Potential of gamma/delta T cells for solid tumor immunotherapy.

Gamma/delta T (γδ T)cells possess a unique mechanism for killing tumors, making them highly promising and distinguished among various cell therapies for tumor treatment. This review focuses on the major histocompatibility complex (MHC)-independent recognition of antigens and the interaction between γδ T cells and solid tumor cells. A comprehensive review is provided regarding the classification of human gamma-delta T cell subtypes, the characteristics and mechanisms underlying their functions, as well as their r545egulatory effects on tumor cells. The involvement of γδ T cells in tumorigenesis and migration was also investigated, encompassing potential therapeutic targets such as apoptosis-related molecules, the TNF receptor superfamily member 6(FAS)/FAS Ligand (FASL) pathways, butyrophilin 3A-butyrophilin 2A1 (BTN3A-BTN2A1) complexes, and interactions with CD4, CD8, and natural killer (NK) cells. Additionally, immune checkpoint inhibitors such as programmed cell death protein 1/Programmed cell death 1 ligand 1 (PD-1/PD-L1) have the potential to augment the cytotoxicity of γδ T cells. Moreover, a review on gamma-delta T cell therapy products and their corresponding clinical trials reveals that chimeric antigen receptor (CAR) gamma-delta T therapy holds promise as an approach with encouraging preclinical outcomes. However, practical issues pertaining to manufacturing and clinical aspects need resolution, and further research is required to investigate the long-term clinical side effects of CAR T cells. In conclusion, more comprehensive studies are necessary to establish standardized treatment protocols aimed at enhancing the quality of life and survival rates among tumor patients utilizing γδ T cell immunotherapy.

Irinotecan plus raltitrexed as second-line treatment in locally advanced or metastatic colorectal cancer patients: a prospective open-label, single-arm, multi-center, phase II study.

BACKGROUND: Colorectal cancer is the third most common cancer and the second leading cause of cancer death. There are limited therapeutic options for the treatment of locally advanced or metastatic colorectal cancers which fail first-line chemotherapy. Phase I/II studies showed that the combined application of the raltitrexed and irinotecan has significant synergistic effect and acceptable toxicity. However, most of these previous studies have relatively small sample size. METHODS: This is a prospective open-label, single-arm, multi-center, Phase II trial. Brief inclusion criteria: patients were aged 18 to 75 years with locally advanced or metastatic colorectal cancer after failure of 5-FU and oxaliplatin therapy. Enrolled patients received raltitrexed (3 mg/m2, d1) and irinotecan (180 mg/m2, d1) each 21-day cycle until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival, and the secondary endpoints were disease control rate, objective response rate, overall survival and safety. RESULTS: A total of 108 patients were enrolled between September 2016 and May 2020. The median age was 61 years, ECOG 1 score accounts for 67.6%, the rest were ECOG 0. A total of 502 cycles were completed, with an average of 4.6 cycles and a median of 4 cycles. 108 patients were evaluated, with an objective response rate of 17.6%, and disease control rate of 76.9%. The median follow-up time was 27 months (range:3.1-61.0 m) at data cut-off on March 2023. Median progression-free survival was 4.9 months (95% CI 4.1-5.7) and median overall survival was 13.1 months (95% CI 12.2-15.5). The most common adverse events that were elevated are alanine aminotransferase increased, aspartate aminotransferase increased, fatigue, diarrhoea, neutrocytopenia, thrombocytopenia, hypohemoglobin, and leukocytopenia. Most of the adverse events were Grade I/II, which were relieved after symptomatic treatment, and there were no treatment-related cardiotoxicities and deaths. CONCLUSIONS: The combination of raltitrexed and irinotecan as second-line treatment for mCRC could be a reliable option after failure of standard 5-Fu-first-line chemotherapy in locally advanced or metastatic colorectal cancers, especially for patients with 5-FU intolerance (cardiac events or DPD deficiency patients). TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03053167, registration date was 14/2/2017.

