The transcription factor MYC is a key regulator of cellular proliferation and metabolism and is frequently dysregulated in malignancies such as multiple myeloma (MM). Despite its clinical relevance, direct therapeutic targeting of MYC remains limited, emphasizing the need to identify upstream regulators that control endogenous MYC expression. To systematically uncover such regulators, we developed a genome-wide CRISPR-Cas9 loss-of-function screening approach, employing a custom-engineered MM reporter cell line (RPMI8226-F11), in which oncogenic MYC protein was endogenously tagged with EGFP (referred to as GFP). This fluorescent readout enabled a direct, quantitative assessment of endogenous MYC expression levels. A pooled genome-wide sgRNA library was introduced, and cells were sorted based on GFP fluorescent intensity to reflect varying MYC levels. Next-generation sequencing of sgRNA distributions across sorted populations enabled the identification of candidate MYC regulators. Validation of screen hits, including the established MYC activator IRF4 and repressor FBXW7, confirmed the reliability of our system. To further dissect regulatory networks, we performed an overrepresentation analysis of target genes, which revealed the enrichment of Mediator complex subunits among MYC activators and ubiquitin-proteasome pathway components among MYC repressors. Functional validation of prioritized hits-MED30 (Mediator complex) and UBE3C (E3 ubiquitin ligase)-demonstrated a strong impact on endogenous MYC levels. Notably, the knockout of UBE3C markedly increased MYC expression, whereas its paralogs, UBE3A and UBE3B, showed no measurable effect, suggesting a specific regulatory role for UBE3C in MM cells. Together, our study provides a comprehensive CRISPR screen-based resource for the discovery of MYC regulators and highlights UBE3C as a potential therapeutic node for modulating MYC expression in MM.

Small cell lung cancer (SCLC) patients frequently experience a remarkable response to first-line therapy. Follow up maintenance treatments aim to control residual tumor cells, but generally fail due to cross-resistance, inefficient targeting of tumor vulnerabilities, or dose-limiting toxicity, resulting in relapse and disease progression. Here we show that SCLC cells, similar to their cells of origin, pulmonary neuroendocrine cells, exhibit low activity in pathways protecting against reactive oxygen species (ROS). When exposed to a thioredoxin reductase 1 (TXNRD1) inhibitor, these cells quickly exhaust their ROS-scavenging capacity, regardless of their molecular subtype or resistance to first-line therapy. Importantly, unlike non-cancerous cells, SCLC cells cannot adapt to drug-induced ROS stress due to the suppression of ROS defense mechanisms by multiple layers of gene regulation. By exploiting this difference in oxidative stress management, we safely increase the therapeutic dose of TXNRD1 inhibitors in vivo by pharmacological activation of the NRF2 stress response pathway. This results in improved tumor control without added toxicity to healthy tissues. These findings underscore the therapeutic potential of TXNRD1 inhibitors for maintenance therapy in SCLC.

Accurate detection of low-frequency DNA mutations in body fluids is essential for cancer monitoring and treatment evaluation. However, the high abundance of wild-type DNA often masks rare mutant signals, making sensitive detection particularly challenging.

Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.

HER2 plays a crucial role in breast cancer (BC) progression, with the D769H and D769Y mutations significantly influencing its structural integrity, drug-binding dynamics, and therapeutic response. This study employs molecular docking and molecular dynamics simulations (MDS), with trajectories propagated for 1000 ns, to examine their distinct effects. Root mean square deviation (RMSD) analysis indicates increased conformational deviations in mutant structures, signifying heightened instability, while root mean square fluctuation (RMSF) reveals enhanced flexibility near the mutation site. Solvent accessible surface area (SASA) calculations highlight changes in solvent exposure, directly affecting ligand accessibility, while radius of gyration (Rg) assessments suggest structural loosening or tightening in response to mutation-induced alterations. Binding free energy calculations using MM-PBSA indicate variability in drug affinity, with mutations disrupting hydrogen-bonding networks and altering ligand stability. Principal Component Analysis (PCA) delineates distinct motion trajectories in mutant proteins, revealing shifts in conformational behavior. Umbrella sampling simulations indicate that while the wild-type HER2-drug complex requires 150 ps to reach equilibrium, the D769H mutant stabilizes within 100 ps, suggesting diminished drug retention. Conversely, the D769Y mutation enhances ligand binding, surpassing wild-type interaction strength. These findings elucidate mutation-specific effects on HER2 structural dynamics and drug interactions, underscoring the need for mutation-tailored therapeutic strategies to mitigate the impact of these variants.

