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[Image: see text] Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes that control a wide variety of cellular functions such as cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking. PI3Kγ plays a critical role in mediating leukocyte chemotaxis as well as mast cell degranulation, making it a potentially interesting target for autoimmune and inflammatory diseases. We previously disclosed a novel series of PI3Kγ inhibitors derived from a benzothiazole core. The truncation of the benzothiazole core led to the discovery of a structurally diverse alkynyl thiazole series which displayed high PI3Kγ potency and subtype selectivity. Further medicinal chemistry optimization of the alkynyl thiazole series led to identification of compounds such as 14 and 32, highly potent, subtype selective, and CNS penetrant PI3Kγ inhibitors. Compound 14 showed robust inhibition of PI3Kγ mediated neutrophil migration in vivo.

The lipid kinase phosphoinositide 3-kinase γ (PI3Kγ) has attracted attention as a potential target to treat a variety of autoimmune disorders, including multiple sclerosis, due to its role in immune modulation and microglial activation. By minimizing the number of hydrogen bond donors while targeting a previously uncovered selectivity pocket adjacent to the ATP binding site of PI3Kγ, we discovered a series of azaisoindolinones as selective, brain penetrant inhibitors of PI3Kγ. This ultimately led to the discovery of 16, an orally bioavailable compound that showed efficacy in murine experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis.

A series of high affinity second-generation thiazolopiperidine inhibitors of PI3Kγ were designed based on some general observations around lipid kinase structure. Optimization of the alkylimidazole group led to inhibitors with higher levels of PI3Kγ selectivity. Additional insights into PI3K isoform selectivity related to sequence differences in a known distal hydrophobic pocket are also described.

Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.

The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.

While several therapeutic options exist, the need for more effective, safe, and convenient treatment for a variety of autoimmune diseases persists. Targeting the Janus tyrosine kinases (JAKs), which play essential roles in cell signaling responses and can contribute to aberrant immune function associated with disease, has emerged as a novel and attractive approach for the development of new autoimmune disease therapies. We screened our compound library against JAK3, a key signaling kinase in immune cells, and identified multiple scaffolds showing good inhibitory activity for this kinase. A particular scaffold of interest, the 1H-pyrrolo[2,3-b]pyridine series (7-azaindoles), was selected for further optimization in part on the basis of binding affinity (Ki) as well as on the basis of cellular potency. Optimization of this chemical series led to the identification of VX-509 (decernotinib), a novel, potent, and selective JAK3 inhibitor, which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). On the basis of these findings, it appears that VX-509 offers potential for the treatment of a variety of autoimmune diseases.

Cytokines, growth factors, and other chemical messengers rely on a class of intracellular nonreceptor tyrosine kinases known as Janus kinases (JAKs) to rapidly transduce intracellular signals. A number of these cytokines are critical for lymphocyte development and mediating immune responses. JAK3 is of particular interest due to its importance in immune function and its expression, which is largely confined to lymphocytes, thus limiting the potential impact of JAK3 inhibition on nonimmune physiology. The aim of this study was to evaluate the potency and selectivity of the investigational JAK3 inhibitor VX-509 (decernotinib) [(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide] against JAK3 kinase activity and inhibition of JAK3-mediated signaling in vitro and JAK3-dependent physiologic processes in vivo. These results demonstrate that VX-509 potently inhibits JAK3 in enzyme assays (Ki = 2.5 nM + 0.7 nM) and cellular assays dependent on JAK3 activity (IC50 range, 50-170 nM), with limited or no measurable potency against other JAK isotypes or non-JAK kinases. VX-509 also showed activity in two animal models of aberrant immune function. VX-509 treatment resulted in dose-dependent reduction in ankle swelling and paw weight and improved paw histopathology scores in the rat collagen-induced arthritis model. In a mouse model of oxazolone-induced delayed-type hypersensitivity, VX-509 reduced the T cell-mediated inflammatory response in skin. These findings demonstrate that VX-509 is a selective and potent inhibitor of JAK3 in vitro and modulates proinflammatory response in models of immune-mediated diseases, such as collagen-induced arthritis and delayed-type hypersensitivity. The data support evaluation of VX-509 for treatment of patients with autoimmune and inflammatory diseases such as rheumatoid arthritis.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor γ, PPARγ), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Objective T lymphocytes can be activated by soluble factors such as cytokines or through direct cell-cell interactions. Although cytokine receptors are known to signal through STAT family transcription factors, the mechanisms by which other cell-surface molecules, such as CD2, transduce signals is unclear. The goal of this study was to determine whether stimulation of T cells through CD2 recapitulates aspects of cytokine-induced T-cell activation by use of STAT transcription factors. Materials and methods T cells were treated with anti-CD2 antibodies or cells bearing the natural CD2 ligand CD58, after which signaling through STAT transcription factors was assessed. Results Stimulation of CD2 on primary T lymphocytes leads to the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1. In contrast to stimulation by cytokines, the activation of STAT1 in response to CD2 ligation is delayed and does not involve Jak kinases. Furthermore, while STAT phosphorylation induced by cytokines is generally transient, STAT1 phosphorylation following CD2 stimulation persists for a period of days. Transcription of key target genes such as IRF1 and c-fos proceeds with delayed kinetics following CD2 stimulation, suggesting that this unique pattern of STAT activation may lead to a distinct cellular response following CD2 ligation. This pathway appears to be restricted to T cells, as stimulation of CD2 on NK cells does not lead to STAT1 activation. Conclusion Stimulation of T cells through cell-surface molecules such as CD2 involves activation of STAT transcription factors, thus recapitulating elements of cytokine signaling.

