MOTAI: A Novel Method for the Study of O-GalNAcylation and Complex O-Glycosylation in Cancer.

The Tn antigen, an immature truncated O-glycosylation, is a promising biomarker for cancer detection and diagnosis. However, reliable methods for analyzing O-GalNAcylation and complex O-glycosylation are lacking. Here, we develop a novel method, MOTAI, for the sequential analysis of O-glycosylation using different O-glycoproteases. MOTAI conjugates glycopeptides on a solid support and releases different types of O-glycosylation through sequential enzymatic digestion by O-glycoproteases, including OpeRATOR and IMPa. Because OpeRATOR has less activity on O-GalNAcylation, MOTAI enriches O-GalNAcylation for subsequent analysis. We demonstrate the effectiveness of MOTAI by analyzing fetuin O-glycosylation and Jurkat cell lines. We then apply MOTAI to analyze colorectal cancer and benign colorectal polyps. We identify 32 Tn/sTn-glycoproteins and 43 T/sT-glycoproteins that are significantly increased in tumor tissues. Gene Ontology analysis reveals that most of these proteins are ECM proteins involved in the adhesion process of the intercellular matrix. Additionally, the protein disulfide isomerase CRELD2 has a significant difference in Tn expression, and the abnormally glycosylated T345 and S349 O-glycosylation sites in cancer group samples may promote the secretion of CRELD2 and ultimately tumorigenesis through ECM reshaping. In summary, MOTAI provides a powerful new tool for the in-depth analysis of O-GalNAcylation and complex O-glycosylation. It also reveals the upregulation of Tn/sTn-glycoproteins in colorectal cancer, which may provide new insights into cancer biology and biomarker discovery.

A non-invasive nanobody probe for high precision mapping of Lck spatial distribution.

The tyrosine kinase Lck is mandatory for initiating signaling responses downstream the antigenic T cell receptor (TCR). Numerous studies have shown that a prerequisite for efficient and well-balanced Lck regulation and function is its finely orchestrated spatial distribution pattern, especially at the plane of the plasma membrane. There is a wealth of knowledge on Lck localization sites, preference for specialized lipid microenvironments and colocalization partners. However, several questions concerning the spatial organization of its differentially phosphorylated conformers and the dynamics of their juxtaposition in relation to ligated and non-ligated TCRs remain elusive. In this brief report we introduce a non-invasive nanobody-based approach for mapping Lck subcellular allocation with high precision. Our initial data using this methodology, provide insight into the topology of Lck in resting T cells and its confined localization in a strictly delimited environment within the plane of the plasma membrane.

Design, synthesis, pharmacological evaluation, and computational study of benzo[d] isothiazol-based small molecule inhibitors targeting PD-1/PD-l1 interaction.

Blockade of the programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is an attractive strategy for immunotherapy, but the clinical application of small molecule PD-1/PD-L1 inhibitors remains unclear. In this work, based on BMS-202 and our previous work YLW-106, a series of compounds with benzo[d]isothiazol structure as scaffold were designed and synthesized. Their inhibitory activity against PD-1/PD-L1 interaction was evaluated by a homogeneous time-resolved fluorescence (HTRF) assay. Among them, LLW-018 (27c) exhibited the most potent inhibitory activity with an IC50 value of 2.61 nM. The cellular level assays demonstrated that LLW-018 exhibited low cytotoxicity against Jurkat T and MDA-MB-231. Further cell-based PD-1/PD-L1 blockade bioassays based on PD-1 NFAT-Luc Jurkat cells and PD-L1 TCR Activator CHO cells indicated that LLW-018 could interrupt PD-1/PD-L1 interaction with an IC50 value of 0.88 µM. Multi-computational methods, including molecular docking, molecular dynamics, MM/GBSA, MM/PBSA, Metadynamics, and QM/MM MD were utilized on PD-L1 dimer complexes, which revealed the binding modes and dissociation process of LLW-018 and C2-symmetric small molecule inhibitor LCH1307. These results suggested that LLW-018 exhibited promising potency as a PD-1/PD-L1 inhibitor for further investigation.

Circular RNA circPHF16 enhances IL-17A expression and secretion by sequestering miR-378a-3p to activate the IL6ST axis in Graves' disease.