Integrated Analysis of Clinical Outcome of Mesenchymal Stem Cell-related Genes in Pan-cancer.

Background: Although the application of mesenchymal stem cells (MSCs) in engineered medicine, such as tissue regeneration, is well known, new evidence is emerging that shows that MSCs can also promote cancer progression, metastasis, and drug resistance. However, no large-scale cohort analysis of MSCs has been conducted to reveal their impact on the prognosis of cancer patients. Objectives: We propose the MSC score as a novel surrogate for poor prognosis in pan-cancer. Methods: We used single sample gene set enrichment analysis to quantify MSC-related genes into a signature score and identify the signature score as a potential independent prognostic marker for cancer using multivariate Cox regression analysis. TIDE algorithm and neural network were utilized to assess the predictive accuracy of MSC-related genes for immunotherapy. Results: MSC-related gene expression significantly differed between normal and tumor samples across the 33 cancer types. Cox regression analysis suggested the MSC score as an independent prognostic marker for kidney renal papillary cell carcinoma, mesothelioma, glioma, and stomach adenocarcinoma. The abundance of fibroblasts was also more representative of the MSC score than the stromal score. Our findings supported the combined use of the TIDE algorithm and neural network to predict the accuracy of MSC-related genes for immunotherapy. Conclusion: We comprehensively characterized the transcriptome, genome, and epigenetics of MSCs in pan-cancer and revealed the crosstalk of MSCs in the tumor microenvironment, especially with cancer-related fibroblasts. It is suggested that this may be one of the key sources of resistance to cancer immunotherapy.

Icotinib in a lung adenocarcinoma patient with acquired EGFR 19del/C797S mutation-mediated resistance to osimertinib: a case report.

Based on the FLAURA and AURA III trials, compared to first- and second-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), osimertinib provides a longer overall survival benefit for patients with untreated EGFR mutated non-small cell lung cancer. Similar to other EGFR-TKIs, drug resistance is, however, inevitable. The most common mechanism of acquired resistance to first-line osimertinib therapy is the C797S mutation, which accounts for 6% of cases. In view of the current challenges of the development of the next generation of EGFR inhibitors, the mechanism of third-generation targeted drug resistances and targeted strategies are key for further exploration. Our case report discusses a female patient with advanced lung adenocarcinoma carrying the EGFR exon19 E746_A750delinsIP mutation who received osimertinib as first-line therapy and acquired C797S resistance during treatment. The patient was then treated with icotinib for 8 months until the disease progressed. Icotinib may be effective in patients with the EGFR 19del-C797S resistant mutation acquired after osimertinib treatment.

Serum cytokines and creatinine/cystatin C ratio as prognostic biomarkers in advanced cancer patients treated with anti-PD-1/PD-L1 therapy.

OBJECTIVE: Immune checkpoint inhibitors (ICIs), specifically targeting the programmed cell death protein-1 or its ligand (PD-1/PD-L1), have been extensively used in the treatment of a spectrum of malignancies, although the predictive biomarkers remain to be elucidated. This study aims to investigate the association between baseline circulating levels of cytokines and the creatinine/cystatin C ratio (CCR) with the treatment outcomes of ICIs in patients with advanced cancer. METHODS: The pre-treatment circulating levels of 10 cytokines (PD-L1, CTLA4, CXCL10, LAG3, HGF, CCL2, MIG, GRANB, IL-18, and IL-6) were measured via automated capillary-based immunoassay platform in the serum of 65 advanced cancer patients treated with anti-PD-1/PD-L1-based systemic therapy and 10 healthy volunteers. The levels of cytokines and CCR were quantified and categorized into high and low groups based on the median value. The associations of serum cytokines and CCR with response to treatment, survival, and immune-related adverse events were assessed. RESULTS: Elevated circulating levels of 6 cytokines (PD-L1, CXCL10, HGF, CCL2, MIG, and IL-6) were observed in cancer patients compared with that in healthy volunteers. The correlation coefficients between cytokines, CCR and nutritional risk index were also calculated. In the cancer cohort (N = 65), low circulating HGF (P = 0.023, P = 0.029), low IL-6 (P = 0.002, P < 0.001), and high CCR (P = 0.031, P = 0.008) were associated with significantly improved progression-free survival (PFS) and overall survival (OS). Multi-variable COX analyses adjusted for clinicopathological factors revealed that low HGF, low IL-6, and high CCR were independent favorable prognostic factors for PFS (P = 0.028, P = 0.010, and P = 0.015, respectively) and OS (P = 0.043, P = 0.003, and P = 0.026, respectively). Grade 2 irAEs occurred more frequently in patients with low levels of circulating CCL2 and LAG3. CONCLUSIONS: Pre-treatment circulating levels of serum IL-6, HGF, and CCR may serve as independent predictive and prognostic biomarkers in advanced cancer patients treated with ICIs-based systemic therapy. These findings might help to identify potential patients who would benefit from these therapies.