Pituitary Neuroendocrine Tumors (PitNETs) are the most frequently diagnosed intracranial neoplasms in adults. The World Health Organization's 2022 fifth edition classification of pituitary neuroendocrine tumours (PitNETs) maintains an immunohistochemistry based taxonomy that places molecular biology at the centre of diagnosis. This framework provides a solid basis for subtype classification and therapy development. Methylation, defined as the transfer of a methyl group (CH₃) to DNA bases, histone side chains, or RNA nucleotides, is an epigenetic modification that has emerged as a key mechanism in neoplastic transformation. In this review, we synthesise current knowledge on histone, DNA, and RNA methylation in pituitary tumourigenesis, describing their distinct roles and mutual molecular crosstalk. By examining how these epigenetic modifications promote tumour initiation and progression, we assess their potential as drug targets and their translational applicability. Our objective is to propose new research directions and precision treatment strategies that exploit methylation related vulnerabilities, with the goal of improving clinical outcomes for patients with PitNETs.

The phosphorylation dependent regulation of transcription factors, transcriptional co-regulators and chromatin remodelling factors influences transcription. Aberrant transcriptional regulations driven by chromatin-associated oncogenic factors are a feature of various cancers; however, their phosphorylation-dependent regulation remains poorly characterised. ATPase family AAA domain-containing protein 2 (ATAD2) is a chromatin-associated factor implicated in oncogenic transcriptions. Here, we present a comprehensive phosphosite-centric analysis of ATAD2 by integrating data from multiple phosphoproteomic studies, encompassing 859 profiling and 285 differential datasets. From the class 1 differentially regulated phosphosites, four predominant phosphosites-S327, S337, S342, and T1152, emerged as consistently regulated and were frequently detected in diverse tumour datasets. The co-differentially phosphorylated proteins, including their interactors and potential upstream kinases (HASPIN, STK10, CDK12, PRP4K, CDK13, PAK4), were involved in cell cycle, chromatin remodelling, transcription, and DNA repair. Several phosphosites in transcription factors were found to be coregulated along with ATAD2 phosphosites. Phosphorylation at S327 and S342 was broadly upregulated and positively associated with transcriptional activators, suggesting a role in promoting transcription. In contrast, phosphorylation at S337 and T1152 correlated with proteins involved in transcriptional repression, indicating its involvement in inhibitory function. Collectively, these findings indicate the involvement of the ATAD2 phosphoregulatory network in transcriptional regulation and provide insights into the regulatory landscape of ATAD2, laying the groundwork for its potential therapeutic targeting in cancers.

Malignant tumors currently pose a significant threat to global health. Tumor progression is jointly regulated by genetic mutations and the mechanical properties of the tumor microenvironment (TME), including increased tissue stiffness, elevated fluid pressure, and mechanical compression experienced by circulating tumor cells (CTCs) within microvessels. These mechanical signals are transmitted through mechanosensitive pathways, with the Piezo channel family (Piezo1/Piezo2) serving as a core mediator.With their propeller-like trimeric structure, Piezo channels sense membrane tension, mediate calcium influx, and activate downstream signaling pathways (e.g., MAPK, PI3K/AKT/mTOR, YAP/TAZ), thereby regulating tumor cell proliferation, migration, immune microenvironment remodeling, and cancer stem cell-like transformation. Its expression exhibits tissue specificity and correlates with tumor staging, invasiveness, and pro gnosis.

Coiled-coil domain-containing proteins (CCDCs) play pivotal roles in tumorigenesis by regulating gene transcription, apoptosis, and cell cycle progression. This study focuses on the function and mechanisms of CCDC137 in acute myeloid leukemia (AML). Our findings revealed that CCDC137 is significantly overexpressed in AML and is closely associated with poor patient prognosis. Functional experiments demonstrated that CCDC137 promotes cell proliferation and accelerates the cell cycle, thereby driving AML progression. Mechanistically, co-immunoprecipitation (Co-IP) experiments confirm the interaction between CCDC137 and S100A6, which significantly enhanced S100A6 protein stability. Stable S100A6 then activates the PI3K/AKT signaling pathway, thereby mediating the oncogenic effects of CCDC137. This study revealed the mechanism by which CCDC137 drives AML progression by stabilizing S100A6 and activating the PI3K/AKT pathway, thus providing a novel target for AML-specific therapy.