Interleukin-17A (IL-17A) plays a pivotal role in the pathogenesis of Graves' disease (GD), an autoimmune disorder affecting thyroid function, but the detailed regulatory mechanisms remain elusive. Circular RNAs (circRNAs) have emerged as key regulators of IL-17A expression and secretion in autoimmune diseases, yet their specific role in GD, especially within CD4 + T lymphocytes, are not well understood. In this study, a circRNA, circPHF16 (hsa_circ_0090364) was found to be highly expressed in the peripheral blood mononuclear cells and serum of GD patients. In vitro experiments in Jurkat T cells revealed that silencing of circPHF16 suppressed IL-17A expression and secretion, while overexpression of circPHF16 had the opposite effect. Furthermore, bioinformatics analysis demonstrated a circPHF16/miR-378a-3p/IL6ST pathway, in which circPHF16 regulates IL6ST expression, which, in turn, influences IL-17A expression and secretion by interacting with miR-378a-3p. In vivo studies in a mouse model of GD showed similar trends in molecular expression levels, consistent with competitive endogenous RNA interactions. Together the results of the study identify circPHF16 as a potential target in the development of new strategies for GD diagnosis and treatment, and thus, offer a theoretical foundation for clinical therapeutic approaches in GD.

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.

Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5 min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Cytotoxic and viricidal effects of human semen on mumps virus-infected lymphocytes: In vitro studies.

Viruses in human semen may be sexually transmitted via free and cell-mediated viral infection. The potential effects of semen on the infection and sexual transmission of most viruses in semen remain largely unclear. The present study elucidated the inhibitory effects of human seminal plasma (SP) on Jurkat cell (JC)-mediated mumps virus (MuV) infection. We demonstrated that MuV efficiently infected JCs and that the JCs infected by MuV (JC-MuV) mediated MuV infection of HeLa cells. Remarkably, SP was highly cytotoxic to JCs and inhibited JC-MuV infection of HeLa cells. The cytotoxic factor possessed a molecular weight of less than 3 kDa, whereas that of the viricidal factor was over 100 kDa. The cooperation of cytotoxic and viricidal factors was required for the SP inhibition of JC-MuV infection, and prostatic fluid (PF) was responsible for both the cytotoxic and viricidal effects of SP. The cytotoxic effects we observed were resistant to the treatment of PF with boiling water, proteinase K, RNase A, and DNase I. Our results provide novel insights into the antiviral properties of SP, which may limit cell-mediated sexual viral transmission.

CD147 regulates the formation and function of immune synapses.

CD147 is a T cell activation-associated molecule which is closely involved in the formation of the immune synapse (IS). However, the precise role of CD147 in T cell activation and IS formation remains unclear. In the present study, we demonstrated that CD147 translocated to the IS upon T cell activation and was primarily distributed in the peripheral super molecular cluster (p-SMAC). The knock down of CD147 expression in T cells, but not in B cells, impaired IS formation. CD147 participated in IS formation between T cells and different types of antigen-presenting cells (APCs), including macrophages and dendritic cells. Ligation of CD147 with its monoclonal antibody (mAb) HAb18 effectively inhibited T cell activation and IL-2 secretion. CD98, a critical molecule interacting with CD147, was distributed in IS in a CD147-dependent way. Phosphorylation levels of T cell receptor (TCR) related molecules, like ZAP-70, ERK, and cJun, were down-regulated by CD147 ligation, which is crucial for the interaction of CD147 and TCR signaling transduction. CD147 is indispensable for the formation of immune synapses and plays an important role in the regulation of its function.

Retention of E-selectin functionalized liposome fanny packs on Jurkat cells following invasion through collagen.

