Predicting immune checkpoint therapy response in three independent metastatic melanoma cohorts.

While Immune checkpoint inhibition (ICI) therapy shows significant efficacy in metastatic melanoma, only about 50% respond, lacking reliable predictive methods. We introduce a panel of six proteins aimed at predicting response to ICI therapy. Evaluating previously reported proteins in two untreated melanoma cohorts, we used a published predictive model (EaSIeR score) to identify potential proteins distinguishing responders and non-responders. Six proteins initially identified in the ICI cohort correlated with predicted response in the untreated cohort. Additionally, three proteins correlated with patient survival, both at the protein, and at the transcript levels, in an independent immunotherapy treated cohort. Our study identifies predictive biomarkers across three melanoma cohorts, suggesting their use in therapeutic decision-making.

Complex treatment of residual metastatic germ cell cancer: A single center experience.

BACKGROUND: Testicular cancer is the most common solid malignancy among men aged 15-35. Radical orchiectomy and platinum-based chemotherapy (BEP) are curative in the majority of patients, including advanced, metastatic cases. According to current urooncology guidelines all non-seminoma patients harbouring post-chemotherapy residual masses of ≥ 1 cm should undergo salvage retroperitoneal lymph node dissection (RPLND). However, only 10% of residual tumors contain viable disease. OBJECTIVE: To assess patient outcomes and complications considering different treatment regimens and clinical characteristics. MATERIALS AND METHODS: In a retrospective cross-sectional study patients (n=127) who underwent postchemotherapy RPLND between 2007 and 2023 at our referral center were evaluated. The patients received systemic treatment at various oncology centers. The number of BEP cycles received were occasionally different from standard. Only patients with normal postchemotherapy serum tumor markers and primary testicular or extragonadal germ cell neoplasms were included. Treatment groups were established according to the number of BEP cycles received, and the extent of RPLND (bilateral or modified template). Treatment outcomes and complications were assessed. RESULTS: Standard 3-4 courses of BEP were received by 100 (78,7%) patients, while 11 (8,7%) patients underwent less, and 16 (12,6%) more courses than standard. On histopathologic evaluation viable germ cell tumor, teratoma, and necrosis/fibrosis was present in 26 (20,5%), 67 (52,7%) and 34 (26,8%) of specimen, respectively. In the 5-6 BEP series subgroup high rate of viable disease (37,5%) was found and significantly more nephrectomies were performed, than other chemotherapy subgroups. Extratesticular GCT, viable disease in residual mass or progression after RPLND indicated lower survival. Mild (Clavien-Dindo I-II) or no postoperative complications were reported in 93,7% of cases. CONCLUSIONS: The study suggests no significant benefit from exceeding 3-4 courses of BEP. Timely salvage RPLND should be performed in high volume centers for optimal treatment outcomes with acceptable complication rates. Adherence to the Heidenreich criteria is advisable where practical.

Licensing effects of inflammatory factors and TLR ligands on the regenerative capacity of adipose-derived mesenchymal stem cells.

Introduction: Adipose tissue-derived mesenchymal stem cells are promising contributors to regenerative medicine, exhibiting the ability to regenerate tissues and modulate the immune system, which is particularly beneficial for addressing chronic inflammatory ulcers and wounds. Despite their inherent capabilities, research suggests that pretreatment amplifies therapeutic effectiveness. Methods: Our experimental design exposed adipose-derived mesenchymal stem cells to six inflammatory factors for 24 h. We subsequently evaluated gene expression and proteome profile alterations and observed the wound closure rate post-treatment. Results: Specific pretreatments, such as IL-1ß, notably demonstrated an accelerated wound-healing process. Analysis of gene and protein expression profiles revealed alterations in pathways associated with tissue regeneration. Discussion: This suggests that licensed cells exhibit potentially higher therapeutic efficiency than untreated cells, shedding light on optimizing regenerative strategies using adipose tissue-derived stem cells.

Identifying Ferroptosis Inducers, HDAC, and RTK Inhibitor Sensitivity in Melanoma Subtypes through Unbiased Drug Target Prediction.

