Tacrolimus Related Acute Pancreatitis: An Observational, Retrospective, Pharmacovigilance Study.

PURPOSE: Recent case reports have drawn attention to the emergence of acute pancreatitis, a potentially life-threatening complication associated with tacrolimus. This study uses the Food and Drug Administration Adverse Event Reporting System (FAERS) to investigate the risk signal of acute pancreatitis associated with calcineurin inhibitors (CNIs), with a focus on tacrolimus. METHODS: We conducted an observational retrospective pharmacovigilance study utilizing the FAERS database, encompassing data from its inception to the third quarter of 2023. The assessment of the association between CNIs and acute pancreatitis was carried out using the Information Component (IC) and Reporting Odds Ratio (ROR). Logistic regression analysis was employed to elucidate factors contributing to fatal outcomes. All analyses were performed using R version 3.2.5. FINDING: We identified 221 cases of acute pancreatitis linked to CNIs. The median age of individuals experiencing acute pancreatitis induced by tacrolimus was 43, with a predominant occurrence among male patients. Our study showed a significant association between CNIs and acute pancreatitis (ROR 1.82 [1.60-2.08], IC 0.85 [3.66-3.92]). Comparing tacrolimus and cyclosporine, the signal for tacrolimus seemed to be higher. Further analysis revealed that, with the exception of patients aged 60 and above, the signal for tacrolimus remained stable. Contrastingly, the signal for cyclosporine was unstable and limited to the male group and individuals aged less than 20 years. In cases of CNIs-related acute pancreatitis, the mortality rate was 31.67% (70/221 cases). Logistic regression analysis indicated that a younger age acts as a protective factor for death due to CNIs-related acute pancreatitis (OR 0.943, 95% CI 0.915-0.972, P = 0.000). IMPLICATIONS: Our study has identified a safety signal for tacrolimus in relation to acute pancreatitis. Additionally, we observed advanced age as a significant risk factor for tacrolimus-related acute pancreatitis, leading to mortality. Given the widespread use of tacrolimus, it is crucial for healthcare providers to be vigilant and informed about the potential association with acute pancreatitis.

Immune checkpoint inhibitor related myositis: an observational, retrospective, pharmacovigilance study.

BACKGROUND: Immune checkpoint inhibitors (ICIs) show promise in cancer treatment, but recent cases highlight myositis as a serious complication. RESEARCH DESIGN AND METHODS: We did a retrospective study on drug safety using FAERS data up to Q3 2022, focusing on immune checkpoint inhibitors (ICIs) and myositis. We used IC and ROR to assess the association. Logistic regression in R 3.2.5 helped identify factors linked to fatal outcomes. RESULTS: We identified 558 cases of ICIs-associated myositis. Our study found a significant link between ICIs and myositis (ROR 15.54 [14.23-16.96], IC 3.79 [3.66-3.92], see Figure 1). Notably, myositis was more common in patients on ICI combination therapy compared to monotherapy (ROR 1.72 [1.39-2.11], IC 0.63 [0.30-0.93]). Age increased the risk of ICI-associated myositis and was also a factor in fatality (p = 0.011). Common accompanying adverse events included myocarditis (21.33%), severe myasthenia gravis (16.49%), and malignant neoplasm progression (8.06%). Fatal cases were more common when myositis was accompanied by myocarditis, severe myasthenia gravis, or malignant neoplasm progression. CONCLUSIONS: Clinicians must note the risk of ICI-associated myositis, especially dangerous in older patients or when combined with other issues like myocarditis or severe myasthenia gravis.

Wip1 inhibits neutrophil extracellular traps to promote abscess formation in mice by directly dephosphorylating Coronin-1a.

