Integrated pan-cancer profiling and experimental validation identify CCDC59 as a key driver and therapeutic biomarker in liver hepatocellular carcinoma.

Epidermal Growth Factor Receptor/KIT-Linked Proliferative Bias in Normal Breast Lobules from Matched Non-Hispanic Black and White Women Is Rapidly Reversible by Receptor Tyrosine Kinase Inhibition.

Palbociclib Enhances Radiotherapy Efficacy by Promoting Apoptosis and Immune Modulation in Oral Squamous Cell Carcinoma.

Dissecting placental host-pathogen interactions: Rift Valley fever virus infection in early human trophoblast stem cells.

Chondrosarcoma (CHS), the second most common primary malignant cartilage tumor, is largely resistant to conventional therapies, making surgical resection the standard treatment. Proton therapy offers a physical advantage through the Bragg peak, enabling targeted irradiation while sparing surrounding tissues. However, differential biological responses between malignant and normal cartilage cells remain poorly understood. In this study, CHS SW1353 cells and normal chondrocytes (MC615) were exposed to proton irradiation. Biological responses were assessed via clonogenic survival, cell viability, apoptosis (caspase 3/7), micronucleus formation, cell cycle profiling, and oxidative stress markers. Proteomic changes were analyzed using mass spectrometry and bioinformatics. CHS cells exhibited higher radioresistance (D10 = 6.45 Gy) than normal chondrocytes (D10 = 5.08 Gy), oxidative stress adaptation, G1 arrest and proteomic plasticity, whereas normal chondrocytes displayed increased oxidative stress, extracellular matrix fragility and impaired integrin signaling. Notably, the tumor-specific increased levels of Tyrosine-protein kinase Fyn and Yes1-associated transcriptional regulator (YAP1) signaling suggest molecular drivers of radioresistance. Overall, proton irradiation elicits distinct biological and proteomic responses in malignant versus normal cartilage cells. These findings highlight potential radiosensitization targets, including Fyn/Src and YAP1/Hippo pathways, while underscoring the need to optimize proton therapy to enhance tumor control while minimizing damage to healthy cartilage.

IntS11, the core catalytic subunit of the Integrator (INT), functions in various stages of transcription. However, its specific function in orchestrating neural lineage progression remains poorly understood. Using a Drosophila model, we found that the absence of IntS11 leads to reduced brain size, which is not attributable to neuroblast (NB) loss or apoptosis but is due to impaired NB proliferation. MARCM (mosaic analysis with a repressible cell marker) revealed impaired clonal expansion, characterized by a significant decrease in progeny cell number. Live imaging and FUCCI analysis further confirmed a G1 arrest in IntS11-deficient NBs. Single-cell RNA-seq revealed downregulation of cell cycle regulators (aurB, CycE, Cdk4) in NBs and differentiation genes in GMCs, which were confirmed as candidates by ChIP-qPCR. Approximately 80% of neuronal morphogenesis-associated genes exhibiting 3'UTR shortening were downregulated. We further established that IntS11 physically binds to these loci to maintain lengthened 3'UTR isoforms and ensure mRNA stability in larval brains. Collectively, our findings establish IntS11 as a stage-specific regulator of neural lineage progression that sustains NB proliferation through transcriptional control of cell-cycle genes and stabilizes differentiation programs by maintaining long 3'UTR isoforms.