Circulating immune cells are an appealing candidate to serve as carriers of therapeutic cargo via nanoparticles conjugated to their surface, for several reasons: these cells are highly migratory and can squeeze through small pores of diameter smaller than their resting size; they are easily accessible in the peripheral blood via minimally invasive IV injection of particles, or can be harvested, processed ex vivo, and reintroduced to the body; they are adept at traveling through the circulation with minimal destruction and thus have access to various tissue beds of the body; and immune cells have built-in signal transduction machinery which allows them to actively engage in chemotaxis and home to regions of the tissue containing tumors, invading microorganisms, or injuries in need of wound healing. In this study, we sought to examine and quantify the degree to which nanoscale liposomes, functionalized with E-selectin adhesion receptor, could bind to a model T cell line and remain on the surface of the cells as they migrate through collagen gels of varying density in a transwell cell migration chamber. It is demonstrated that physiological levels of fluid shear stress are necessary to achieve optimal binding of the E-selectin liposomes to the cell surface as expected, and that CD3/CD28 antibody activation of the T cells was not necessary for effective liposome binding. Nanoscale liposomes were successfully conveyed by the migrating cells across a layer of rat tail type 1 collagen gel ranging in composition from 1 to 3 mg/mL. The relative fraction of liposomes carried through the collagen decreased at higher collagen density, likely due to the expected decrease in average pore size, and increased fiber content in the gels. Taken together, these results support the idea that T cells could be an effective cellular carrier of therapeutic molecules either attached to the surface of nanoscale liposomes or encapsulated within their interior.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.

CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147KO THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147KO THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147KO THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (KD: 2.05 ± 0.30 × 10-9 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147KO THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147KO THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147KO THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147KO monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.

Development of Degraders of Cyclin-Dependent Kinases 4 and 6 Based on Rational Drug Design.

Degradation of target proteins has been considered to be a promising therapeutic approach, but the rational design of compounds for degradation remains a challenge. In this study, we reasonably designed and synthesized only 10 compounds to discover effective CDK4/6 protein degraders. Among the newly synthesized compounds, 7f achieved dual degradation of CDK4/6 protein, with DC50 values of 10.5 and 2.5 nM, respectively. Compound 7f also exhibited inhibitory proliferative activity against Jurkat cells with an IC50 value of 0.18 µM. Furthermore, 7f induced cell apoptosis and G1 phase cell cycle arrest in a dose-dependent manner in Jurkat cells. In conclusion, these findings demonstrate the potential of 7f as a CDK4/6 degrader and a potential therapeutic strategy against cancer, thereby expanding the potential of CDK4/6 dual PROTACs.

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) as a CD45 Protein Stability Regulator.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a binding partner of the phosphatase CD45, but its function remains poorly understood. Its close interaction with CD45 suggests that LPAP may potentially regulate CD45, but direct biochemical evidence for this has not yet been obtained. We found that in the Jurkat lymphoid cells the levels of LPAP and CD45 proteins are interrelated and well correlated with each other. Knockout of LPAP leads to the decrease in the surface expression of CD45, while its overexpression, on the contrary, caused its increase. No such correlation was found in the non-lymphoid K562 cells. We hypothesize that LPAP regulates expression level of CD45 and thus can affect lymphocyte activation.

The Influence of N-Acetyl-selenomethionine on Two RONS-Generating Cancer Cell Lines Compared to N-Acetyl-methionine.

N-acetyl-selenomethionine (NASeLM), a representative of the selenium compounds, failed to convince in clinical studies and cell cultures that it neither inhibits cancer growth nor has a chemoprotective effect. This study aims to find out whether NASeLM shows a growth-inhibiting property compared to the carrier substance N-Acetyl-L-methionine (NALM) on two different cancer cells, namely Jurkat cells and MTC-SK cells. METHODS: Jurkat and MTC-SK cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of NASeLM and NALM solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of Jurkat and MTC-SK cells. RESULTS: Both substances, NASeLM and NALM, were similarly able to inhibit cell growth and mitochondrial activity of Jurkat cells in a concentration-dependent and time-dependent manner up to 70%. Only the determination of caspase activity showed that only NASeLM was able to increase this to almost 40% compared to the control as well as the same lack of NALM. However, the experiments on MTC-SK cells showed a clear difference in favor of NASeLM compared to NALM. While NASeLM was able to reduce cell growth to up to 55%, the same amount of NALM was only at around 15%, which turned out to be highly significant (p < 0.001). The same could also be measured for the reduction in MTC-SK mitochondrial activity. Time dependence could also be recognized: the longer both substances, NASeLM and NALM, were incubated, the higher the effect on cell growth and mitochondrial activity, in favour of NASeLM. Only NASeLM was able to increase caspase-3 activity in MTC-SK cells: at 250 µg/mL NASeLM, caspase-3 activity increased significantly to 28% after 24 and 48 h compared to the control (14%) or the same NALM concentration (14%). After 72 h, this could still increase to 37%. A further increase in the NASeLM concentration did not result in higher caspase-3 activity. CONCLUSION: NASeLM could clearly increase caspase-3 activity in both cell types, Jurkat or MTC-SK cells, and thus induce cell death. NALM and NASeLM showed a reduction in cell growth and mitochondrial activity in both cell lines: While NALM and NASeLM showed almost identical measurements on Jurkat cells, NASeLM was much more effective on MTC-SK than the non-selenium-containing carrier, indicating that it has additional anti-chemoprotective effects.