CEACAM6 expression and function in tumor biology: a comprehensive review.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily protein primarily expressed on epithelial surfaces and myeloid cells. It plays a significant role in cancer progression by inhibiting apoptosis, promoting drug resistance, and facilitating cancer cell invasion and metastasis. Overexpression of CEACAM6 has been observed in various cancers, including lung, breast, colorectal, and hepatocellular cancers, and is associated with poorer overall survival and disease-free survival. Its differential expression on tumor cell surfaces makes it a promising cancer marker. This review aims to provide a comprehensive summary of CEACAM6's role in different cancer types, its involvement in signaling pathways, and recent advancements in CEACAM6-targeted treatments.

Extensive Stage Small-Cell Lung Cancer with Cystic Brain Metastases: A Report of Two Cases.

Objective: Cystic brain metastases (BMs) are rare in small cell lung cancer (SCLC), and there are limited data on the treatment and prognosis of cystic BMs. Whole brain radiotherapy has been the mainstay for BMs since several years. Immune checkpoint inhibitors in extensive stage small cell lung cancer (ES-SCLC) have been shown to be suitable for patients who experienced better overall survival and progress-free survival and have been approved as the first-line treatment for ES-SCLC. In this report, we described two ES-SCLC patients developed cystic BMs after immunotherapy, after which the patients continued to treat the primary lesion with immune checkpoint inhibitors and the cystic BMs with radiotherapy. Case Description: Two male patients were diagnosed with ES-SCLC at the first admission and were subsequently treated with immunotherapy plus platinum therapy, during which cystic BMs developed. One patient received whole brain radiotherapy and the other received whole brain radiotherapy and Gamma knife radiosurgery (GKRS). Immunotherapy was continued after the brain lesions were controlled. It has been 33 months since the first patient was diagnosed and is now in stable condition. The other patient achieved an overall survival of 30 months. Conclusion: This report describes two patients with cystic brain metastases in ES-SCLC. Whole brain radiotherapy has a good effect on local control of cystic brain metastases in small cell lung cancer and can significantly improve the symptoms of patients. At the same time, we treat immunotherapy as the first-line treatment, and then perform cross-immunotherapy after disease progression, combined with anti-vascular targeting drugs. The patient did not develop severe iRAEs.

Investigation of the molecular mechanism of Danggui Buxue tang in treating lung cancer using network pharmacology and molecular docking techniques.

This study used network pharmacology and molecular docking techniques to investigate the molecular targets and pathways of Danggui Buxue Tang (DBT) in treating lung cancer. The compound-target network was constructed using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), and a lung cancer-specific network was created using the GEO database and Cytoscape software. GO and KEGG pathway analyses were performed to understand the biological processes associated with DBT. The key compounds from Astragalus, kaempferol, and quercetin, and the potential targets are IL-6, IL-1ß, FOS, ICAM1, and CCL2. GO enrichment analysis revealed numerous biological process-related entries, while KEGG pathway analysis highlighted the TNF and IL-17 signalling pathways. Molecular docking confirmed the stable binding activity between the main active compounds of DBT and the target proteins. Overall, these findings shed light on the molecular mechanism of DBT in treating lung cancer, providing insights into targets, pathways, and biological processes involved.

