Genome-wide CRISPR screens have systematically identified genes required for cancer cell survival, yet these studies are typically performed under standardized conditions that do not fully recapitulate the physiological stresses encountered within the tumor microenvironment. In a recent issue of Nature Genetics, Cheruiyot and colleagues perform genome-wide loss-of-function screens under inflammatory conditions induced by interferon-β (IFN-β), interferon-γ (IFN-γ), and tumor necrosis factor (TNF), revealing that distinct cytokines impose different genetic requirements for tumor cell survival. The study shows that inflammatory signaling reshapes genetic dependency landscape in a cytokine-specific manner. Mechanistic analyses identify the glycosylphosphatidylinositol (GPI) transamidase complex and FITM2 as representative examples of genes that become selectively required under inflammatory stress by maintaining membrane protein maturation, endoplasmic reticulum homeostasis, and resistance to oxidative stress. These findings broaden our understanding of how inflammatory cytokines influence tumor cell biology beyond transcriptional regulation and immune recognition. More broadly, the study highlights the value of incorporating physiologically relevant conditions into functional genetic screens, suggesting that conventional dependency maps capture only part of the genetic requirements for tumor survival. Applying similar approaches to other microenvironmental stresses-including hypoxia, metabolic competition, extracellular matrix remodeling, and stromal signaling-may uncover additional therapeutic opportunities for cancer immunotherapy.
Aging and cancer reflect distinct but interconnected manifestations of cellular stress responses shaped by context-specific molecular and environmental factors. Senescence initially restrains malignancy through stable cell-cycle arrest and immune clearance, yet persistence of senescent cells and the senescence-associated secretory phenotype (SASP) paradoxically drives tumor progression, therapeutic resistance, and immune evasion via chronic inflammation and metabolic reprogramming. Functional genomic screening has evolved from bulk RNA interference to precision clustered regularly interspaced short palindromic repeats (CRISPR) modalities, including knockout, interference, activation, and base editing, and onward to single-cell and in vivo platforms. These technological advances have transformed our capacity to dissect cellular state transitions and network dependencies with unprecedented resolution. Here, we trace this technological evolution and synthesize its impact on aging and cancer research, with emphasis on chromatin regulation, metabolic rewiring, the SASP, nucleocytoplasmic transport, therapeutic response, and the tumor microenvironment. We emphasize that the current evidence derives predominantly from preclinical functional screens and computational predictions. We propose shifting from gene-centric discovery toward context-dependent network analysis. Integrating precise editing, in vivo screening, single-cell multi-omics, and emerging artificial intelligence (AI)-assisted design may provide information and a design basis for future combined strategies that simultaneously target vulnerabilities in senescent cells and malignant populations.
Bo Fan, Aiwei Wu, Xue Pan et al.· Ageing and Cancer Research &...· 0 citations
Unresolved replication stress (RS) or DNA damage, and inflammation often lead to genomic instability – a feature of many diseases including cancer. Therefore, understanding the molecular foundation of these phenomena is crucial for identifying potential biomarkers or therapeutic targets. Our lab has identified an uncharacterized protein that is symmetrically dimethylated by PRMT5 under hydroxyurea (HU)-induced RS. Therefore, we hypothesize that C11ORF68 is involved in inflammatory and RS response.
MCF10A normal breast epithelial cells was used. Depletion of C11ORF68 using siRNA and dicer siRNA (dsiRNA) systems were used to evaluate its function. RNA-seq, qPCR, western blotting and immunofluorescence were used to evaluate the basal and HU-induced inflammatory response.
RNA-seq of C11ORF68-depleted cells displayed a basal and RS-induced upregulation of several inflammatory response pathways including interferon alpha and gamma response, and TNF signaling via NF-kB. Selected genes such as CXCL10 and IFIT1 were validated to be upregulated at both basal and RS conditions. This increase in inflammatory gene transcription may be due accumulation of dsDNA in the cytoplasm of C11ORF68-depleted cells. The mechanism of dsDNA leakage into the cytoplasm is currently being investigated. Surprisingly, the observation of cytoplasmic dsDNA was not accompanied with increased DNA damage as there was no basal increase in DNA damage or RS markers such as γH2AX and 53BP1. However, C11ORF68-depleted cells exhibited attenuated activation of DNA damage/RS pathways such as lowered PIKK activation and less repair foci formation after addition of DNA damaging/RS agents.
Our data suggests that C11ORF68 potentially acts as a repressor of both basal and RS-induced inflammatory response. C11ORF68 may also be a mediator of early DDR. Mechanisms behind how a nucleolar protein plays role in regulating these phenomena are underway, which may help us understand the involvement of a nucleolar protein in controlling inflammatory and DNA damage/RS responses.
Toh Chin Min Clarissa, Hoang Mai Phuong, Hong Liang, Anand Jeyasekharan. C11ORF68 is a repressor of inflammatory response under normal and replication stress conditions [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr P92.
Toh Chin Min Clarissa, H. M. Phuong, Hong Liang et al.· Cancer Research· 0 citations
Chemotherapy resistance remains a major challenge in cancer treatment, driven by cancer cells' ability to acquire adaptive properties, rewire signaling pathways, and alter chromatin structure to evade drug-induced cytotoxicity. Because these processes rely heavily on epigenetic mechanisms that regulate chromatin organization and transcriptional plasticity, epigenetic regulators have emerged as key contributors to chemotherapy resistance. To investigate resistance to paclitaxel, one of the most widely used chemotherapeutic agents in triple-negative breast cancer (TNBC), we employed an epigenome-focused knockout library (EPIKOL), a CRISPR-Cas9-based library, designed to systematically disrupt genes involved in chromatin regulation. Chemoresistant cell lines were generated through a stepwise dose-escalation protocol that recapitulates clinically relevant drug adaptation. However, these resistant cells exhibit a multidrug-resistant (MDR) phenotype, posing significant challenges for efficient viral transduction and the selection of stable cell populations. In this study, we describe key methodological steps for achieving high-efficiency lentiviral transduction and selection, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models. Following the described protocol, an epigenome-wide CRISPR screen was conducted on chemoresistant TNBC cells, and novel epigenetic regulators of chemoresistance were identified. This protocol provides a robust framework for identifying epigenetic regulators that contribute to acquired paclitaxel resistance using a CRISPR-based loss-of-function approach.