[Clinical Significance of USP5 Expression Level in Acute Myeloid Leukemia and Its Regulatory Effects on AKT/mTOR/4EBP1 Signaling Pathway].

OBJECTIVE: To investigate the clinical significance, functional role and potential downstream mechanism of USP5 in acute myeloid leukemia (AML). METHODS: The expression of USP5 in AML and normal tissues and its correlation with patients' survival were analyzed based on TCGA database. USP5 was knocked down and overexpressed in Jurkat and HL-60 cells using lentivirus. USP5 mRNA and protein expression were detected by RT-qPCR and Western blot, respectively. Cell proliferation and growth were measured by CCK-8 and methylcellulose colony-forming assay. Flow cytometry was used to analyze cell cycle and apoptosis. RESULTS: USP5 was highly expression in AML compared with normal tissues. Up-regulation of USP5 was negatively correlated with the survival of AML patients. USP5 knockdown and overexpression inhibited and promoted the proliferation and colony growth of AML cells, respectively. Cell cycle arrest and apoptosis were induced in USP5 knockdown Jurkat and HL-60 cells. Furthermore, USP5 knockdown inhibited the phosphrylation of AKT, mTOR and 4EBP1. CONCLUSION: Overexpression of USP5 predicts poor survival of AML patients. Targeting USP5 suppresses AKT/mTOR/4EBP1 signaling and reduces the proliferation and growth of AML cells.

Formaldehyde exacerbates inflammation and biases T helper cell lineage commitment through IFN-γ/STAT1/T-bet pathway in asthma.

The correlation between formaldehyde (FA) exposure and prevalence of asthma has been widely reported. However, the underlying mechanism is still not fully understood. FA exposure at 2.0 mg/m3 was found to exacerbate asthma in OVA-induced murine models. IFN-γ, the cytokine produced by T helper 1 (Th1) cells, was significantly induced by FA in serum and bronchoalveolar lavage fluid (BALF) of asthmatic mice, which was different from cytokines secreted by other Th cells. The observation was also confirmed by mRNA levels of Th marker genes in CD4+ T cells isolated from BALF. In addition, increased production of IFN-γ and expression of T-bet in Jurkat T cells primed with phorbol ester and phytohaemagglutinin were also observed with 100 µM FA treatment in vitro. Upregulated STAT1 phosphorylation, T-bet expression and IFN-γ production induced by FA was found to be restrained by STAT1 inhibitor fludarabine, indicating that FA promoted Th1 commitment through the autocrine IFN-γ/STAT1/T-bet pathway in asthma. This work not only revealed that FA could bias Th lineage commitment to exacerbate allergic asthma, but also identified the signaling mechanism of FA-induced Th1 differentiation, which may be utilized as the target for development of interfering strategies against FA-induced immune disorders.

PDL1 targeting by miR-138-5p amplifies anti-tumor immunity and Jurkat cells survival in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) has constituted over 80% of the lung cancer population with a poor prognosis. Over the past decade, immunotherapy has been constructed in the enlargement of immune checkpoint inhibitors as a promising approach for NSCLC treatment. Evading the immune system using the PD-1/PD-L1 axis is an intelligent way for cancers, and T cells cannot respond fully and confront cancer. Recently, the miR-138 was reported as a PD-L1 regulator in NSCLC. However, its inhibitory impact on T-cell exhaustion has not been characterized. The present study aims to impair PD-L1 (B7-H1) expression in Adenocarcinoma cell lines using miR-138-5p and determines how it prevents Jurak cell exhaustion. To gain the purpose, first, 18 highly significant dysregulated miRNAs containing hsa-miR-138 and CD274-mRNA network were detected in NSCLC based on bioinformatics analysis. Moreover, our study revealed a high level of miR-138-5p could make significant changes like PDL1 downregulation, proliferation, and mortality rate in A549/Calu6 cells. We also simulate cancer environmental conditions by culturing Jurak cells and NSCLC cell lines under the influence of stimulator cytokines to show how miR-138-5p survives Jurak cells by targeting PD-L1/PD-1pathway.