Updated overall survival and circulating tumor DNA analysis of ensartinib for crizotinib-refractory ALK-positive NSCLC from a phase II study.

BACKGROUND: The initial phase II stuty (NCT03215693) demonstrated that ensartinib has shown clinical activity in patients with advanced crizotinib-refractory, anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC). Herein, we reported the updated data on overall survival (OS) and molecular profiling from the initial phase II study. METHODS: In this study, 180 patients received 225 mg of ensartinib orally once daily until disease progression, death or withdrawal. OS was estimated by Kaplan‒Meier methods with two-sided 95% confidence intervals (CIs). Next-generation sequencing was employed to explore prognostic biomarkers based on plasma samples collected at baseline and after initiating ensartinib. Circulating tumor DNA (ctDNA) was detected to dynamically monitor the genomic alternations during treatment and indicate the existence of molecular residual disease, facilitating improvement of clinical management. RESULTS: At the data cut-off date (August 31, 2022), with a median follow-up time of 53.2 months, 97 of 180 (53.9%) patients had died. The median OS was 42.8 months (95% CI: 29.3-53.2 months). A total of 333 plasma samples from 168 patients were included for ctDNA analysis. An inferior OS correlated significantly with baseline ALK or tumor protein 53 (TP53) mutation. In addition, patients with concurrent TP53 mutations had shorter OS than those without concurrent TP53 mutations. High ctDNA levels evaluated by variant allele frequency (VAF) and haploid genome equivalents per milliliter of plasma (hGE/mL) at baseline were associated with poor OS. Additionally, patients with ctDNA clearance at 6 weeks and slow ascent growth had dramatically longer OS than those with ctDNA residual and fast ascent growth, respectively. Furthermore, patients who had a lower tumor burden, as evaluated by the diameter of target lesions, had a longer OS. Multivariate Cox regression analysis further uncovered the independent prognostic values of bone metastases, higher hGE, and elevated ALK mutation abundance at 6 weeks. CONCLUSION: Ensartinib led to a favorable OS in patients with advanced, crizotinib-resistant, and ALK-positive NSCLC. Quantification of ctDNA levels also provided valuable prognostic information for risk stratification.

A Five-gene Signature based on MicroRNA for Predicting Prognosis and Immunotherapy in Stomach Adenocarcinoma.

AIMS: We aimed to classify molecular subtypes and establish a prognostic gene signature based on miRNAs for the prognostic prediction and therapeutic response in Stomach adenocarcinoma (STAD). BACKGROUND: STAD is a common diagnosed gastrointestinal malignancy and its heterogeneity is a big challenge that influences prognosis and precision therapies. Present study was designed to classify molecular subtypes and construct a prognostic gene signature based on miRNAs for the prognostic prediction and therapeutic response in STAD. OBJECTIVE: The objective of this study is to investigate the molecular subtypes and prognostic model for STAD. METHODS: A STAD specific miRNA-messenger RNA (mRNA) competing endogenous RNA (ceRNA) network was generated using the RNA-Seq and miRNA expression profiles from The Cancer Genome Atlas (TCGA) database, in which miRNA-related mRNAs were screened. Molecular subtypes were then determined using miRNA-related genes. Through univariate Cox analysis and multivariate regression analysis, a prognostic model was established in GSE84437 Train dataset and validated in GSE84437 Test, TCGA, GSE84437 and GSE66229 datasets. Immunotherapy datasets were employed for assessing the performance of the risk model. Finally, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to validate the expression of hub genes used for the risk score signature. RESULTS: We constructed a ceRNA network containing 84 miRNAs and 907 mRNAs and determined two molecular subtypes based on 26 genes from the intersection of TCGASTAD and GSE84437 datasets. Subtype S2 had poor prognosis, lower tumor mutational burden, higher immune score and lower response to immunotherapy. Subtype S1 was more sensitive to Sorafenib, Pyrimethamine, Salubrinal, Gemcitabine, Vinorelbine and AKT inhibitor VIII. Next, a five-gene signature was generated and its robustness was validated in Test and external datasets. This risk model also had a good prediction performance in immunotherapy datasets. CONCLUSION: This study promotes the underlying mechanisms of miRNA-based genes in STAD and offers directions for classification. A five-gene signature accurately predicts the prognosis and helps therapeutic options.

