Intracerebral hemorrhage (ICH) causes severe neurological deficits, and secondary neuronal injury is a major barrier to recovery. Cuproptosis, a copper-dependent form of regulated cell death, has been linked to metabolic stress, but its role in axonal injury after ICH remains unclear. Here, we used in vivo rat ICH models and in vitro ICH-like neuronal injury models to examine changes in copper homeostasis, cuproptosis-associated molecular alterations, and axonal injury following ICH. Copper levels were markedly increased in perihematomal brain tissue and injured neurons, accompanied by reduced mitochondrial protein lipoylation and increased dihydrolipoamide S-acetyltransferase oligomerization, consistent with cuproptosis-associated molecular abnormalities. Specificity protein 1 (Sp1) and the copper transporter 1 (CTR1) were also significantly upregulated and showed similar temporal patterns. In parallel, ICH induced axonal injury-related molecular changes, including increased amyloid precursor protein and decreased growth-associated protein 43 expression. Pharmacological inhibition of Sp1 with plicamycin reduced CTR1 expression, attenuated copper overload, ameliorated cuproptosis-associated molecular abnormalities, and improved the measured axonal injury-related molecular and morphological changes in vivo and in vitro. These findings suggest that Sp1/CTR1-associated copper uptake regulation may contribute to secondary neuronal injury after ICH and may represent a potential therapeutic entry point for further investigation.

Plicamycin (PLI) is a small-molecule aureolic acid antibiotic that has been approved for cancer treatment since the 1970s. However, its interaction with cardiovascular biological systems during myocardial ischemia-reperfusion injury (MIRI) remains largely unknown. This study examines its cardioprotective effects and the mechanisms associated with cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in a murine MIRI model. PLI, administered intraperitoneally to mice at 150 μg/kg/day for seven days before cardiac ischemia-reperfusion, attenuated the rise in myocardial infarct size and serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels. PLI also improved cardiac function in MIRI mice, as reflected by higher ejection fraction (EF) and fractional shortening (FS) values. Biochemical assays and dihydroethidium (DHE) staining analyses showed that PLI administration prevented oxidative stress imbalance within the ischemic myocardium following reperfusion. PLI also prevented cardiomyocyte apoptosis within ischemic myocardial tissues by downregulating caspase-3, caspase-9, and Bax and upregulating Bcl-2 expression in MIRI mice. Further investigation showed that I/R injury reduced the expression levels of phospho-CREB and BDNF within the ischemic myocardium, both of which were prevented by PLI. More importantly, pharmacological suppression of CREB by 666-15 (10 mg/kg/day) or BDNF signaling by K252a (25 μg/kg/day) diminished the cardioprotective effects of PLI. Overall, our data suggest that PLI prevents I/R-induced cardiac injury by activating CREB-BDNF signaling, accompanied by suppression of nitro-oxidative stress.

TP53 abnormalities contribute to treatment resistance and poor prognosis in multiple myeloma (MM), yet their functional consequences remain unclear. Here, we integrate ex vivo drug sensitivity profiling, genomics, transcriptomics, and proteomics across 167 CD138[+] bone marrow patient samples to characterize TP53-associated vulnerabilities. Genome-wide CRISPR-Cas9 and RNAi screening identify vulnerabilities in TP53-mutated MM, with or without del(17p), highlighting the dependency on spindle organization, mitotic regulation, DNA synthesis, and transcriptional and metabolic regulation, but independence from MDM2. CD138[+] cells with TP53 mutation exhibit increased sensitivity to chemotherapeutics, HDAC, HSP90, IGF1R, and PI3K/AKT/mTOR inhibitors as well as RNA synthesis inhibitor plicamycin, with distinct drug response profiles of MM with del(17p) and WT TP53. Our study provides new insights into refining TP53 classification to optimize treatment strategies for high-risk MM.