NUP98-BPTF promotes oncogenic transformation through PIM1 upregulation.

INTRODUCTION: Nucleoporin 98 (NUP98) fusion proteins are recurrently found in leukemia and are associated with unfavorable clinical outcomes. They are distributed to the nucleus and contribute to leukemogenesis via aberrant transcriptional regulation. We previously identified NUP98-BPTF (NB) fusion in patients with T-cell acute lymphoblastic leukemia (T-ALL) using next-generation sequencing. The FG-repeat of NUP98 and the PHD finger and bromodomain of bromodomain PHD finger transcription factor (BPTF) are retained in the fusion. Like other NUP98 fusion proteins, NB is considered to regulate genes that are essential for leukemogenesis. However, its target genes or pathways remain unknown. MATERIALS AND METHODS: To investigate the potential oncogenic properties of the NB fusion protein, we lentivirally transduced a doxycycline-inducible NB expression vector into mouse NIH3T3 fibroblasts and human Jurkat T-ALL cells. RESULTS: NB promoted the transformation of mouse NIH3T3 fibroblasts by upregulating the proto-oncogene Pim1, which encodes a serine/threonine kinase. NB transcriptionally regulated Pim1 expression by binding to its promoter and activated MYC and mTORC1 signaling. PIM1 knockdown or pharmacological inhibition of mTORC1 signaling suppressed NB-induced NIH3T3 cell transformation. Furthermore, NB enhanced the survival of human Jurkat T-ALL cells by inactivating the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). CONCLUSION: We demonstrated the pivotal role of NB in cell transformation and survival and identified PIM1as a key downstream target of NB. These findings propose a promising therapeutic strategy for patients with NB fusion-positive leukemia.

Synergistic Effect of Venetoclax and Bendamustine in Early T-cell Precursor Acute Lymphoblastic Leukemia.

BACKGROUND/AIM: To date, therapeutic options for T-cell acute lymphoblastic leukemia (T-ALL) remain very limited. This study evaluated the efficacy of monotherapies and combination therapies including a selective BCL-2 inhibitor for T-ALL cell lines, namely Jurkat, CCRF-CEM, and Loucy. MATERIALS AND METHODS: Loucy is an early T-precursor ALL (ETP-ALL) cell line characterized by an immature phenotype, whereas Jurkat and CCRF-CEM are late T-cell progenitor ALL (LTP-ALL) cell lines. Monotherapy was conducted with venetoclax, cytarabine, bendamustine, or azacytidine, whereas combination therapy was performed with venetoclax plus cytarabine, venetoclax plus bendamustine, or venetoclax plus azacytidine. Cell viability assay was conducted after 48 h using Trypan blue and the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Statistical analysis for evaluating synergistic interactions between anticancer drugs was performed by using the SynergyFinder Plus and drc R package. RESULTS: Adding venetoclax to cytarabine, bendamustine, or azacitidine achieved an additive effect, with Loewe synergic scores ranging from -10 to 10 in Jurkat and CCRF-CEM. Conversely, the combination of venetoclax and cytarabine displayed an additive effect (Loewe synergic score: 8.45 and 5.82 with MTS and Trypan blue assays, respectively), whereas venetoclax plus bendamustine or azacitidine exhibited a synergistic effect (Loewe synergic score >10 with MTS assay) in Loucy. Remarkably, the Bliss/Loewe score revealed that the combination of venetoclax and bendamustine was the most synergistic, yielding a score of 13.832±0.55. CONCLUSION: The combination of venetoclax and bendamustine demonstrated the greatest synergistic effect in suppressing ETP-ALL cell proliferation. Further studies are warranted to determine the mechanisms for the synergism between venetoclax and bendamustine in high-risk T-ALL.