Current Situation and Prospect of Adoptive Cellular Immunotherapy for Malignancies.

Adoptive cell immunotherapy (ACT) is an innovative promising treatment for tumors. ACT is characterized by the infusion of active anti-tumor immune cells (specific and non-specific) into patients to kill tumor cells either directly or indirectly by stimulating the body's immune system. The patient's (autologous) or a donor's (allogeneic) immune cells are used to improve immune function. Chimeric antigen receptor (CAR) T cells (CAR-T) is a type of ACT that has gained attention. T cells from the peripheral blood are genetically engineered to express CARs that rapidly proliferate and specifically recognize target antigens to exert its anti-tumor effects. Clinical application of CAR-T therapy for hematological tumors has shown good results, but adverse reactions and recurrence limit its applicability. Tumor infiltrating lymphocyte (TIL) therapy is effective for solid tumors. TIL therapy exhibits T cell receptor (TCR) clonality, superior tumor homing ability, and low targeted toxicity, but its successful application is limited to a number of tumors. Regardless, TIL and CAR-T therapies are effective for treating cancer. Additionally, CAR-natural killer (NK), CAR-macrophages (M), and TCR-T therapies are currently being researched. In this review, we highlight the current developments and limitations of several types of ACT.

NPLOC4 is a potential target and a poor prognostic signature in lung squamous cell carcinoma.

Few prognostic biomarkers exist for lung squamous cell carcinoma (LUSC), which has a poor five-year survival rate. Using bioinformatics, this study evaluated NPLOC4 as a prognostic marker for patients with lung squamous cell carcinoma. Shorter survival periods and tumor growth were linked to high NPLOC4 expression.Disulfiram (DSF) combined with copper (Cu) targets NPLOC4 to achieve antitumor effects in lung squamous cell carcinoma. Thus, we investigated the effects of DSF with Cu in LUSC. Gene-set enrichment analysis identified ubiquitin-mediated proteolysis as the NPLOC4-associated mechanism influencing LUSC prognosis. In SK-MES-1 cell lines, DSF + Cu increased K48-linked ubiquitinated protein expression and apoptosis. This study identified NPLOC4 as a prognostic biomarker and a potential therapeutic target for LUSC.

Current status and prospects of GREM1 research in cancer (Review).

GREM1 is a secreted protein that antagonizes bone morphogenetic proteins (BMPs) and participates in critical biological processes, including embryonic development, organogenesis and tissue differentiation. Gremlin 1 (GREM1) is also an inhibitor of TGF-ß and a ligand for vascular endothelial growth factor receptor 2. In addition, GREM1 can induce cells, participate in the process of epithelial-mesenchymal transition, and then participate in tumor development. GREM1 has a variety of biological functions and can participate in the malignant progression of a variety of tumors through the BMP signaling pathway. GREM1 also can inhibit TGF-ß in some tumors, thereby inhibiting tumors, and its involvement in tumor development varies in different types of cancer. The present review examines the role and function of GREM1 in tumors. GREM1 is expressed in a variety of tumor types. GREM1 expression can affect the epithelial-mesenchymal transformation of tumor cells. GREM1 has been studied in breast and colon cancer, and its potential role is to promote cancer. However, in pancreatic cancer, which was found to act differently from other cancer types, overexpression of GREM1 inhibits tumor metastasis. The present review suggests that GREM1 can be a diagnostic and prognostic indicator. In future studies, the study of GREM1 based on single-cell sequencing technology will further clarify its role and function in tumors.