Glioblastoma (GBM) is recognized as one of the most treatment-resistant malignancies, owing to its reinforced DNA repair systems and limited drug accessibility across the blood-brain barrier. This study, identifies, daidzin (DZN), a naturally derived isoflavone, as a potent redox-active DNA intercalator that intrinsically combines physical intercalation with chemical reactivity to breach this resistance. Unlike traditional intercalators, DZN autonomously triggers destabilization of DNA helices by inducing torsional strain, thereby producing convergent strand and base lesions through photo-independent redox pathways involving deoxyribose cleavage and C8 guanine oxidation. Additionally, DZN demonstrates pronounced glioma-specific cytotoxicity by initiating1O2-driven oxo-cation formation and concomitant H2O2 production. This redox burst results in DNA strand scission activating robust DDR signaling and oxidative base lesions, which cripple tumor survival. Enhanced membrane fluidity in glioma cells likely facilitates superior DZN permeability, intracellular accumulation, thereby allowing DZN to initiate this robust DNA damage responses, culminating in G1 arrest and apoptosis in GBM cells while sparing normal glia. In vivo, DZN markedly suppresses tumor growth and surpasses temozolomide efficacy, current clinical option for GBM treatment. This work, thus, establishes a previously unrecognized paradigm of DNA intercalation-driven redox chemistry, presenting DZN as a promising therapeutic capable of exploiting the genomic frailties to overcome therapeutic resistance in glioma.

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue sarcoma marked by substantial treatment-related morbidity and poor outcomes, particularly in metastatic or recurrent disease. Although diagnostic and therapeutic approaches have improved, survival remains low for patients with high-risk molecular features, including tumors harboring the PAX3::FOXO1 fusion. This fusion protein drives an aberrant transcriptional program that promotes tumor progression and resistance to therapy, underscoring the need for new treatment strategies. In this work, we investigated therapeutic vulnerabilities created by inhibiting the transcription-regulatory kinases CDK8/19. Using a high-throughput drug combination screen, we tested several CDK8/19 inhibitors against a library of 2,803 preclinical, investigational, and approved small molecules. The screen identified a strong synergistic interaction between CDK8/19 inhibitors and dihydrofolate reductase (DHFR) inhibitors. Unsupervised hierarchical clustering showed that multiple DHFR inhibitors formed a distinct cluster with highly similar patterns of synergy, supporting the robustness of this interaction. Follow-up viability assays confirmed synergy in both PAX3::FOXO1-positive and fusion-negative RMS cell lines. To define the biological basis of this synergy, we examined effects on transcriptional regulation, cell-cycle dynamics, and apoptosis. Annexin-V/DAPI staining demonstrated that combined treatment produced significantly higher apoptosis and G1 arrest than either drug alone. EdU incorporation assays showed a strong decrease in DNA replication, indicating impaired nucleotide synthesis. Transcriptomic profiling, including gene set enrichment analysis, revealed that CDK8/19 inhibitors upregulated PAX3::FOXO1 fusion targets, MYC-regulated genes, and oxidative phosphorylation pathways, whereas DHFR inhibitors primarily induced immune-related and KRAS signaling programs. The transcriptional responses to each drug class were largely distinct, and combination treatment reversed expression patterns driven by single agents. Importantly, DHFR inhibition triggered compensatory activation of pyrimidine biosynthesis genes, a response that was suppressed by CDK8/19 inhibition, providing a mechanistic basis for the observed synergy. ChIP-seq and proteomic data further supported these findings. Additionally, CDK8/19 inhibition amplified DHFR inhibitor-induced DNA damage, reflected by elevated γH2AX accumulation. Overall, these results highlight a therapeutic strategy that concurrently disrupts PAX3::FOXO1-driven transcriptional programs and nucleotide-dependent DNA repair. The combination of CDK8/19 and DHFR inhibitors may therefore overcome resistance mechanisms and enhance treatment efficacy in RMS. Citation Format: Ukhyun Jo, Ying Wu, Lisa M Jenkins, Tapan Kumar Maity, Seth P Zimmerman, Chris M. Counter, Assil Fahs, Corinne Linardic, Craig J. Thomas, John F. Shern. High-throughput screening identifies synergistic drug interactions between CDK8/19 and DHFR inhibitors in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1155.

Hijacking by sequestration: An hCMV lncRNA reshapes the host transcriptome.

Lomitapide mesylate and lomitapide target ALDOA to inhibit growth and enhance gemcitabine efficacy in PDAC.