The utilization of PD1 and CTLA4 inhibitors has revolutionized the treatment of malignant melanoma (MM). However, resistance to targeted and immune-checkpoint-based therapies still poses a significant problem. Here we mine large scale MM proteogenomic data integrating it with MM cell line dependency screen, and drug sensitivity data to identify druggable targets and forecast treatment efficacy and resistance. Leveraging protein profiles from established MM subtypes and molecular structures of 82 cancer treatment drugs, we identified nine candidate hub proteins, mTOR, FYN, PIK3CB, EGFR, MAPK3, MAP4K1, MAP2K1, SRC and AKT1, across five distinct MM subtypes. These proteins serve as potential drug targets applicable to one or multiple MM subtypes. By analyzing transcriptomic data from 48 publicly accessible melanoma cell lines sourced from Achilles and CRISPR dependency screens, we forecasted 162 potentially targetable genes. We also identified genetic resistance in 260 genes across at least one melanoma subtype. In addition, we employed publicly available compound sensitivity data (Cancer Therapeutics Response Portal, CTRPv2) on the cell lines to assess the correlation of compound effectiveness within each subtype. We have identified 20 compounds exhibiting potential drug impact in at least one melanoma subtype. Remarkably, employing this unbiased approach, we have uncovered compounds targeting ferroptosis, that demonstrate a striking 30x fold difference in sensitivity among different subtypes. This implies that the proteogenomic classification of melanoma has the potential to predict sensitivity to ferroptosis compounds. Our results suggest innovative and novel therapeutic strategies by stratifying melanoma samples through proteomic profiling, offering a spectrum of novel therapeutic interventions and prospects for combination therapy. Highlights: (1) Proteogenomic subtype classification can define the landscape of genetic dependencies in melanoma (2) Nine proteins from molecular subtypes were identified as potential drug targets for specified MM patients (3) 20 compounds identified that show potential effectiveness in at least one melanoma subtype (4) Proteogenomics can predict specific ferroptosis inducers, HDAC, and RTK Inhibitor sensitivity in melanoma subtypes.

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System.

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a "zero set" of potential reference gene candidates.

BC-miR: Monitoring Breast Cancer-Related miRNA Profile in Blood Sera-A Prosperous Approach for Tumor Detection.

Breast cancer is the most frequent cancer with a high fatality rate amongst women worldwide. Diagnosing at an early stage is challenging, and due to the limitations of the currently used techniques, including mammography and imaging diagnostics, it still remains unascertained. Serum biomarkers can be a solution for this as they can be isolated in a less painful, more cost-effective, and minimally invasive manner. In this study, we shed light on the relevant role of multiple microRNAs (miRNAs) as potential biomarkers in breast cancer diagnosis. We monitored the expressional changes of 15 pre-selected miRNAs in a large cohort, including 65 patients with breast cancer and 42 healthy individuals. We performed thorough statistical analyses on the cohort sample set and determined the diagnostic accuracy of individual and multiple miRNAs. Our study reveals a potential improvement in diagnostics by implicating the monitoring of miR-15a+miR-16+miR-221 expression in breast cancer management.

The role of p53 in the DNA damage-related ubiquitylation of S2P RNAPII.

DNA double-strand breaks are one of the most deleterious lesions for the cells, therefore understanding the macromolecular interactions of the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. We previously demonstrated that following transcription block, p53 plays a pivotal role in transcription elongation by interacting with S2P RNAPII. In the current study, we reveal that p53 is involved in the fine-tune regulation of S2P RNAPII ubiquitylation. Furthermore, we emphasize the potential role of p53 in delaying the premature ubiquitylation and the subsequent chromatin removal of S2P RNAPII as a response to transcription block.

Usp5, Usp34, and Otu1 deubiquitylases mediate DNA repair in Drosophila melanogaster.

Ubiquitylation is critical for preventing aberrant DNA repair and for efficient maintenance of genome stability. As deubiquitylases (DUBs) counteract ubiquitylation, they must have a great influence on many biological processes, including DNA damage response. To elucidate the role of DUBs in DNA repair in Drosophila melanogaster, systematic siRNA screening was applied to identify DUBs with a reduced survival rate following exposure to ultraviolet and X-ray radiations. As a secondary validation, we applied the direct repeat (DR)-white reporter system with which we induced site-specific DSBs and affirmed the importance of the DUBs Ovarian tumor domain-containing deubiquitinating enzyme 1 (Otu1), Ubiquitin carboxyl-terminal hydrolase 5 (Usp5), and Ubiquitin carboxyl-terminal hydrolase 34 (Usp34) in DSB repair pathways using Drosophila. Our results indicate that the loss of Otu1 and Usp5 induces strong position effect variegation in Drosophila eye following I-SceI-induced DSB deployment. Otu1 and Usp5 are essential in DNA damage-induced cellular response, and both DUBs are required for the fine-tuned regulation of the non-homologous end joining pathway. Furthermore, the Drosophila DR-white assay demonstrated that homologous recombination does not occur in the absence of Usp34, indicating an indispensable role of Usp34 in this process.

SerpinB10, a Serine Protease Inhibitor, Is Implicated in UV-Induced Cellular Response.

UV-induced DNA damage response and repair are extensively studied processes, as any malfunction in these pathways contributes to the activation of tumorigenesis. Although several proteins involved in these cellular mechanisms have been described, the entire repair cascade has remained unexplored. To identify new players in UV-induced repair, we performed a microarray screen, in which we found SerpinB10 (SPB10, Bomapin) as one of the most dramatically upregulated genes following UV irradiation. Here, we demonstrated that an increased mRNA level of SPB10 is a general cellular response following UV irradiation regardless of the cell type. We showed that although SPB10 is implicated in the UV-induced cellular response, it has no indispensable function in cell survival upon UV irradiation. Nonetheless, we revealed that SPB10 might be involved in delaying the duration of DNA repair in interphase and also in S-phase cells. Additionally, we also highlighted the interaction between SPB10 and H3. Based on our results, it seems that SPB10 protein is implicated in UV-induced stress as a "quality control protein", presumably by slowing down the repair process.