Neutrophil extracellular traps (NETs) participate in the rapid inhibition and clearance of pathogens during infection; however, the molecular regulation of NET formation remains poorly understood. In the current study, we found that inhibition of the wild-type p53-induced phosphatase 1 (Wip1) significantly suppressed the activity of Staphylococcus aureus (S. aureus) and accelerated abscess healing in S. aureus-induced abscess model mice by enhancing NET formation. A Wip1 inhibitor significantly enhanced NET formation in mouse and human neutrophils in vitro. High-resolution mass spectrometry and biochemical assays demonstrated that Coro1a is a substrate of Wip1. Further experiments also revealed that Wip1 preferentially and directly interacts with phosphorylated Coro1a than compared to unphosphorylated inactivated Coro1a. The phosphorylated Ser426 site of Coro1a and the 28-90 aa domain of Wip1 are essential for the direct interaction of Coro1a and Wip1 and for Wip1 dephosphorylation of p-Coro1a Ser426. Wip1 deletion or inhibition in neutrophils significantly upregulated the phosphorylation of Coro1a-Ser426, which activated phospholipase C and subsequently the calcium pathway, the latter of which promoted NET formation after infection or lipopolysaccharide stimulation. This study revealed Coro1a to be a novel substrate of Wip1 and showed that Wip1 is a negative regulator of NET formation during infection. These results support the potential application of Wip1 inhibitors to treat bacterial infections.

Distinct genomic features between osteosarcomas firstly metastasing to bone and to lung.

Background: Osteosarcoma initially metastasing to bone only shows distinct biological features compared to osteosarcoma that firstly metastasizes to the lung, which suggests us underlying different genomic pathogenetic mechanism. Methods: We analyzed whole-exome sequencing (WES) data for 38 osteosarcoma with paired samples in different relapse patterns. We also sought to redefine disease subclassifications for osteosarcoma based on genetic alterations and correlate these genetic profiles with clinical treatment courses to elucidate potential evolving cladograms. Results: We investigated WES of 12/38 patients with high-grade osteosarcoma (31.6%) with initial bone metastasis (group A) and 26/38 (68.4%) with initial pulmonary metastasis (group B), of whom 15/38 (39.5%) had paired samples of primary lesions and metastatic lesions. We found that osteosarcoma in group A mainly carries single-nucleotide variations displaying higher tumor mutation burden and neoantigen load and more tertiary lymphoid structures, while those in group B mainly exhibits structural variants. High conservation of reported genetic sequencing over time in their evolving cladograms. Conclusions: Osteosarcoma with mainly single-nucleotide variations other than structural variants might exhibit biological behavior predisposing toward bone metastases as well as better immunogenicity in tumor microenvironment.

Identification of IRAK1BP1 as a candidate prognostic factor in lung adenocarcinoma.

Introduction: Lung cancer is one of the major causes of cancer-related mortality worldwide. High-throughput RNA sequencing (RNA-seq) of surgically removed tumors has been used to identify new biomarkers of lung cancer; however, contamination by non-tumor cells in the tumor microenvironment significantly interferes with the search for novel biomarkers. Tumor organoids, as a pre-clinical cancer model, exhibit similar molecular characteristics with tumor samples while minimizing the interference from other cells. Methods and Results: Here we analyzed six RNA-seq datasets collected from different organoid models, in which cells with oncogenic mutations were reprogrammed to mimic lung adenocarcinoma (LUAD) tumorigenesis. We uncovered 9 LUAD-specific biomarker genes by integrating transcriptomic data from multiple sources, and identified IRAK1BP1 as a novel predictor of LUAD disease outcome. Validation with RNA-seq and microarray data collected from multiple patient cohorts, as well as patient-derived xenograft (PDX) and lung cancer cell line models confirmed that IRAK1BP1 expression was significantly lower in tumor cells, and had no correlation with known markers oflung cancer prognosis. In addition, loss of IRAK1BP1 correlated with the group of LUAD patients with worse survival; and gene-set enrichment analysis using tumor and cell line data implicated that high IRAK1BP1 expression was associated with suppression of oncogenic pathways. Discussion: In conclusion, we demonstrate that IRAK1BP1 is a promising biomarker of LUAD prognosis.