Background and Objectives: Combining osimertinib (OSI) with pemetrexed (PEM) can enhance antitumor efficacy; however, the benefit is schedule-dependent. Our previous pharmacodynamic (PD) study showed that concurrent PEM + OSI is limited by OSI-induced G1 arrest, attenuating early PEM cytotoxicity. In contrast, sequential PEM→OSI allows PEM to fully induce S-phase arrest and DNA damage but elicits a pro-survival EGFR rebound; subsequent OSI suppresses this rebound and promotes apoptosis of damaged cells, yielding strong synergy. Here, we investigated whether pharmacokinetic (PK) drug-drug interactions (DDIs) contribute to this synergy and predicted the relative advantage of PEM→OSI versus PEM + OSI under clinically relevant conditions using a PK/PD approach. Method and Results: Potential PK-DDIs were assessed at cellular uptake, plasma exposure, and intratumoral distribution levels. No meaningful PK-DDIs were observed, supporting a primary PD-driven synergy. We integrated mouse PK with in vitro/in vivo PD data to build a mechanistic Quantitative System Pharmacology (QSP)-PK-PD model linking drug disposition to folate biology, Epidermal Growth Factor Receptor (EGFR) signaling, and tumor growth inhibition. The model recapitulated schedule dependence and explained PEM→OSI superiority: PEM initiates damage and EGFR compensatory rebound, after which OSI suppresses EGFR signaling and enhances apoptosis. In contrast, concurrent PEM + OSI induced G1 arrest, reduced the pool of damaged apoptosis-susceptible cells, and weakened the synergy. Global sensitivity analysis identified intrinsic OSI sensitivity and the pro-apoptotic protein Bim as key determinants; reduced OSI sensitivity or Bim activity diminished the advantage of the sequential strategy. The simulations indicated that OSI can start 48 h after PEM exposure (no extended drug holiday is needed) and that the PEM→OSI benefit remains robust across heterogeneity, including BIM-deletion polymorphisms and inter-individual variability in tumor drug sensitivity. Conclusions: This mechanism-based QSP-PK-PD framework connects whole-body PK to core PD processes, explains schedule-dependent synergy, and supports optimization of sequencing intervals and identification of likely responders.

The Golgi complex undergoes dynamic remodeling during the cell cycle, as ribbon unlinking in G2 is required for proper mitotic progression. Failure to fragment the ribbon leads to G2 arrest, whereas forced mitotic entry with intact ribbons results in multipolar spindle formation. Phosphorylation of the Golgi matrix protein GRASP65 at serine 277 (S277) in rat (S274 in human) by JNK2 is essential for ribbon unlinking, but its upstream regulation has remained unclear. Here, we generated and validated a phospho-specific antibody recognizing human GRASP65 phosphorylated at S274, enabling accurate detection of this modification. Using this tool, we identify protein kinase D2 (PKD2) as a critical upstream regulator required for GRASP65 phosphorylation and Golgi unlinking. GRASP65-S274 phosphorylation increases during G2 and is markedly reduced upon PKD2 inhibition or depletion, resulting in decreased Golgi unlinking and delayed G2/M transition. Conversely, PKD2-activating stimuli, including phorbol esters and nocodazole, enhance GRASP65 phosphorylation in a PKD2-dependent manner. These findings define PKD2 as a key regulator of the JNK2-GRASP65 signaling axis controlling Golgi disassembly at the G2/M transition. Moreover, the phospho-specific GRASP65 antibody described here provides a valuable tool to dissect upstream signaling mechanisms and to identify the initial triggers driving Golgi unlinking at G2 entry.

