Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma.

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3ß Signaling-Dependent Notch Pathway.

In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4(+) T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4(+) T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1-mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3ß. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK-GSK3ß signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses.

Long-Term Real-Time In Vivo Drug Release Monitoring with AIE Thermogelling Polymer.

A new drug concentration meter is developed. In vivo drug release can be monitored precisely via a self-indicating drug delivery system consisting of a new aggregation-induced emission thermoresponsive hydrogel. By taking the advantage of a self-indicating system, one can easily detect the depletion of drugs, and reinject to maintain a dosage in the optimal therapeutic window.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

Selective Induction of Intrinsic Apoptosis in Retinoblastoma Cells by Novel Cationic Antimicrobial Dodecapeptides.

Host defense peptides represent an important component of innate immunity. In this work, we report the anticancer properties of a panel of hyper-charged wholly cationic antimicrobial dodecapeptides (CAPs) containing multiple canonical forms of lysine and arginine residues. These CAPs displayed excellent bactericidal activities against a broad range of pathogenic bacteria by dissipating the cytoplasmic membrane potential. Specifically, we identified two CAPs, named HC3 and HC5, that effectively killed a significant number of retinoblastoma (WERI-Rb1) cells (p ≤ 0.01). These two CAPs caused the shrinkage of WERI-Rb1 tumor spheroids (p ≤ 0.01), induced intrinsic apoptosis in WERI-Rb1 cells via activation of caspase 9 and caspase 3, cleaved the PARP protein, and triggered off the phosphorylation of p53 and γH2A.X. Combining HC3 or HC5 with the standard chemotherapeutic drug topotecan showed synergistic anti-cancer activities. Overall, these results suggest that HC3 and HC5 can be exploited as potential therapeutic agents in retinoblastoma as monotherapy or as adjunctive therapy to enhance the effectiveness of currently used treatment modalities.

Robust Antitumor Activity and Low Cytokine Production by Novel Humanized Anti-CD19 CAR T Cells.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

Targeted Gene Silencing in Malignant Hematolymphoid Cells Using GapmeR.

Delivery of conventional antisense oligonucleotides or small interfering RNA (siRNA) molecules into hematolymphoid cells for targeted gene silencing has been proven to be difficult. Here, we describe a simple protocol to knockdown specific gene(s) in malignant hematolymphoid cells using "GapmeR." This protocol could be applicable to a wide range of cell-types and thus solves an important problem for researchers working with cell lines or primary cells derived from patients with hematolymphoid malignancies.

The Citrus Flavanone Hesperetin Induces Apoptosis in CTCL Cells via STAT3/Notch1/NFκB-Mediated Signaling Axis.

BACKGROUND: Hesperetin is a natural compound known for its cholesterol-lowering effect and a wide range of pharmacological activities. OBJECTIVES: Investigating the potential anticancer activities of Hesperetin in malignant hematolymphoid cell lines HuT78 and MJ, derived from patients with Cutaneous T-Cell Lymphomas (CTCL). METHODS: The cytotoxic effect of Hesperetin on two different CTCL cell lines, HuT78 and MJ, was assessed by MTS-based colorimetric assay. Apoptosis, cell cycle, ROS (Reactive Oxygen Species) and molecular analysis were performed using flow-cytometry and immunoblotting. RESULTS: Hesperetin-treated CTCL cells were arrested at the sub-G1 phase of cell cycle with the concomitant decrease in the expression of the cell cycle regulator protein cyclin B. In addition, the study found that the cellular treatment with Hesperetin caused an induction of apoptosis, which was independent of ROS generation. Hesperetin caused a significant decrease in the expression level of anti-apoptotic protein Bcl-xL and an increase in cleaved caspase-3 and PARP proteins in CTCL cells. Furthermore, Hesperetin treatment in CTCL cells down-regulated the expression of Notch1 and phosphorylation of STAT3 (Tyr705) and inhibited NFκBp65. CONCLUSION: This study highlights the anticancer properties of Hesperetin. Which induces apoptosis in CTCL cells via STAT3/Notch1/NFκB mediated signaling pathway, suggesting that further development of this novel class of flavonoid may contribute to new drug discovery for certain hematolymphoid malignancies.

Quantitative Real-Time PCR for Evaluating Transcriptional Changes in T-Lymphocytes.

The real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an indispensable technology that enables reliable transcriptional analysis routinely used in molecular biology studies. The qRT-PCR technique quantifies mRNA by taking advantage of the reverse transcriptase-dependent conversion of RNA into cDNA and subsequent amplification of the cDNA using PCR. The amount of PCR product is directly proportional to the initial starting quantity of mRNA. The straightforward but complex methodologies used in this technique involve multiple sequential steps that include isolation of mRNA, conversion of mRNA into cDNA, amplification of the cDNA, and quantification of amplicons. In this chapter, we describe an optimized protocol for performing qRT-PCR in human T-lymphocytes.

Isolation of Human Peripheral Blood T-Lymphocytes.

Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays.

GapmeR-Mediated Gene Silencing in Motile T-Cells.

Gene silencing is an important method to study gene functions in health and diseases. While there are various techniques that are applied to knockdown specific gene(s) of interest, they have certain limitations in application to T-lymphocytes. T-cells are "hard-to-transfect" cells and are recalcitrant to transfection reagents. Here, we describe the use of novel cell-permeating antisense molecules, called "GapmeR", to knockdown specific gene(s) in human primary T-cells.

A Laboratory Model to Study T-Cell Motility.

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLß2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.

Centrosome- and Golgi-Localized Protein Kinase N-Associated Protein Serves As a Docking Platform for Protein Kinase A Signaling and Microtubule Nucleation in Migrating T-Cells.

Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 µm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.

Screening of ferrocenyl-phosphines identifies a gold-coordinated derivative as a novel anticancer agent for hematological malignancies.

The development of new organometallic compounds as anticancer agents is currently an active area of research. Here, we report the design, synthesis and characterization of a panel of 10 new ferrocenyl-phosphine derivatives (FD1-FD10) and the analysis of their anti-proliferative activities in hematolymphoid cells representing non-Hodgkin cutaneous T-cell lymphoma (CTCL). The gold-coordinated ferrocenyl-phosphine complex FD10 exhibited a significant and dose-dependent cytotoxicity in 4 different CTCL cell lines - HuT78, HH, MJ and MyLa. FD10 concentrations causing 50% cell growth inhibition (IC50) of HuT78, HH, MJ and MyLa cells at 24 h were recorded to be 5.55 ± 0.20, 7.80 ± 0.09, 3.16 ± 0.10 and 6.46 ± 0.24 µM respectively. Further mechanistic studies showed that FD10 induced apoptosis in CTCL cells by an intrinsic pathway mediated via the activation of caspase-3 and poly(ADP-ribose)polymerase. It suppressed the expression and activity of STAT3 oncoprotein in CTCL cells. FD10 caused robust G0/G1 phase cell cycle arrest and reduced the expression levels of Akt S473 phosphorylation and c-Myc, both are key cell cycle regulator proteins. Taken together, this study highlights anticancer properties of the ferrocenyl-phosphine gold organometallic complex FD10 and suggests that further development of this novel class of molecule may contribute to new drug discovery for certain hematolymphoid malignancies.

GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells.

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to "hard-to-transfect" primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called "GapmeR", is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.