Deciphering intercellular signaling complexes by interaction-guided chemical proteomics.

Indirect cell-cell interactions mediated by secreted proteins and their plasma membrane receptors play essential roles for regulating intercellular signaling. However, systematic profiling of the interactions between living cell surface receptors and secretome from neighboring cells remains challenging. Here we develop a chemical proteomics approach, termed interaction-guided crosslinking (IGC), to identify ligand-receptor interactions in situ. By introducing glycan-based ligation and click chemistry, the IGC approach via glycan-to-glycan crosslinking successfully captures receptors from as few as 0.1 million living cells using only 10 ng of secreted ligand. The unparalleled sensitivity and selectivity allow systematic crosslinking and identification of ligand-receptor complexes formed between cell secretome and surfaceome in an unbiased and all-to-all manner, leading to the discovery of a ligand-receptor interaction between pancreatic cancer cell-secreted urokinase (PLAU) and neuropilin 1 (NRP1) on pancreatic cancer-associated fibroblasts. This approach is thus useful for systematic exploring new ligand-receptor pairs and discovering critical intercellular signaling events.

Integrated and High-Throughput Approach for Sensitive Analysis of Tyrosine Phosphoproteome.

Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 µg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

Comparison of efficacy and safety of ripertamab (SCT400) versus rituximab (Mabthera® ) in combination with CHOP in patients with previously untreated CD20-positive diffuse large B-cell lymphoma: A randomized, single-blind, phase III clinical trial.

This study compared the efficacy, safety and immunogenicity of ripertamab (SCT400) and rituximab (Mabthera® ) combined with CHOP as the first-line treatment for Chinese patients with CD20-positive diffuse large B cell lymphoma (DLBCL). This is a randomized, patient-blind, multicenter, active-control, non-inferiority study with parallel design. Patients were randomly (2:1) to receive ripertamab combined with CHOP (S-CHOP) or rituximab (Mabthera® ) combined with CHOP (R-CHOP) for up to 6 cycles. The primary endpoint was the Independent Review Committee (IRC) assessed objective response rate (ORR) in full analysis set (FAS) and the per protocol set (PPS). A total of 364 patients (243 in the S-CHOP and 121 in the R-CHOP groups) were enrolled in this study. In FAS, IRC-assessed ORRs were 93.8% (95% confidence interval (CI) 90.0%, 96.5%) and 94.2% (95% CI: 88.4%, 97.6%) in the S-CHOP and R-CHOP groups (p = 0.9633), respectively. The ORR difference between the two groups -0.4% (95% CI: -5.5%, 4.8%) met the pre-specified non-inferiority margin of -12%. There were no significant differences between the S-CHOP and R-CHOP groups in 1-year progression-free survival rates (81.1% vs. 83.2%, p = 0.8283), 1 year event-free survival rates (56.2% vs. 58.1%, p = 0.8005), and 3-year overall survival rates (81.0% vs. 82.8%, p = 0.7183). The results in PPS were consistent with those in FAS. The rates of treatment-emergent adverse events (TEAEs) and ≥ grade 3 TEAEs were 97.9% and 99.2%, 85.2% and 86.0% in the S-CHOP and R-CHOP groups, respectively in safety set. The percentage of anti-drug antibodies positive patients in the S-CHOP group was numerically lower than the R-CHOP group (10.9% vs. 16.0%). This study demonstrated that S-CHOP was not inferior to R-CHOP in the first-line treatment of Chinese patients with CD20-positive DLBCL in efficacy, safety and immunogenecity. S-CHOP could be an alternative first-line standard treatment regimen for this patient population.

Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

Disulfiram/Copper induces antitumor activity against gastric cancer via the ROS/MAPK and NPL4 pathways.

Disulfiram (DSF) is an anti-alcoholism medication with superior antitumor activity and clinical safety; its antitumor mechanisms in gastric cancer (GC) have not been fully explored. In the present work, low nontoxic concentrations of copper (Cu) ions substantially enhanced DSF's antitumor activity, inhibiting the proliferation and growth of GC cell lines. DSF/Cu elevated the generation of reactive oxygen species (ROS), and apoptosis was induced in an ROS-dependent manner. This process might involve primary inhibition GC by DSF/Cu through induction of apoptosis via the ROS/mitogen-activated protein kinase pathway. Disordering transportation of ubiquitinated protein may also fuel the process. In summary, we found that DSF exerts antitumor effects on GC. DSF/Cu should be considered as adjunctive therapy for GC.

Disulfiram/copper induces antitumor activity against gastric cancer cells in vitro and in vivo by inhibiting S6K1 and c-Myc.

PURPOSE: Disulfiram (DSF) is an approved drug for the treatment of alcohol dependence. Accumulating evidence indicates that DSF, alone or in combination with copper (Cu), possesses strong antitumor activity in various malignancies. This study investigated the effects of DSF on gastric cancer (GC) and the potential mechanisms involved. METHODS: GC cell proliferation and apoptosis upon treatment with DSF with or without copper were analyzed using CCK-8 assay, colony formation assay, and flow cytometry. Glucose metabolism was investigated using glucose consumption and lactate production assays. The expression of caspase-3, Bcl-2, LC-3, P62, S6K1, c-Myc, GLUT1, PKM2, and LDHA was analyzed using western blot assay. In vivo nude mice studies were performed to verify the findings from in vitro analyses. RESULTS: Our study showed that DSF was highly toxic to GC cells in a Cu-dependent manner. Nontoxic concentrations of Cu enhanced the inhibitory effects of DSF on cell viability and colony formation. DSF also induced apoptotic and autophagic cell death in the presence of Cu. In addition, DSF/Cu inhibited glycolysis and xenograft growth of GC cells by suppressing the expression of S6K1, c-Myc, and their downstream molecules, including GLUT1, PKM2, and LDHA. CONCLUSION: Our study demonstrated that DSF/Cu exerted antitumor activity against GC cells both in vitro and in vivo. DSF/Cu may represent a promising therapeutic strategy for the treatment of GC.

Case Report: Detection of Double ROS1 Translocations, SDC4-ROS1 and ROS1-GK, in a Lung Adenocarcinoma Patient and Response to Crizotinib.

ROS1 rearrangement, identified in ~2% of non-small cell lung cancer (NSCLC), has defined a distinctive molecular subtype. Patients with ROS1 fusion have been shown to be highly sensitive to treatment with crizotinib. However, the efficacy of crizotinib in NSCLC patients with double ROS1 fusions remains to be elucidated. Here, we report a 40-year-old male diagnosed with stage IIIA lung adenocarcinoma. Two ROS1 fusions [SDC4-ROS1 (EX2:EX32) and ROS1-GK (EX31:EX13)] were detected simultaneously in tumor tissue of this patient by next-generation sequencing. Crizotinib was administered, and the patient showed a partial response in lung lesions. Nevertheless, a brain lesion was found at 8 months after treatment. The slightly short duration of response may be related to the presence of ROS1-GK rearrangement. This case proved that patients with SDC4-ROS1 and ROS1-GK fusions may be sensitive to crizotinib, but short progression-free survival of this case showed that the presence of ROS1-GK rearrangement may affect the efficacy of crizotinib. A large-scale investigation on the efficacy of ROS1 inhibitors in patients with complex ROS1 fusions should be conducted in the future.