In brief: The preimplantation embryonic development progress is accompanied by dynamic changes of transcriptome, and many transcription factors have been found to responsible for this progress. This study shows that SP1 is necessary for proper zygotic genome activation and morula-to-blastocyst transition by regulating transcriptome establishment and histone modifications in mouse preimplantation embryos. Abstract: Specificity protein 1 (SP1) is the most active member of the specificity protein and Krüppel-like factor (Sp/KLF) family and is widely expressed across all mammalian cell types. However, more detailed studies on the role of SP1 in preimplantation embryonic development are needed. Here, we analyzed the role of SP1 in the development of preimplantation mouse embryos through supplementation with a small molecular inhibitor (plicamycin) and microinjection of Sp1 siRNA. We found that SP1 was indispensable for zygotic genome activation (ZGA) and the morula-to-blastocyst transition. Plicamycin supplementation arrested embryo development at the 2-cell stage and resulted in aberrant RNA polymerase II preconfiguration. Sp1 expression knockdown by Sp1 siRNA microinjection caused most embryos to arrest at the morula stage, and the expression of NANOG, POU5F1, and CDX2 significantly decreased. Both plicamycin supplementation and Sp1 siRNA microinjection decreased embryonic H3K4me3 levels and increased H3K9me3 levels. Moreover, when Sp1 was overexpressed, the embryos were arrested at the 2-cell stage, the H3K4me3 level increased, and the H3K9me3 level decreased. In conclusion, our findings demonstrate that SP1 is crucial for mouse preimplantation embryonic development through the regulation of gene expression and histone modifications.

Postovulatory oocyte aging (POA) is a key factor contributing to the decline in female fertility and the success rate of assisted reproductive technology. Currently, most studies on POA have focused on downstream phenotypes such as mitochondrial dysfunction and oxidative stress, while little is known about its key upstream regulatory factors. Here, we show that the downregulation of transcription factor Early Growth Response 1 (EGR1) is a key upstream event driving porcine oocyte aging. Microtranscriptome sequencing combined with experimental validation verified a notable reduction in EGR1 protein abundance in aged oocytes. We found that Ursodeoxycholic Acid (UDCA) upregulated EGR1, which in turn promoted the expression of the autophagy-related protein LC3B and the lysosomal protein LAMP1, while reducing P62 accumulation. Furthermore, UDCA enhanced the expression of mitophagy core proteins PINK1, VDAC1 and promoted mitochondrial-lysosomal colocalization, thereby improving mitophagy and restoring the quality of aged oocytes. Crucially, treatment with the EGR1 inhibitor plicamycin completely blocked UDCA's ability to enhance the developmental potential of aged oocytes, confirming that EGR1-mediated mitophagy was the core pathway underlying UDCA's effects. Collectively, this study innovatively identified EGR1 as a key bridge linking oocyte aging and decreased mitophagy, and clarified the novel mechanism by which UDCA exerts its protective effects through the "UDCA-EGR1-mitophagy" axis. Our findings advanced the research on oocyte aging from phenotypic observation to the upstream transcriptional regulation level, providing a novel theoretical target and experimental basis for fundamentally intervening in reproductive aging.

