TIM-3 Engagement Promotes Effector Memory T Cell Differentiation of Human Antigen-Specific CD8 T Cells by Activating mTORC1.

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.

TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

SHOC2 and CRAF mediate ERK1/2 reactivation in mutant NRAS-mediated resistance to RAF inhibitor.

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF(V600E) melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF(V600E)/NRAS(Q61K) co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF(V600) cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF(V600E)/NRAS(Q61K) cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF(V600E)/NRAS(Q61K) cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.

Hierarchical phosphorylation within the ankyrin repeat domain defines a phosphoregulatory loop that regulates Notch transcriptional activity.

The Notch signal transduction pathway mediates important cellular functions through direct cell-to-cell contact. Deregulation of Notch activity can lead to an altered cell proliferation and has been linked to many human cancers. Casein kinase 2 (CK2), a ubiquitous kinase, regulates several cellular processes by phosphorylating proteins involved in signal transduction, gene expression, and protein synthesis. In this report we identify Notch(ICD) as a novel target of phosphorylation by CK2. Using mapping and mutational studies, we identified serine 1901, located in the ankyrin domain of Notch, as the target amino acid. Interestingly, phosphorylation of serine 1901 by CK2 appears to generate a second phosphorylation site at threonine 1898. Furthermore, threonine 1898 phosphorylation only occurs when Notch forms a complex with Mastermind and CSL. Phosphorylation of both threonine 1898 and serine 1901 resulted in decreased binding of the Notch-Mastermind-CSL ternary complex to DNA and consequently lower transcriptional activity. These data indicate that the phosphorylation of serine 1901 and threonine 1898 negatively regulates Notch function by dissociating the complex from DNA. This study identifies a new component involved in regulation of Notch(ICD) transcriptional activity, reinforcing the notion that a precise and tight regulation is required for this essential signaling pathway.

Mechanisms of resistance to RAF inhibitors in melanoma.

The recent RAF inhibitor trial with PLX4032/RG7204 in late-stage mutant B-RAF melanoma patients has been lauded as a success story for personalized cancer therapy since short-term clinical responses were observed in the majority of patients. However, initial responses were followed by subsequent tumor re-growth, and a subset of patients showed intrinsic resistance. Bi-directional translational efforts are now essential to determine the mechanisms underlying acquired/secondary and intrinsic resistance to RAF inhibitors.

Assembly of a Notch transcriptional activation complex requires multimerization.

Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (N(ICD)) from the plasma membrane results in the accumulation of deregulated nuclear N(ICD) that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N(ICD) forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that N(ICD) forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate that N(ICD) multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, we demonstrate that the assembly is mediated by N(ICD) multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA.

The prolyl-isomerase Pin1 is a Notch1 target that enhances Notch1 activation in cancer.

Signalling through Notch receptors requires ligand-induced cleavage to release the intracellular domain, which acts as a transcriptional activator in the nucleus. Deregulated Notch1 signalling has been implicated in mammary tumorigenesis; however the mechanisms underlying Notch activation in breast cancer remain unclear. Here, we demonstrate that the prolyl-isomerase Pin1 interacts with Notch1 and affects Notch1 activation. Pin1 potentiates Notch1 cleavage by gamma-secretase, leading to an increased release of the active intracellular domain and ultimately enhancing Notch1 transcriptional and tumorigenic activity. We found that Notch1 directly induces transcription of Pin1, thereby generating a positive loop. In human breast cancers, we observed a strong correlation between Pin1 overexpression and high levels of activated Notch1. Thus, the molecular circuitry established by Notch1 and Pin1 may have a key role in cancer.

Multifactorial refractory heterotopic ossification.

Ectopic bone formation or "heterotopic ossification" can follow surgery, trauma, or neurologic injury, but the process is usually self-limited, localized to the site of injury, and responds to surgical treatment when necessary. Aggressive, systemic forms of heterotopic ossification exist that generate lesions that often resist surgical treatment and produce a high rate of recurrence. These entities typically manifest during infancy as genetic syndromes such as fibrodysplasia ossificans progressiva or progressive osseous heteroplasia. The authors describe a case of aggressive, systemic heterotopic ossification in an adult that followed a motor vehicle accident and multiple surgeries. The patient developed a large nonhealing wound around a focus of ectopic bone. Skin grafts failed as a result of the recurrence of ectopic bone, and the patient eventually required aggressive debridement and delayed rotational flap closure. A brief review of the clinical features and surgical treatment of heterotopic ossification is outlined.