PARylation During Transcription: Insights into the Fine-Tuning Mechanism and Regulation.

Transcription is a multistep, tightly regulated process. During transcription initiation, promoter recognition and pre-initiation complex (PIC) formation take place, in which dynamic recruitment or exchange of transcription activators occur. The precise coordination of the recruitment and removal of transcription factors, as well as chromatin structural changes, are mediated by post-translational modifications (PTMs). Poly(ADP-ribose) polymerases (PARPs) are key players in this process, since they can modulate DNA-binding activities of specific transcription factors through poly-ADP-ribosylation (PARylation). PARylation can regulate the transcription at three different levels: (1) by directly affecting the recruitment of specific transcription factors, (2) by triggering chromatin structural changes during initiation and as a response to cellular stresses, or (3) by post-transcriptionally modulating the stability and degradation of specific mRNAs. In this review, we principally focus on these steps and summarise the recent findings, demonstrating the mechanisms through which PARylation plays a potential regulatory role during transcription and DNA repair.

dTAF10- and dTAF10b-Containing Complexes Are Required for Ecdysone-Driven Larval-Pupal Morphogenesis in Drosophila melanogaster.

In eukaryotes the TFIID complex is required for preinitiation complex assembly which positions RNA polymerase II around transcription start sites. On the other hand, histone acetyltransferase complexes including SAGA and ATAC, modulate transcription at several steps through modification of specific core histone residues. In this study we investigated the function of Drosophila melanogaster proteins TAF10 and TAF10b, which are subunits of dTFIID and dSAGA, respectively. We generated a mutation which eliminated the production of both Drosophila TAF10 orthologues. The simultaneous deletion of both dTaf10 genes impaired the recruitment of the dTFIID subunit dTAF5 to polytene chromosomes, while binding of other TFIID subunits, dTAF1 and RNAPII was not affected. The lack of both dTAF10 proteins resulted in failures in the larval-pupal transition during metamorphosis and in transcriptional reprogramming at this developmental stage. Surprisingly, unlike dSAGA mutations, dATAC subunit mutations resulted in very similar changes in the steady state mRNA levels of approximately 5000 genes as did ablation of both dTaf10 genes, indicating that dTAF10- and/or dTAF10b-containing complexes and dATAC affect similar pathways. Importantly, the phenotype resulting from dTaf10+dTaf10b mutation could be rescued by ectopically added ecdysone, suggesting that dTAF10- and/or dTAF10b-containing complexes are involved in the expression of ecdysone biosynthetic genes. Indeed, in dTaf10+dTaf10b mutants, cytochrome genes, which regulate ecdysone synthesis in the ring gland, were underrepresented. Therefore our data support the idea that the presence of dTAF10 proteins in dTFIID and/or dSAGA is required only at specific developmental steps. We propose that distinct forms of dTFIID and/or dSAGA exist during Drosophila metamorphosis, wherein different TAF compositions serve to target RNAPII at different developmental stages and tissues.

Acetylations of Ftz-F1 and histone H4K5 are required for the fine-tuning of ecdysone biosynthesis during Drosophila metamorphosis.

The molting during Drosophila development is tightly regulated by the ecdysone hormone. Several steps of the ecdysone biosynthesis have been already identified but the regulation of the entire process has not been clarified yet. We have previously reported that dATAC histone acetyltransferase complex is necessary for the steroid hormone biosynthesis process. To reveal possible mechanisms controlled by dATAC we made assumptions that either dATAC may influence directly the transcription of Halloween genes involved in steroid hormone biosynthesis or it may exert an indirect effect on it by acetylating the Ftz-F1 transcription factor which regulates the transcription of steroid converting genes. Here we show that the lack of dATAC complex results in increased mRNA level and decreased protein level of Ftz-F1. In this context, decreased mRNA and increased protein levels of Ftz-F1 were detected upon treatment of Drosophila S2 cells with histone deacetylase inhibitor trichostatin A. We showed that Ftz-F1, the transcriptional activator of Halloween genes, is acetylated in S2 cells. In addition, we found that ecdysone biosynthetic Halloween genes are transcribed in S2 cells and their expression can be influenced by deacetylase inhibitors. Furthermore, we could detect H4K5 acetylation at the regulatory regions of disembodied and shade Halloween genes, while H3K9 acetylation is absent on these genes. Based on our findings we conclude that the dATAC HAT complex might play a dual regulatory role in Drosophila steroid hormone biosynthesis through the acetylation of Ftz-F1 protein and the regulation of the H4K5 acetylation at the promoters of Halloween genes.

The Not5 subunit of the ccr4-not complex connects transcription and translation.

Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.