Standard neoadjuvant therapy for early HER2-positive breast cancer consists of 18 weeks of carboplatin, docetaxel, trastuzumab, and pertuzumab. However, treatment intensity may limit feasibility in frail patients and exceed therapeutic needs in selected early-stage disease. We report here real-world clinical outcomes of patients receiving a shortened 12-week neoadjuvant regimen of weekly paclitaxel and carboplatin administered with trastuzumab and pertuzumab (12wTCHP).

Somatostatin receptor 2 (SSTR2) protein expression is aberrantly upregulated in various tumors and can be visualized using scintigraphy or radiological techniques and therapeutically targeted using octreotide-based molecules.

Immune modulatory vaccines (IMVs) are an emerging class of immunotherapies designed to expand anti-regulatory T cells (anti-Tregs) that selectively target immunosuppressive elements within the tumor microenvironment (TME). Unlike conventional cancer vaccines aimed at tumor-associated antigens on malignant cells, IMVs target tumor microenvironment antigens (TMAs), such as indoleamine 2,3-dioxygenase (IDO), PD-L1, arginase-1 (ARG1), and transforming growth factor-β (TGF-β), which are expressed by malignant, myeloid, regulatory, endothelial, and stromal populations. IMVs elicit both CD8⁺ and CD4⁺ T-cell responses: CD8⁺ T cells can mediate cytotoxic elimination of TMA-expressing suppressive cells, whereas CD4⁺ T cells can induce proinflammatory cytokine programs that reprogram myeloid and stromal compartments toward immune-permissive states. Through these combined cytolytic and modulatory mechanisms, IMVs remodel suppressive cellular networks, improve antigen presentation, enhance immune infiltration, and amplify endogenous tumor-specific immunity. Early-phase clinical studies targeting IDO and PD-L1 have shown robust immunogenicity, favorable tolerability, and encouraging activity across multiple solid tumors, particularly in combination with immune checkpoint blockade. A phase III study in first-line advanced melanoma recently demonstrated that a therapeutic vaccine, when combined with anti-PD-1 therapy, can improve progression-free survival in patients with metastatic disease. The strongest signal was observed in PD-1-naïve disease and in PD-L1-negative tumors. Next-generation IMVs directed against ARG1 and TGF-β aim to address immune exclusion and desmoplastic stroma and are being developed across peptide- and mRNA-based platforms with favorable safety profiles that support evaluation in earlier-stage settings. Beyond oncology, analogous microenvironment antigens are induced in chronic and acute infections, suggesting that IMV principles may generalize to settings where regulatory circuits constrain pathogen clearance.

To investigate the role of 3-hydroxybutyrate dehydrogenase 2 (BDH2) in regulating ferroptosis and its impact on the metastasis of lung adenocarcinoma (LUAD). Expression levels of BDH2 were modulated in LUAD cell lines (A549, PC9) using pcDNA-BDH2 plasmid transfection. Cell motility was assessed by Transwell assays, while ferroptosis-associated markers, including Fe2+, malondialdehyde (MDA), lipid reactive oxygen species (ROS), ACSL4, and GPX4, were evaluated by biochemical assays, flow cytometry, and Western blotting. The involvement of the Nrf2/HO-1 signaling axis was analyzed by Western blotting and RT-qPCR. Furthermore, a xenograft mouse model was established to confirm the effect of BDH2 on tumor progression and metastasis in vivo. Overexpression of BDH2 significantly inhibited LUAD cell migration and invasion. BDH2 upregulation enhanced ferroptosis, effects that were reversed by the ferroptosis inhibitor Fer-1. Mechanistically, BDH2 suppressed the activation of the Nrf2/HO-1 pathway, thereby enhancing sensitivity to ferroptosis. In vivo, BDH2 overexpression markedly reduced tumor growth and metastasis in nude mice, while inhibition of ferroptosis attenuated these effects. BDH2 suppresses metastasis in LAUD by promoting ferroptosis via suppression of the Nrf2/HO-1 pathway, highlighting BDH2 as a potential therapeutic target for LUAD.