Asymmetric Cell Division and Tumor Heterogeneity.

Asymmetric cell division (ACD) gives rise to two daughter cells with different fates after mitosis and is a fundamental process for generating cell diversity and for the maintenance of the stem cell population. The cancer stem cell (CSC) theory suggests that CSCs with dysregulated self-renewal and asymmetric cell division serve as a source of intra-tumoral heterogeneity. This heterogeneity complicates the diagnosis and treatment of cancer patients, because CSCs can give rise to aggressive clones that are metastatic and insensitive to multiple drugs, or to dormant tumor cells that are difficult to detect. Here, we review the regulatory mechanisms and biological significance of asymmetric division in tumor cells, with a focus on ACD-induced tumor heterogeneity in early tumorigenesis and cancer progression. We will also discuss how dissecting the relationship between ACD and cancer may help us find new approaches for combatting this heterogeneity.

Costimulation of γδTCR and TLR7/8 promotes Vδ2 T-cell antitumor activity by modulating mTOR pathway and APC function.

BACKGROUND: Gamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy. METHODS: We developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies. RESULTS: We find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt)-the mammalian target of rapamycin (mTOR) pathway and the TLR7/8-MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function. CONCLUSIONS: Vδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.

TGF-ß Signaling and Resistance to Cancer Therapy.

The transforming growth factor ß (TGF-ß) pathway, which is well studied for its ability to inhibit cell proliferation in early stages of tumorigenesis while promoting epithelial-mesenchymal transition and invasion in advanced cancer, is considered to act as a double-edged sword in cancer. Multiple inhibitors have been developed to target TGF-ß signaling, but results from clinical trials were inconsistent, suggesting that the functions of TGF-ß in human cancers are not yet fully explored. Multiple drug resistance is a major challenge in cancer therapy; emerging evidence indicates that TGF-ß signaling may be a key factor in cancer resistance to chemotherapy, targeted therapy and immunotherapy. Finally, combining anti-TGF-ß therapy with other cancer therapy is an attractive venue to be explored for the treatment of therapy-resistant cancer.

Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma.

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.

Dichotomous and stable gamma delta T-cell number and function in healthy individuals.

BACKGROUND: Gamma-delta (γδ) T lymphocytes are primed to potently respond to pathogens and transformed cells by recognizing a broad range of antigens. However, adoptive immunotherapy with γδT cells has exhibited mixed treatment responses. Better understanding of γδT cell biology and stratifying healthy donors for allogeneic adoptive therapy is clinically needed to fully realize the therapeutic potential of γδT cells. METHODS: We examine 98 blood samples from healthy donors and measure their expansion capacity after zoledronate stimulation, and test the migration and cytotoxic effector function of expanded γδT cells in 2D culture, 3D tumor spheroid and patient-derived melanoma organoid assays. RESULTS: We find that γδT cell expansion capacity is independent of expansion methods, gender, age and HLA type. Basal γδT cell levels in Peripheral blood mononuclear cell (PBMC) correlate well with their expansion, migration and cytotoxic effector capacity in vitro. Circulating γδT cells with lower expression of PD-1, CTLA-4, Eomes, T-bet and CD69, or higher IFN-γ production expand better. γδT cells with central memory and effector memory phenotypes are significantly more abundant in good expanders. A cut-off level of 0.82% γδT cells in PBMC stratifies good versus poor γδT cell expansion with a sensitivity of 97.78%, specificity of 90.48% and area under the curve of 0.968 in a healthy individual. Donors with higher Vδ2 Index Score in PBMC have greater anti-tumor functions including migratory function and cytotoxicity. CONCLUSIONS: Our results demonstrate that the interindividual γδT cell functions correlate with their circulating levels in healthy donors. Examination of circulating γδT cell level may be used to select healthy donors to participate in γδT-based immunotherapies.