Variants in OTUD5 are associated with neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain unclear. This study aimed to investigate the pathogenicity of a novel OTUD5 variant (c.697G > A, p.Val233Met) and elucidate its regulatory role in neural progenitor cell (NPC) proliferation and differentiation, thereby uncovering the function of OTUD5 in neurodevelopment. The OTUD5 variant was identified in two NDD patients via exome sequencing. Patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9-corrected isogenic controls were generated. NPC proliferative activity was assessed by Ki67 immunofluorescence staining, cell-cycle distribution was analyzed by flow cytometry, and neuronal differentiation was evaluated by Tuj1/MAP2 immunofluorescence staining. Substrate screening was conducted in HEK293T cells using co-immunoprecipitation (Co-IP) and mass spectrometry. Deubiquitination capacity and protein stability were validated through ubiquitination assays and cycloheximide (CHX) chase experiments. The p.Val233Met variant, located within the catalytic OTU domain, induced a marked conformational alteration in the OTUD5 protein. Functionally, the variant caused aberrant NPC proliferation (1.8-fold increase in Ki67+ cells, accompanied by release of G1 arrest) and impaired neuronal differentiation (60% reduction in Tuj+ cells). Mechanistically, wild-type OTUD5 stabilized GSK3β by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3β degradation and shortening its half-life by 40%. This study establishes a novel disease mechanism whereby OTUD5 mutations disrupt NPC homeostasis through GSK3β destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3β-targeted therapies in OTUD5-related NDDs.

Activating mutations in KRAS drive pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). Although mutant-selective KRAS inhibitors and pan-RAS inhibitors provide clinical benefits, the development of resistance limits durable response. Transcriptomic and proteomic analyses reveal that, despite effective suppression of mutant KRAS signaling, resistant cells sustain cell cycle progression. Distinct orthogonal mitogenic pathways are engaged in a context-dependent manner to bypass KRAS inhibition. While these pathways can be broadly inhibited using the pan-RAS-ON inhibitor RMC-6236, cells remained capable of developing acquired resistance where cell proliferation is uncoupled from RAS signaling. Combinatorial drug screens and genome-wide CRISPR-Cas9 screens reveal that perturbing cell cycle nodes via targeting cyclin dependent kinases CDK4/6 and CDK2 could restore sensitivity to KRAS/RAS inhibitors. Co-targeting CDK4/6 induces G1 arrest and suppresses E2F-regulated proteins across all resistant models. In contrast, co-targeting CDK2 exerts a broader effect by impairing DNA replication, inducing G2 arrest, preventing mitotic entry, and yielding a more durable cytostatic response that delays cellular outgrowth after drug withdrawal. Finally, concurrent inhibition of KRAS with either CDK4/6 or CDK2 yields durable tumor control in vivo in xenografts derived from acquired resistant models. In conclusion, our findings identify sustained cell cycle activity as a defining feature of resistance to KRAS-directed therapies and establish cell cycle co-targeting as an effective strategy to overcome KRAS/RAS inhibitor resistance.

Tropomyosin receptor kinase (TRK) is an important therapeutic target for tumors driven by NTRK gene fusions. However, the clinically approved TRK inhibitors, including Larotrectinib and Entrectinib, are limited by insufficient efficacy and resistance due to kinase mutations. Here, we report DZX19 (C02), a novel phenyl thiophene-3-carboxamide TRK inhibitor developed via a pharmacophore-guided scaffold-hopping approach combined with structure-based design, based on the key binding features of Entrectinib. DZX19 demonstrated stronger in vitro activity and an improved resistance profile against Entrectinib-resistant TRKA mutants, including G595R, F589L, and G667C. In the TPM3-NTRK1 fusion-positive Km-12 cell line, DZX19 induced G1 arrest, promoted apoptosis, and suppressed TRK downstream signaling. DZX19 displayed excellent plasma stability and moderate microsomal stability. In the Km-12 xenograft model, DZX19 significantly suppressed tumor growth. These results indicate that DZX19 serves as a novel TRK-targeting lead compound for further investigation.