The high mortality rate of metastatic cutaneous melanoma (SKCM) remains a major challenge in clinical treatment. This study used single-cell RNA sequencing (scRNA-Seq) technology to compare the differences between metastatic and primary tumour cells. By manually annotating cell types, significant disparities in cell communication patterns and functional pathways between the two groups were identified. Combined with transcriptomic data, differential gene analysis was performed to screen out a core gene set associated with tumour metastasis. To achieve accurate prediction of tumour metastasis, this study innovatively constructed a binary classification algorithm (PSO-SVM) integrating particle swarm optimisation (PSO) and support vector machines (SVMs). This model optimises SVM parameters via the PSO algorithm, addressing the limitations of traditional machine learning models such as insufficient accuracy and poor generalization ability in tumour metastasis prediction. Verified by comparison with mainstream machine learning methods, the PSO-SVM model exhibited superior classification performance and successfully identified five key metastasis-related genes: SFN, S100A8, KLF5, ARL4D and TINCR. Furthermore, the expression differences of these genes in the metastatic group were verified at the single-cell level, clarifying their regulatory roles in different cell types and states. Through an innovative analytical strategy integrating single-cell and transcriptomic data, this study elucidated the core molecular mechanisms of SKCM metastasis and key regulatory pathways in the tumour microenvironment, providing potential biomarkers and therapeutic targets for the early diagnosis and targeted treatment of SKCM metastasis. This PSO-SVM-integrated analysis method also offers new insights for research on metastasis mechanisms of other cancers.

Current spatial T cell receptor (TCR) profiling approaches lack the resolution needed to link clonal identity, transcriptional state, and spatial positioning of individual T cells in the tumor microenvironment. Here, we introduce a spatial TCR profiling strategy that resolves individual T cell clones together with their transcriptional states at single-cell resolution and applied the method to human head and neck squamous cell carcinoma. Presumed tumor-specific T cells were broadly dispersed throughout the tumor microenvironment, and cells of the same clone occupied distinct transcriptional states in different locations: Immune-rich regions contained more plastic or progenitor cells, whereas tumor-dense regions were enriched for exhausted states. Patients exhibited notably different spatial architectures of antitumor T cell responses, revealing variation that was not captured by high-resolution, spatially agnostic methods such as spectral flow cytometry and single-cell RNA sequencing. These results provide a blueprint for dissecting antigen-specific T cell states in human tumors and reveal how T cell states are spatially coordinated with local cues across the tumor microenvironment.

Although pancreatic cancer is generally refractory to immune checkpoint blockade, recent studies of tumor-infiltrating T cells in human tumor samples demonstrated the presence of in vivo expanded, tumor-reactive T cell receptor (TCR) clonotypes. Here, we explored the T cell repertoire in a murine pancreatic cancer model by combining single-cell transcriptomics with functional TCR characterization. This uncovered a substantial diversity of tumor-reactive TCR clonotypes. Whereas some of these were exclusively reactive against the autologous tumor, most TCRs reacted against syngeneic tumor cells of diverse tissue origin. Immunopeptidome analyses revealed three T cell epitopes reflecting distinct tumor antigen classes also found in human cancers: a mutanome-encoded neoantigen, an epitope encoded by an ectopically expressed endogenous retroviral provirus, and an epitope derived from a cell stress-induced autoantigen. These findings underline the importance of uncovering the antigen specificity of the natural tumor-reactive TCR repertoire to assess its therapeutic potential and safety with regard to personalized immunotherapy.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality and is largely driven by metabolic disorders such as obesity and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a master regulator of metabolism, and its activation has been proposed as a therapeutic strategy for treating metabolic disorders. However, although AMPK activity is down-regulated in HCC, the precise role of AMPK in HCC development has not been clearly delineated. Here, we investigated the ability of constitutive AMPK activation to prevent HCC development using a constitutively active AMPK transgenic mouse model and a pharmacological AMPK activator. We observed that AMPK activation substantially reduced tumor formation in both diethylnitrosamine (DEN)-induced and streptozocin-induced (STAM) models of HCC via altered bile acid metabolism and inhibition of hepatic nuclear factor alpha (HNF4α) signaling. These findings provide mechanistic insights into AMPK biology and highlight the potential of AMPK as a therapeutic target, emphasizing the intricate interplay between metabolic dysregulation and cancer development.