O. Yedier-Bayram, Elif Ayca Guvener, T. Bagci-Onder· Journal of Visualized Experi...· 0 citations
Polyploid giant cancer cells (PGCCs) are increasingly recognized as major drivers of therapy resistance and tumor relapse, yet the conserved molecular programs underlying their persistence remain incompletely defined. To identify genes consistently deregulated across eight independent datasets, we performed an integrative transcriptomic analysis of PGCCs derived from prostate, ovarian, and breast cancers. By focusing on consistently up- or down-regulated genes that were expressed in at least five datasets and showed a concordant direction of expression across more than 70% of datasets and met a significance threshold of adjusted p < 0.05, we defined the core regulatory architecture stabilizing the PGCC state under therapeutic stress. Our analysis reveals that PGCCs exhibit a paradoxical ranscriptomic signature consistent with cytolytic activity alongside reduced immune detection. These cells activated pro-inflammatory cytokine and chemokine signaling while simultaneously engaging immune-evasion mechanisms, including PD-L1-associated and virus-like escape programs. Concurrently, PGCCs displayed transcriptional features characteristic of immune-privileged cellular states, including embryonic development, reproductive programs, senescence-associated survival, apoptosis resistance, and deep dormancy marked by coordinated suppression of major housekeeping processes. Notably, PGCCs also activated neuronal differentiation and neurodegeneration-associated pathways, including axon guidance, neurogenesis, and calcium signaling. This neuron-like, calcium-dependent stress adaptation program may further enhance immune privilege and long-term survival capacity. We propose that PGCCs represent an immune-adaptive polyploid survival state in which inflammatory and ontogenetic pathways are repurposed to support immune evasion and tumor persistence. By identifying actionable vulnerabilities within calcium signaling, neuronal mimicry, and checkpoint-associated pathways, this study provides a framework for therapeutic strategies aimed at dismantling the PGCC reservoir and preventing tumor relapse.
O. Anatskaya, Alexander E. Vinogradov· International Journal of Mol...· 0 citations
Oncogenic contexts shape synthetic lethal vulnerabilities and suppressor interactions that underlie drug resistance mechanisms, yet this context-dependence remains poorly characterized. Combinatorial CRISPR screens offer a principled approach to surface these interactions at scale with direct implications for therapeutic targeting and drug combination strategies. Using the In4mer 4-plex Cas12a knockout platform, we screened pairwise genetic interactions across RTK signaling and glycosylation networks in oncogene-stratified cancer cells (Lin et al., BioRxiv 2025), identifying hits enriched in the glycosylation machinery. Building on these findings, focused validation screens across cancer cell lines with KRAS-, BRAF-, and EGFR-drivers confirm high-effect genetic interactions concentrated in the glycosylation module across oncogenic backgrounds, revealing both synthetic lethal dependencies and suppressor interactions. Synthetic lethal candidates linking ER stress regulation and glycosyltransferase activity were supported by dependency-expression correlations in cancer cell lines, and recurrent amplification of glycosylation machinery in patient tumors is associated with poor clinical outcomes. Preliminary cell-based validation assays further corroborate a suppressor interaction, implicating a potential resistance mechanism to EGFR inhibition. Together, these findings demonstrate the N-glycosylation module as a recurrent hub of context-dependent genetic interactions and combinatorial CRISPR screening as a scalable approach for identifying therapeutic targets and resistance mechanisms in oncogene-driven cancers.
Subin Kim, Chenchu Lin, Sabriyeh Alibai, Iulia Veronica Gheorghe, Jui Hsuan Chou, Traver Hart. Combinatorial CRISPR screening reveals synthetic lethal and suppressor interactions in RTK signaling and glycosylation networks [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B093.
Subin Kim, Chenchu Lin, Sabriyeh Alibai et al.· Clinical Cancer Research· 0 citations
The cGAS/STING pathway is a central innate immune DNA-sensing system that links aberrant DNA species to innate immune and stress-response transcriptional programs and has emerged as a key regulator of tumor-immune interactions. In cancer, pathway outputs are shaped by interconnected downstream signaling modules, including type I IFN, NF-κB, autophagy, and stress-metabolic checkpoints, as well as by stringent spatial and biochemical regulation of both cGAS and STING. When activation is acute and appropriately compartmentalized, cGAS/STING signaling promotes antitumor immunity across multiple cellular compartments in the tumor microenvironment, supporting DC cross-priming and cytotoxic lymphocyte responses. In contrast, chronic or dysregulated activation rewires downstream signaling toward stress-adaptive and inflammatory programs that promote tumor progression, metastasis, and immune dysfunction, including deleterious effects in lymphocytes and the induction of suppressive myeloid and B cell populations. Here, we examine how context determines the consequences of cGAS/STING activation in cancer, review emerging therapeutic strategies that modulate this pathway, and discuss how its antitumor potential can be maximized while minimizing systemic toxicity and immune dysregulation.
Yi Wang, J. Angulo-Lozano, Yueqi Wang et al.· Journal of Clinical Investig...· 0 citations