Activated ras causes increased activity of several signal transduction systems, including the mitogen-activated protein kinase kinase (MAPKK) pathway and the phosphoinositol-3-kinase (PI-3-K. pathway. We have previously shown that the PI-3-K pathway plays a major role in regulation of ras-mediated tumor angiogenesis in angiosarcoma cells. However, the contribution of the MAPKK pathway to tumorigenesis and angiogenesis is not fully understood. Overexpression of constitutively active forms of MAPKK has previously been shown to transform nonmalignant NIH3T3 fibroblasts, but the effect of down-regulation of MAPKK on tumorigenesis and angiogenesis in a well established tumor has not been fully explored. We introduced a dominant negative MAPKK gene into SVR murine angiosarcoma cells. Introduction of a dominant negative MAPKK causes a significant decrease in proliferation rate in vitro and morphological reversion. Cells expressing the dominant negative MAPKK have a greatly decreased ability to form colonies in soft agar compared with wild-type cells. Despite the decreased cell growth in vitro and inability to grow in soft agar, the cells were equally tumorigenic in nude mice. Our results suggest that the MAPKK pathway is required for soft agar growth of angiosarcoma cells, and separates the phenotypes of soft agar growth versus in vivo tumorigenicity. These findings have implications in the development of signal transduction modulators as potential antineoplastic agents.

Objective TEL/PDGFβR is a tyrosine kinase fusion protein associated with the pathogenesis of chronic myelomonocytic leukemia. The following experiments were undertaken to understand the mechanisms whereby TEL/PDGFβR transforms cells. Materials and Methods Activation of JAK and STAT proteins was studied in an interleukin 3 (IL-3)-dependent cell line, Ba/F3, transformed to IL-3 independence by TEL/PDGFβR. Results TEL/PDGFβR activates STAT1 and STAT5 in transformed Ba/F3 cells through a JAK-independent pathway. Activation of STAT proteins requires the kinase activity of TEL/PDGFβR. JAK1, JAK2, JAK3, and TYK2 are not phosphorylated by TEL/PDGFβR. However, TEL/PDGFβR can phosphorylate STAT5 in transiently transfected COS cells, suggesting that TEL/PDGFβR may itself be the kinase involved in tyrosine phosphorylation of STAT proteins. In contrast, native PDGFβR stimulated by PDGF ligand does not activate STAT proteins to a significant degree in this hematopoietic context. STAT1 and STAT5 also are activated by TEL/ABL and TEL/JAK2 fusion proteins associated with human leukemia. Conclusions STAT activation may be a common mechanism of transformation by leukemogenic tyrosine kinase fusion proteins.

Constitutive activation of the EPO/JAK2 signaling cascade has recently been implicated in a variety of myeloproliferative disorders including polycythemia vera, essential thrombocythemia and myelofibrosis. In an effort to uncover therapeutic potential of blocking the EPO/JAK2 signaling cascade, we sought to discover selective inhibitors that block the kinase activity of JAK2. Herein, we describe the discovery and structure based optimization of a novel series of 2-amino-pyrazolo[1,5-a]pyrimidines that exhibit potent inhibition of JAK2.

