homologous recombination deficiency associated genomic aberration based molecular signatures

BackgroundConsistent with their assumed mechanism of action, PARP inhibitors show significant therapeutic efficacy in breast, ovarian and prostate cancer, which are the solid tumor types most often associated with the loss of function of key homologous recombination genes. It remains unknown, however, how frequent homologous recombination deficiency (HRD) is in other solid tumor types. Since it is well established, that HRD induces specific DNA aberration profiles and genomic scars that can be captured by various next-generation sequencing (NGS) based biomarkers, it is possible to assess the presence or absence of this DNA repair pathway aberration in any given tumor biopsy. MethodsWe derived the various HRD associated mutational signatures from whole genome and whole exome sequencing data in the lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) cases from TCGA, in a patient of ours with stage IVA lung cancer with exceptionally good response to platinum-based therapy and in lung cancer cell lines. ResultsWe have found evidence that a subset of the investigated cases shows robust signs of HR deficiency, some of which exhibiting similar patterns to those with a complete loss of function of either BRCA1 or BRCA2 genes, however, without any signs of genetic alterations being present in either of those genes. The extreme platinum responder case also showed a robust HRD associated genomic mutational profile. HRD associated mutational signatures were also associated with PARP inhibitor sensitivity in lung cancer cell lines. ConclusionsLung cancer cases with high levels of HRD associated mutational signatures could be candidates for PARP inhibitor treatment, and in general, the prioritization of patients for clinical trials might be achieved using the combined analysis of the HRD-related next-generation sequencing-based mutational signatures.

cancer biology↗