We recently reported that GADD34, an integrated stress response (ISR)-associated protein expressed at low levels in many cell lines, functions as a novel HIV-1 restriction factor. To elucidate the mechanism underlying GADD34-mediated inhibition of HIV replication, we investigated the role of long non-coding RNAs (lncRNAs). Here, we identify and characterize a novel lncRNA, GRHAL1 (GADD34-regulated HIV accessory lncRNA-1), which is highly expressed in GADD34-knockout (GADD34-KO) cells relative to WT cells. GRHAL1 is expressed at low basal levels in Jurkat and MT-2 CD4+ T cell lines and in primary human CD4+ T cells and is upregulated following HIV-1 infection. T cell activation signals that promote HIV-1 replication also induced GRHAL1 expression; however, this induction was independent of IFN and ISR signalling. Transfection of in vitro - synthesized GRHAL1 significantly enhanced HIV-1 LTR-driven gene expression from both integrated and unintegrated promoters. GRHAL1 stimulated both Tat-independent and Tat-dependent LTR activation, with a more pronounced effect observed at suboptimal Tat levels, indicating functional synergy between GRHAL1 and Tat under limiting Tat conditions. Mutational analysis of the LTR demonstrated that GRHAL1-mediated activation requires an intact Tat-responsive TAR element and upstream Sp1-binding sites, linking GRHAL1 activity to Tat and Sp1 in transcriptional regulation. This synergy was further confirmed using a minimal HIV-1 promoter containing Sp1 sites and a TAR sequence. Moreover, GRHAL1 alone was sufficient to activate a heterologous promoter containing Sp1-binding elements. The Sp1-selective inhibitor mithramycin suppressed both Tat-independent LTR activation and the cooperative activation mediated by GRHAL1 and Tat. Electrophoretic mobility shift assays, RNA immunoprecipitation and RNA-pulldown experiments demonstrated that GRHAL1 directly interacts with Sp1 and that Tat enhances this interaction, suggesting that GRHAL1 modulates Sp1 activity through direct binding. In summary, we identify GRHAL1 as a novel HIV-1-induced lncRNA that regulates viral gene expression by interacting with Sp1 and synergizing with Tat to enhance HIV-1 transcription.

Metabolic reprogramming is a hallmark of cancer, yet the critical drivers and mechanisms in hepatocellular carcinoma (HCC) remain incompletely understood. Through a metabolism-focused CRISPR screen, we identified phosphomevalonate kinase (PMVK), a mevalonate pathway enzyme, as a key regulator of HCC stemness and progression. PMVK directly phosphorylates the transcription factor SP1 at Thr[355], which enhances SP1's DNA binding affinity and promotes its interaction with the master lipid regulator SREBP1/2, driving a transcriptional program essential for de novo cholesterol and fatty acid synthesis. Clinically, PMVK levels are positively correlated with phospho-SP1 (Thr[355]) levels and predicate poor prognosis in patients with HCC. Functionally, we demonstrate that genetic or pharmacologic inhibition of the PMVK-SP1 axis, including using the SP1 inhibitor mithramycin A (MTA), significantly suppresses HCC tumorigenesis. In conclusion, our findings uncover the PMVK-SP1 axis as a central driver of lipid metabolic reprogramming and hepatocarcinogenesis, highlighting it as a compelling therapeutic target in HCC.

Background/Objectives: The orphan nuclear receptors 4A1 (NR4A1) and NR4A2 are overexpressed in multiple solid tumors, and both receptors exhibit tumor promoter-like activities. A recent study reported that luteolin, a flavonoid that binds NR4A1, decreased the expression of the pro-oncogenic receptor tyrosine kinase MerTK in colon cancer cells. Methods/Results: In this study, we observed that MerTK protein was expressed in human SW480 and HCT116 and mouse CT26 colon cancer cell lines, and was significantly downregulated after treatment with 1,1-bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) compounds, which are dual NR4A1/NR4A2 ligands. Moreover, knockdown of NR4A1 and NR4A2 also decreased MerTK protein expression and DIM-3,5 ligands, and receptor knockdown also decreased MerTK RNA levels expression. MerTK expression was also downregulated by knockdown of Sp1, Sp3, or Sp4 and by treatment with mithramycin. Subsequent studies using chromatin immunoprecipitation and transfection of a MERTK (promoter)-luciferase construct containing transcriptionally active GC-rich promoter elements indicated that MerTK expression in colon cancer cells was regulated by NR4A/Sp complexes, including NR4A1, NR4A2, Sp1, Sp3, and Sp4 transcription factors. Conclusions: The participation of NR4A1 and NR4A2 in the regulation of MerTK indicates that DIM-3,5 ligands represent a novel class of agents that can be used to inhibit MerTK expression in cancer cells by acting as dual NR4A1 and NR4A2 inverse agonists.