Ewing sarcoma remains partially uncontrollable even after treatment with chemotherapy, surgery, and radiation therapy due to its high malignancy. To explore genes that drive Ewing sarcoma cell proliferation, we conducted a comprehensive analysis of mRNA expression. Based on cDNA array results, we identified consistently elevated expression of Myc and S-phase kinase-associated protein 2 (Skp2) across all five Ewing sarcoma cell lines examined. The functional roles of Myc and Skp2 were assessed by siRNA-mediated knockdown and overexpression assays, followed by cell proliferation, cell cycle, and protein expression analyses. Ubiquitination of p27 and activation of the CCNE/CDK2 complex were evaluated by immunoprecipitation and western blotting. Finally, the in vivo relevance of Myc and Skp2 knockdown was validated using a xenograft mouse model. Knockdown (KD) using siRNAs specific for Myc and Skp2 resulted in reduced cell growth and an increased proportion of cells in the G0/G1 phase, indicating G1 arrest. In KD cells, we observed decreased CDK2 activity, increased p27 expression, and reduced expression of cyclin E (CCNE). The increased activity of the CCNE/CDK2 complex led to enhanced phosphorylation of p27 at Thr187, accelerating p27 degradation via Skp2-mediated ubiquitination. Concurrently, the CCNE/CDK2 complex promoted phosphorylation of Rb at Ser807/808, which is involved in E2F1 activation. This mechanism was identified through a comprehensive expression analysis aimed at uncovering the drivers of cell cycle acceleration in Ewing sarcoma. The findings offer new insights into therapeutic strategies for this malignancy, which has seen little progress in treatment over several decades. This discovery holds the potential to transform the current landscape, as no effective molecularly targeted therapies have yet been developed.

Autophagy has emerged as a potential drug target for enhancing the efficacy of radiotherapy, but its precise role in cellular responses to ionizing radiation (IR) remains incompletely understood. To address this, we conducted a focused RNA interference (RNAi) screen in Drosophila melanogaster targeting autophagy-related genes. We found that knocking down Pink1 or ref(2)P (Drosophila p62/SQSTM1) in larval eye discs resulted in adult eye defects when the larvae were irradiated during the 3rd instar stage. Cell biological analyses revealed that Pink1RNAi expression did not affect cell proliferation or DNA damage recognition/repair but significantly increased apoptosis following IR exposure. This finding was confirmed using loss-of-function mutants of Pink1 and its partner Parkin and was found to extend to larval wing discs. Increased apoptosis after Pink1 depletion was notably concentrated in the G1-arrested morphogenetic furrow (MF). However, Pink1 mutation does not affect G1 arrest in the MF or induction of reactive oxygen species (ROS) by IR, suggesting that the role of Pink1 in protecting cells from IR-induced apoptosis is not via cell cycle regulation or ROS induction. To our knowledge, this is the first report of a role for Pink1/Park in regulating IR-induced apoptosis in any system.

Scaffold-free biofabrication has emerged as a promising strategy for cartilage repair, which may facilitate improved tissue integration while avoiding exogenous biomaterials. However, reproducible scaffold-free 3D bioprinting is strongly influenced by the robustness of the expanded cell population, particularly when induced pluripotent stem cell (iPSC)-derived neural crest mesenchymal stem cells (iNCMSCs) undergo repeated monolayer expansion. In this study, we tested whether TD-198946 priming during expansion could stabilize cell population quality and improve fabrication outcomes. TD-198946 priming supported iNCMSC expansion, as evidenced by increased cell number and MTT signal, accompanied by reduced G1 arrest and improved cell-cycle progression. These effects were reversed by the NOTCH3 signaling inhibitor DAPT, supporting the involvement of NOTCH3 as a mediator of TD-198946 activity. In parallel, TD-198946 priming increased N-cadherin expression in expanded iNCMSCs, a cell-cell adhesion molecule associated with spheroid cohesion in scaffold-free biofabrication systems. Applied to scaffold-free 3D bioprinting, TD-198946 priming led to dose-dependent increases in spheroid size, glycosaminoglycan deposition, and mechanical strength of the resulting constructs, with optimal construct quality observed at 50 nM. In contrast, excessive TD priming (100 nM) disrupted extracellular matrix production and resulted in inferior mechanical properties, highlighting the importance of dose optimization. This approach improved the robustness of the expanded iNCMSC population, thereby enhancing the consistency of scaffold-free biofabrication and construct maturation.