Protein kinases play a crucial role in regulating cellular processes, and their dysregulation is frequently implicated in various diseases, including cancer. Targeting protein kinases represents a promising therapeutic strategy for cancer treatment. Esophageal squamous cell carcinoma (ESCC) constitutes over 90% of esophageal cancer cases in high-incidence regions, with a global five-year survival rate below 20%. Here, we report that CK2 is aberrantly activated in ESCC, identified through kinase-substrate enrichment analysis (KSEA) of large-scale proteomic and phosphoproteomic data. Functional enrichment revealed the splicing factor SF3B3 as a clinically relevant CK2 substrate. We demonstrated that CK2-mediated phosphorylation of SF3B3 T1200 plays a pivotal role in ESCC progression. Mechanistically, CK2-mediated phosphorylation of SF3B3 enhances its affinity for the deubiquitinase USP7, leading to SF3B3 deubiquitination and subsequent protein stabilization. This stabilization drives ESCC progression by regulating alternative splicing (AS) events, including a critical event involving the inclusion of exon 4 in the EXOSC2 transcript. Furthermore, we demonstrated that SF3B3 T1200 phosphorylation specifically facilitates its incorporation into the U2 snRNP complex, directly promoting the aforementioned EXOSC2 exon 4 inclusion. Crucially, targeting CK2 or USP7, either individually or in combination, effectively suppressed ESCC progression. Our findings uncover a key molecular mechanism underlying SF3B3 stabilization and AS regulation, offering novel therapeutic opportunities for ESCC.

Invasion plasticity allows malignant cells to toggle between collective, mesenchymal, and amoeboid phenotypes while traversing extracellular matrix (ECM) barriers. Current dogma holds that collective and mesenchymal invasion programs trigger the mobilization of proteinases that digest structural barriers dominated by type I collagen, while amoeboid activity allows cancer cells to marshal mechanical forces to traverse tissues independently of ECM proteolysis. Here, we use cancer spheroid-3-dimensional matrix models, single-cell RNA sequencing, and human tissue explants to identify the mechanisms controlling mesenchymal versus amoeboid invasion. Unexpectedly, collective/mesenchymal- and amoeboid-type invasion programs-though distinct-are each characterized by active tunneling through ECM barriers, with expression of matrix-degradative metalloproteinases. CRISPR/Cas9-mediated targeting of a single membrane-anchored collagenase, MMP14/MT1-MMP, ablates tissue-invasive activity while coregulating cancer cell transcriptional programs. Though changes in matrix architecture, nuclear rigidity, and metabolic stress as well as the presence of cancer-associated fibroblasts are proposed to support amoeboid activity, none of these changes restore invasive activity of MMP14-targeted cancer cells. While a requirement for MMP14 is bypassed in low-density collagen hydrogels, invasion by the proteinase-deleted cells is associated with nuclear envelope and DNA damage, highlighting a proteolytic requirement for maintaining nuclear integrity. Nevertheless, when cancer cells confront explants of live human breast tissue, MMP14 is again required to support invasive activity. Corroborating these results, spatial transcriptomic and immunohistological analyses of human breast cancers identified MMP14 expression in tissue-infiltrating carcinoma cells that were further juxtaposed with proteolyzed type I collagen fragments, underlining the pathophysiologic importance of this proteinase in directing invasive activity in vivo.

Intrinsic and acquired resistance to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) remains a major barrier in treating homologous recombination (HR) repair-deficient tumors, including those with germline or somatic BRCA1/2 mutations. Although PARPi are FDA approved for adjuvant treatment of locally advanced or metastatic breast cancer in patients with germline BRCA1/2 mutations, emerging data support their use as monotherapy in the neoadjuvant setting. Promising safety profiles of newer-generation PARPi further support this potential. However, resistance mechanisms specific to the neoadjuvant setting are poorly understood. To address this gap, we leveraged resources from a phase II neoadjuvant clinical trial (NCT03499353), analyzing tumors from patients with germline BRCA1/2 mutant breast tumors before and after six months of talazoparib monotherapy. Whole-transcriptome analyses were performed on these samples. Additionally, we established orthotopic patient-derived xenograft models from a subset of the patient tumors and conducted whole-exome and whole-transcriptome analysis. This integrative approach revealed both known and previously unknown PARPi resistance mechanisms. In one case, overexpression of BRN2, encoding a transcription factor that plays a critical role in neurogenesis, led to activation of ATR/RAD51 and STAT3 pathways, restoring HR repair. BRN2-driven resistance could be reversed with ATR and STAT3 inhibitors, resensitizing cells to talazoparib. In another, an HR repair proficient tumor subclone lacking Shieldin 2 expression expanded during treatment and accounted for intrinsic resistance. Our findings highlight the need to determine intrinsic and anticipate acquired resistance pathways in treatment-naïve tumors and support combining PARPi with targeted agents to improve outcomes in the neoadjuvant setting.