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy characterized by poor prognosis and limited therapeutic response. Cancer stem cells (CSCs) contribute to tumor progression, therapeutic resistance, and tumor recurrence. Among transcriptional regulators potentially involved in these processes, Specificity Protein 1 (SP1) has emerged as a candidate integrator of oncogenic and epigenetic signaling networks. However, its contribution to CSC-associated phenotypes and drug resistance in HCC remains incompletely defined. In this study, we combined transcriptomic analyses of TCGA datasets with functional experiments in HCC cell lines (Huh7 and HepG2). SP1-associated transcriptional programs were targeted pharmacologically using mithramycin A (MIT-A) and genetically using siRNA-mediated knockdown. The effects were assessed by RNA sequencing, RT-qPCR, Western blotting, flow cytometry, and functional assays evaluating proliferation, migration, CSC-associated properties, and response to sorafenib. MIT-A treatment markedly reduced the expression of stemness-associated transcription factors (NANOG, OCT4, SOX2) and CSC markers (CD133, CD24), impaired CSC-related functions including ALDH activity and the Side Population phenotype, and inhibited cell proliferation and migration. MIT-A also sensitized both parental and sorafenib-resistant HCC cells to sorafenib, associated with modulation of apoptotic regulators and reduced transporter-mediated efflux activity. SP1 knockdown partially reproduced several of these effects, supporting a contribution of SP1-dependent transcriptional programs to these phenotypes. Overall, these findings identify SP1-associated transcriptional networks as potential regulators of CSC features and therapeutic resistance in HCC and support targeting SP1-associated transcriptional programs as a strategy to enhance sorafenib efficacy.

While anti-androgen therapies have significantly improved survival outcomes for patients with advanced prostate cancer, their efficacy is often limited by the emergence of resistance. This resistance is largely driven by the expression of AR splice variants (AR-Vs). However, therapeutic strategies to effectively target AR-V-mediated castration-resistant PCa remain an unmet clinical need. Here, we report that prolonged exposure of PCa cells expressing both AR-FL and AR-Vs to enzalutamide induces extensive reprogramming of their transcriptomic and proteomic profiles. Notably, in enzalutamide-resistant cells, EGFR expression was upregulated at both the mRNA and protein levels, despite the absence of EGFR mutations. Downstream of EGFR, the extracellular signal-regulated kinase (ERK) pathway was constitutively activated, promoting tumor cell proliferation. Furthermore, protein array analyses of clinical specimens revealed that high EGFR protein levels-but not EGFR mRNA levels-were associated with poor patient prognosis. In addition, we demonstrated that inhibiting EGFR, either through genetic knockdown or pharmacological intervention, significantly sensitized AR-V-mediated enzalutamide-resistant cells to AR-targeted therapy (Enzalutamide or Mithramycin). Mechanistically, our data indicate that ELF1 transcriptionally regulates EGFR and that ERK1/2 interacts with ELF1, suggesting the existence of a positive ELF1/EGFR/ERK feedback loop that sustains resistance. This study elucidates a possible mechanism of resistance to AR inhibition driven by an ELF1/EGFR/ERK feedback loop in AR-Vs positive cells and provides a rationale for combining EGFR inhibitors with AR-targeted therapy as a potential treatment strategy for patients with advanced, enzalutamide-resistant prostate cancer.

Solitary fibrous tumor (SFT) is a rare and aggressive sarcoma driven by NAB2::STAT6 gene fusions, yet effective targeted therapies remain unavailable. Here, we report that the NAB2ex4::STAT6ex2 fusion variant forms nuclear condensates via liquid-liquid phase separation (LLPS) in engineered fibroblast models and primary SFT cells. These condensates co-localize with BRD4S and EGR1, key transcriptional regulators, and are functionally active, driving widespread transcriptional reprogramming. Treatment with Mithramycin A, a compound that disrupts EGR1-DNA interactions, dissolves NAB2::STAT6 condensates and reverses their aberrant gene expression and chromatin binding signatures. Our findings uncover a previously unrecognized role for NAB2::STAT6 in condensate-mediated oncogenic signaling and provide a mechanistic rationale for condensate-targeted therapy in SFT.