NUT carcinoma (NC) is a rare exceptionally aggressive malignancy, defined by NUTM1 gene translocations, most commonly generating a BRD4::NUTM1 fusion that results in a poor prognosis and limited therapeutic options. Oncolytic virotherapy has emerged as a promising strategy for NC, and the dual bromodomain and extra-terminal domain (BET) and p300/CBP inhibitor NEO2734 has demonstrated potent antiproliferative activity. To investigate multimodal therapeutic approaches that combine epigenetic modulation with immunogenic and cytotoxic effects of oncolytic viruses (OVs), we evaluated two recombinant OVs, including the herpes simplex virus talimogene laherparepvec (T-VEC) and a measles vaccine virus (MeV-GFP), in combination with NEO2734 in four distinct NC cell lines. Viability assays revealed enhanced tumor cell reduction with all combinations, including synergistic effects with T-VEC combinations. Cell cycle analysis showed G1 arrest with NEO2734 alone, whereas its combination with T-VEC resulted in S-phase broadening and reduced G2-phase populations, consistent with replicative stress and increased cytotoxicity. Evaluation of immunogenic cell death (ICD) markers displayed elevated ATP and HMGB1 levels and increased surface calreticulin with T-VEC and NEO2734 combinations. Overall, these findings indicate that combining OVs with BET/p300 inhibitors elicits potent antitumor responses, supports synergistic interactions and immunogenicity, and warrants further investigation in multimodal therapeutic strategies for NC.

Abstract Cyclin-dependent kinase 2 (CDK2) is a critical effector of cell-cycle progression, including during late G1, S, and G2 phases. While CDK4/6 inhibitors have become a backbone in HR+/HER2- breast cancer therapy, resistance frequently emerges through activation of alternative CDK/Cyclin complexes. Notably, the CDK2/Cyclin E axis is frequently associated with resistance to CDK4/6 inhibitors and has driven the development of catalytic CDK2 inhibitors for breast cancer therapy. Here, we sought to: 1, delineate the mechanistic determinants of response to pharmacologic CDK2 inhibition in breast cancer models; 2, define biomarkers predictive of response; and 3, interrogate rational combination strategies to maximize therapeutic efficacy. Using a panel of breast cancer models, it was found that CDK2 catalytic inhibitors induced a bimodal response with two fundamentally distinct phenotypic responses. A minority of cell lines underwent G1 arrest while most accumulated 4N DNA content consistent with a G2/M block. Co-expression of p16INK4A and high Cyclin E1 strongly correlated with the G1 arrest phenotype and marked sensitivity to CDK2 inhibition that was also observed in vivo. Models lacking this co-expression adopted the 4N arrest response and displayed upregulation of phospho-CDK1 (Y15) and Cyclin B1. Critically, this latter phenotype was observed in all HR+/HER2- models tested, including those with engineered resistance to CDK4/6 inhibitors. Using whole-genome CRISPR screens, loss of CDK2 was identified as a top hit conferring resistance to catalytic inhibitors, underscoring the concept that inhibitor binding to CDK2 (rather than simple loss) is essential for the cytostatic effect. Further, deletion of CDK2 reversed the G2/M arrest induced by the inhibitor and restored proliferation. Ahost of potential targets were identified that enhanced sensitivity to CDK2 inhibitors through CRISPR and drug screening approaches. These included CDK4/6 inhibitors that were broadly synergistic with CDK2 inhibitors irrespective of breast cancer subtype. The basis of this cooperation involved arrest in multiple phases of the cell cycle. Several additional combinatorial strategies have recently been defined that further credential CDK2 as a key therapeutic target moving forward. Together this work addresses the complexity of targeting CDK2 for the treatment of breast cancer and multiple opportunity areas for combinations. Citation Format: E. Knudsen, V. Kumarasamy, A. Dommer, J. Wang, A. Witkiewicz. Discrete Vulnerabilities to CDK2 Inhibition in Breast Cancer: Genetic Determinants and Therapeutic Opportunities [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-13-01.