Aradhana Rani

IL-2 Regulates Expression of C-MAF in Human CD4 T Cells

Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R–directed therapies may have utility in the treatment of allergic disorders.

STAT5 in Cancer and Immunity

Signal transducers and activators of transcription 5 (STAT5a and STAT5b) are highly homologous proteins that are encoded by 2 separate genes and are activated by Janus-activated kinases (JAK) downstream of cytokine receptors. STAT5 proteins are activated by a wide variety of hematopoietic and nonhematopoietic cytokines and growth factors, all of which use the JAK-STAT signalling pathway as their main mode of signal transduction. STAT5 proteins critically regulate vital cellular functions such as proliferation, differentiation, and survival. The physiological importance of STAT5 proteins is underscored by the plethora of primary human tumors that have aberrant constitutive activation of these proteins, which significantly contributes to tumor cell survival and malignant progression of disease. STAT5 plays an important role in the maintenance of normal immune function and homeostasis, both of which are regulated by specific members of IL-2 family of cytokines, which share a common gamma chain (γ(c)) in their receptor complex. STAT5 critically mediates the biological actions of members of the γ(c) family of cytokines in the immune system. Essentially, STAT5 plays a critical role in the function and development of Tregs, and consistently activated STAT5 is associated with a suppression in antitumor immunity and an increase in proliferation, invasion, and survival of tumor cells. Thus, therapeutic targeting of STAT5 is promising in cancer.

FRA2 Is a STAT5 Target Gene Regulated by IL-2 in Human CD4 T Cells

Signal transducers and activators of transcription 5(STAT5) are cytokine induced signaling proteins, which regulate key immunological processes, such as tolerance induction, maintenance of homeostasis, and CD4 T-effector cell differentiation. In this study, transcriptional targets of STAT5 in CD4 T cells were studied by Chromatin Immunoprecipitation (ChIP). Genomic mapping of the sites cloned and identified in this study revealed the striking observation that the majority of STAT5-binding sites mapped to intergenic (>50 kb upstream) or intronic, rather than promoter proximal regions. Of the 105 STAT5 responsive binding sites identified, 94% contained the canonical (IFN-γ activation site) GAS motifs. A number of putative target genes identified here are associated with tumor biology. Here, we identified Fos-related antigen 2 (FRA2) as a transcriptional target of IL-2 regulated STAT5. FRA2 is a basic -leucine zipper (bZIP) motif ‘Fos’ family transcription factor that is part of the AP-1 transcription factor complex and is also known to play a critical role in the progression of human tumours and more recently as a determinant of T cell plasticity. The binding site mapped to an internal intron within the FRA2 gene. The epigenetic architecture of FRA2, characterizes a transcriptionally active promoter as indicated by enrichment for histone methylation marks H3K4me1, H3K4me2, H3K4me3, and transcription/elongation associated marks H2BK5me1 and H4K20me1. FRA2 is regulated by IL-2 in activated CD4 T cells. Consistently, STAT5 bound to GAS sequence in the internal intron of FRA2 and reporter gene assays confirmed IL-2 induced STAT5 binding and transcriptional activation. Furthermore, addition of JAK3 inhibitor (R333) or Daclizumab inhibited the induction in TCR stimulated cells. Taken together, our data suggest that FRA2 is a novel STAT5 target gene, regulated by IL-2 in activated CD4 T cells.

HES1 in immunity and cancer

Hairy and enhancer of split homolog-1 (HES1) is a part of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neuronal, endocrine, T-lymphocyte progenitors as well as various cancers. HES1 is a transcription factor which is regulated by the NOTCH, Hedgehog and Wnt signalling pathways. Aberrant expression of these pathways is a common feature of cancerous cells. There appears to be a fine and complicated crosstalk at the molecular level between the various signalling pathways and HES1, which contributes to its effects on the immune response and cancers such as leukaemia. Several mechanisms have been proposed, including an enhanced invasiveness and metastasis by inducing epithelial mesenchymal transition (EMT), in addition to its strict requirement for tumour cell survival. In this review, we summarize the current biology and molecular mechanisms as well as its use as a clinical target in cancer therapeutics.

PRT062607 Achieves Complete Inhibition of the Spleen Tyrosine Kinase at Tolerated Exposures Following Oral Dosing in Healthy Volunteers

The spleen tyrosine kinase (SYK) regulates immune cell activation in response to engagement of a variety of receptors, making it an intriguing target for the treatment of inflammatory and autoimmune disorders as well as certain B‐cell malignancies. We have previously reported on the discovery and preclinical characterization of PRT062607, a potent and highly selective inhibitor of SYK that exhibits robust anti‐inflammatory activity in a variety of animal models. Here we present data from our first human studies aimed at characterizing the pharmacokinetics (PK), pharmacodynamics (PD), and safety of PRT062607 in healthy volunteers following single and multiple oral administrations. PRT062607 demonstrated a favorable PK profile and the ability to completely inhibit SYK activity in multiple whole‐blood assays. The PD half‐life in the more sensitive assays was approximately 24 hours and returned to predose levels by 72 hours. Selectivity for SYK was observed at all dose levels tested. Analysis of the PK/PD relationship indicated an IC50 of 324 nM for inhibition of B‐cell antigen receptor‐mediated B‐cell activation and 205 nM for inhibition of FcεRI‐mediated basophil degranulation. PRT062607 was safe and well tolerated across the entire range of doses. Clinical PK/PD was related to in vivo anti‐inflammatory activity of PRT062607 in the rat collagen‐induced arthritis model, which predicts that therapeutic concentrations may be safely achieved in humans for the treatment of autoimmune disease. PRT062607 has a desirable PK profile and is capable of safely, potently, and selectively suppressing SYK kinase function in humans following once‐daily oral dosing.