Biology subjects

Cingoz, A.

Publications and source records attributed to Cingoz, A..

6 recordsLinked to original sources

bioRxiv2026

Coupling Raman Spectroscopy and Acoustic Microscopy to Characterize Glioblastoma Resistance

Therapy resistance in glioblastoma is usually described in genetic or metabolic terms. Here we ask whether it also has a measurable biophysical form. Combining Raman spectroscopy with scanning acoustic microscopy on TRAIL-sensitive and TRAIL-resistant glioblastoma cells, we find that resistance carries both a biochemical and a mechanical signature: the resistant cells differ at protein, lipid and nucleic-acid bands, contain 4.1-fold more neutral lipid, and hold a high acoustic impedance that does not change when the drug is applied, while sensitive cells stiffen as they die. Because acoustic impedance is an absolute quantity and neither measurement requires a label, the approach offers a route to recognising resistant populations from their physical state alone.

biophysics↗

bioRxiv2025

Functional Genomic Screens Reveal RBBP4 as a Key Regulator of Cell Cycle Progression in TMZ-Resistant Glioblastoma

Temozolomide (TMZ) remains the standard of care for glioblastoma; however, its efficacy is frequently influenced by epigenetic mechanisms, notably the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. While MGMT promoter hypermethylation is associated with enhanced responsiveness to TMZ, additional epigenetic determinants of TMZ resistance remain largely undefined. In this study, we established TMZ-resistant glioblastoma cell lines that consistently maintained their resistant phenotype both in vitro and in vivo. Transcriptomic analyses revealed a marked upregulation of MGMT expression in these models. To systematically investigate the epigenetic regulators governing TMZ resistance and cell survival, we conducted CRISPR/Cas9-based functional genomic screens using our focused Epigenetic Knock-Out Library (EPIKOL), which targets 800 chromatin regulators alongside selected positive and negative controls. These unbiased screens validated MGMT as a primary mediator of TMZ resistance, confirming the robustness of our approach. Moreover, dropout screens across multiple resistant cell line models identified Retinoblastoma Binding Protein 4 (RBBP4) as a critical vulnerability. Notably, RBBP4 knockout significantly impaired cell proliferation without affecting MGMT expression, suggesting a distinct mechanism supporting the survival of TMZ-resistant glioblastoma cells. Subsequent transcriptomic profiling following RBBP4 loss demonstrated significant downregulation of cell cycle pathways, particularly the G2/M checkpoint. Live-cell imaging and immunofluorescence analyses further revealed increased cell size and multinucleation in RBBP4-deficient cells, indicative of disrupted mitotic progression. Collectively, our results identify RBBP4 as a key regulator of cell cycle progression and survival in TMZ-resistant glioblastoma and highlight its potential as a novel epigenetic target for therapeutic intervention in recurrent disease.

cancer biology↗

bioRxiv2024

Chromatin-focused genetic and chemical screens identify BRPF1 as a targetable vulnerability in Taxol-resistant triple-negative breast cancer

Triple-negative breast cancer (TNBC) stands out as a particularly aggressive and frequently recurring form of breast cancer. Due to the absence of hormone receptors, the available treatment avenues are constrained, making chemotherapy the primary approach. Unfortunately, the development of resistance to chemotherapy poses a significant challenge, further restricting the already limited therapeutic alternatives for recurrent cases. Understanding the molecular basis of chemotherapy resistance in TNBC is pivotal for improving treatment outcomes. Here, we generated two different Taxol-resistant TNBC cell lines with a dose-escalation method to mimic chemotherapy resistance in vitro. These cells exhibited hallmark features of resistance, including reduced cell growth, altered morphology, and evasion of apoptosis. Transcriptome analysis uncovered elevated ABCB1 expression and multidrug-resistant phenotype in the resistant cells. To comprehensively investigate the key epigenetic regulators of Taxol resistance, we conducted chromatin-focused genetic and chemical screens and pinpointed Bromodomain and PHD Finger Containing 1 (BRPF1) as a novel regulator of Taxol resistance in TNBC cells. Knockout of BRPF1, the reader protein in the MOZ/MORF histone acetyl-transferase complex, but not the other complex members, sensitized resistant cells to Taxol. Additionally, BRPF1 inhibitors, PFI-4 and OF-1, in combination with Taxol significantly reduced cell viability. Transcriptome analysis upon BRPF1 loss or inhibition revealed a negative impact on ribosome biogenesis-related gene sets, resulting in a global decrease in protein translation in Taxol-resistant cells. Our ChIP-qPCR analysis demonstrated that active BRPF1 directly interacts with the ABCB1 promoter, enhancing its expression towards inducing a multidrug-resistant phenotype. Conversely, knockout or inhibition of BRPF1 leads to decreased ABCB1 expression. This dual mechanism critically sensitizes Taxol-resistant TNBC cells to chemotherapy. Our findings uncover a comprehensive molecular framework, highlighting the pivotal role of epigenetic reader protein BRPF1 in Taxol resistance and providing potential avenues for therapeutic intervention in TNBC.