Author Correction: PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Oncogenic BRAF-Mediated Melanoma Cell Invasion.

Melanoma patients with oncogenic BRAF(V600E) mutation have poor prognoses. While the role of BRAF(V600E) in tumorigenesis is well established, its involvement in metastasis that is clinically observed in melanoma patients remains a topic of debate. Here, we show that BRAF(V600E) melanoma cells have extensive invasion activity as assayed by the generation of F-actin and cortactin foci that mediate membrane protrusion, and degradation of the extracellular matrix (ECM). Inhibition of BRAF(V600E) blocks melanoma cell invasion. In a BRAF(V600E)-driven murine melanoma model or in patients' tumor biopsies, cortactin foci decrease upon inhibitor treatment. In addition, genome-wide expression analysis shows that a number of invadopodia-related genes are downregulated after BRAF(V600E) inhibition. Mechanistically, BRAF(V600E) induces phosphorylation of cortactin and the exocyst subunit Exo70 through ERK, which regulates actin dynamics and matrix metalloprotease secretion, respectively. Our results provide support for the role of BRAF(V600E) in metastasis and suggest that inhibiting invasion is a potential therapeutic strategy against melanoma.

Exo70 isoform switching upon epithelial-mesenchymal transition mediates cancer cell invasion.

Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here, we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2 and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoform expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal-but not the epithelial-isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion.

MicroRNA-9 up-regulates E-cadherin through inhibition of NF-κB1-Snail1 pathway in melanoma.

MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression. Hsa-miR-9 has been shown to have opposite functions in different tumour types; however, the underlying mechanism is unclear. Here we show that hsa-miR-9 is down-regulated in metastatic melanomas compared to primary melanomas. Overexpression of miR-9 in melanoma cells resulted in significantly decreased cell proliferation and migratory capacity with decreased F-actin polymerization and down-regulation of multiple GTPases involved in cytoskeleton remodelling. miR-9 overexpression induced significant down-regulation of Snail1 with a concomitant increase in E-cadherin expression. In contrast, knockdown of miR-9 increased Snail1 expression as well as melanoma cell proliferation and migration capacity. Mechanistically, miR-9 expression down-regulated NF-κB1 in melanoma and the effect was abolished by mutations in the putative miR-9 binding sites within the 3'-untranslated region (UTR) of NF-κB1. Anti-miR-9 miRNA inhibitor also increased the expression of NF-κB1. The effects of miR-9 on Snail1 expression and melanoma cell proliferation and migration were rescued by overexpression of NF-κB1 in these cells. Furthermore, miR-9 overexpression resulted in significantly decreased melanoma growth and metastasis in vivo. In summary, miR-9 inhibits melanoma proliferation and metastasis through down-regulation of the NF-κB1-Snail1 pathway. This study finds a new mechanism that miR-9 utilizes to decrease E-cadherin expression and inhibit melanoma progression. The results suggest that function of microRNAs is context and tumour type-specific.

An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition.

Alternative splicing achieves coordinated changes in post-transcriptional gene expression programmes through the activities of diverse RNA-binding proteins. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) are cell-type-specific regulators of transcripts that switch splicing during the epithelial-mesenchymal transition (EMT). To define a comprehensive programme of alternative splicing that is regulated during the EMT, we identified an extensive ESRP-regulated splicing network of hundreds of alternative splicing events within numerous genes with functions in cell-cell adhesion, polarity, and migration. Loss of this global ESRP-regulated epithelial splicing programme induces the phenotypic changes in cell morphology that are observed during the EMT. Components of this splicing signature provide novel molecular markers that can be used to characterize the EMT. Bioinformatics and experimental approaches revealed a high-affinity ESRP-binding motif and a predictive RNA map that governs their activity. This work establishes the ESRPs as coordinators of a complex alternative splicing network that adds an important post-transcriptional layer to the changes in gene expression that underlie epithelial-mesenchymal transitions during development and disease.