cancer biology↗

bioRxiv2023

HOXB13 alters chromatin accessibility in prostate cancer through interactions with the SWI/SNF complex

HOXB13 is a posterior homeobox protein that is associated with the initiation and growth of prostate cancer (PCa). While most research has focused on the role of HOXB13 on androgen receptor (AR) activity, we demonstrate that HOXB13 is essential to the proliferation of both AR-positive and -negative PCa. Strikingly, HOXB13 is remarkably selective and has almost no effect on non-prostatic tissues. Despite this common essentiality in PCa, HOXB13 activity is markedly different in AR-negative PCa, where interactions with the AP-1 change the HOXB13 cistrome in stem-cell like castration-resistant prostate cancer. We show that HOXB13 activity is commonly mediated by SMARCD2, a member of the mSWI/SNF chromatin remodeling complex. Despite the distinct transcription factor interactions in AR-positive and -negative PCa the HOXB13/SMARCD2 commonly alters chromatin accessibility at HOXB13 binding sites that causes increased proliferation in PCa. Overall, this work demonstrates a novel mechanism of action for HOXB13 and highlights its critical role in AR-negative castration-resistant prostate cancer.

cancer biology↗

bioRxiv2022

Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival

Glioblastoma is the most common and aggressive primary brain tumor with poor prognosis, highlighting an urgent need for novel treatment strategies. In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma.

cancer biology↗

bioRxiv2020

Combined inhibition of KDM6A/B and HDACs exacerbates integrated stress response and mediates therapeutic effects in IDH1-mutant glioma

BackgroundIDH1/2-mutant gliomas are primary brain tumors for which curative treatments are lacking. Mutant IDH-dependent 2-hydroxyglutarate (2-HG) accumulation leads to DNA and histone hypermethylation. Based on this distinct phenotype, we interrogated epigenetic dependencies of IDH-mutant glioma that can be targeted therapeutically. MethodsWe conducted a chemical screen targeting chromatin modifiers in patient derived IDH1-mutant GBM cells. We investigated mechanisms of action of compound hits and their combinations through cell-based functional assays, live-cell imaging, Western blot, CRISPR knockout, RNA-seq and ChIP experiments. The therapeutic concept was validated in vivo using chemical inhibitors GSK-J4 and Belinostat in an orthotopic GBM model. ResultsWe identified the H3K27me3 demethylase (KDM6) inhibitor GSK-J4 and histone deacetylase inhibitor Belinostat as potent, genotype-selective agents against IDH1-mutant glioma. RNA-sequencing on paired wild-type and IDH1R132H cells revealed inhibition of cholesterol biosynthesis and activation of cellular stress in IDH1R132H cells, which were reversible with a mutant IDH1 inhibitor. GSK-J4 caused further repression of cholesterol biosynthesis pathway genes through H3K27me3 deposition and exacerbated the ATF4-mediated integrated stress response. Belinostat inhibited anti-apoptotic pathways through activation of TGF-{beta} signaling and induced cell cycle arrest. Together, the GSK-J4 and Belinostat combination activated DDIT3/CHOP-dependent apoptosis in IDH1-mutant cells and extended survival in an IDH1-mutant orthotopic model in vivo. ConclusionsThese results provide a possible therapeutic approach that exploits epigenetic vulnerabilities of IDH-mutant gliomas. Key points- Combination of GSK-J4 and Belinostat selectively targets IDH1-mutant cells. - GSK-J4 downregulates cholesterol biosynthesis and activates an ATF4-mediated stress response. - Belinostat activates the TGF{beta} pathway, induces G2/M arrest and inhibits anti-apoptotic pathways. Importance of the studyIDH1/2 genes are frequently mutated in low grade glioma and secondary glioblastoma. These tumors exhibit a distinct epigenomic signature with increased DNA and histone methylation; therefore, identifying and exploiting their epigenetic vulnerabilities may lead to effective therapies. We discovered that targeting of KDM6A/6B together with HDACs provides a promising therapeutic approach for IDH1-mutant glioma.

cancer biology↗