Immune checkpoint inhibitors have generated impressive clinical responses, however, a number of patients and cancer types remain resistant to checkpoint blockade. Immunosuppressive cells such as Tregs and MDSCs in the tumor microenvironment are hypothesized to mediate this resistance. The gamma (γ) isoform of PI3K has long been known to modulate myeloid and lymphocyte cell migration and function, including trafficking of monocytic and granulocytic cells into inflamed tissues such as tumors. Here we report the identification and characterization of BVD-723, a PI3Kγ selective small molecule kinase inhibitor which demonstrated single agent and enhanced combination anti-tumor activity[xx]with either an anti-PD-1 or anti-CTLA-4 antibody. In a mouse syngeneic colon cancer model,[xx]BVD-723 in combination with anti-PD-1 or anti-CTLA-4 resulted in complete regression[xx]of tumors in 35% (7/20) and 60% (12/20) of animals treated with each antibody respectively [versus 0% (0/20) and 5% (1/20) with either antibody alone]. Interestingly, the anti-tumor activity was coupled with a decrease in tumor infiltrating Tregs, granulocytic MDSCs, CD4+ T cells and an increase in the CD8+/Treg ratio, suggesting that inhibition of PI3Kγ by BVD-723 promotes immune-reactivation in the tumor microenvironment. Additionally, BVD-723 demonstrated potent synergy with an anti-PD-1 antibody in a syngeneic mouse model of lymphoma. Single agent BVD-723 activity was also observed in syngeneic mouse models of pancreas, colon, lymphoma and bladder cancers. Results from completed safety pharmacology, toxicology and in vivo efficacy studies support clinical evaluation of BVD-723[xx]as a monotherapy or in combination with the checkpoint inhibitors in cancer.[xx] Citation Format: Saurabh Saha, Linping Zhang, Thomas Hoock, Alex Aronov, Sudipta Mahajan, Michael Boyd, Jon Come, Veronique Damagnez, Wojciech Dworakowski, Suvarna Khare-Pandit, Arnaud LeTiran, Cameron Moody, Harwin O'Dowd, Joseph Prezioso, Setu Roday, Xiaoyan M. Zhang. A novel PI3K gamma isoform selective small molecule kinase inhibitor demonstrates single agent anti-tumor activity and enhanced combination activity with checkpoint blockade in syngeneic mouse models of cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B15.

Colorectal carcinoma cells are characterized by over-expression of IL-6 and the IL-6 receptor, an autocrine loop that promotes the development of many tumors. To determine the importance of this pathway, we examined the role that IL-6 plays in the biology of 228 and RKO colorectal tumor cells. IL-6 induced prominent tyrosine phosphorylation of the transcription factor STAT1 in both cell types. Furthermore, IL-6 exerts functional effects in these cells in that it inhibited apoptosis induced by Fas ligation, and up-regulated Bcl-xl, a STAT target gene, which can promote cell survival. Butyrate, a compound formed in the intestines of people who consume a high-fiber diet, may confer protection against the development of colorectal cancer. Given the potential importance of IL-6 in the pathogenesis of colorectal tumors, we tested the hypothesis that butyrate acts by inhibiting IL-6-induced signaling events in colorectal carcinoma cells. Following treatment with butyrate, the activation of STAT1 in response to IL-6, but not interferon-gamma, was completely lost. Butyrate induced a prominent decrease of mRNA and cell surface expression of the IL-6 receptor alpha (IL-6Ralpha) chain. Introduction of a soluble form of the IL-6Ralpha chain restored IL-6-induced STAT1 activation and resistance to apoptosis of butyrate treated cells. These experiments indicate that IL-6 may play an important role in the pathogenesis of colorectal cancers, and that butyrate may exert its protective effect by specifically blocking IL-6-induced signaling events.

Leukemias continue to cause significant mortality in adults and children, and the use of standard cytotoxic chemotherapy has reached a therapeutic plateau. Thus, there is great interest in treatments directed against inappropriately activated cell signaling pathways which stimulate the uncontrolled growth of neoplastic cells. Increasing evidence suggests that the STAT signaling cascade may be one target of these therapies. Signal transducer and activator of transcription (STAT) proteins are critical in mediating the response of hematopoietic cells to a diverse spectrum of cytokines. Constitutive STAT activation is present in many malignancies and has been especially well characterized in acute and chronic leukemias. While STAT activation is a common characteristic of leukemias, the specific pattern of activated STATs and the manner by which STAT activation occurs vary with each disease. STAT tyrosine phosphorylation can occur through inappropriate Jak activation or by direct activation of an oncoprotein such as Bcr/Abl, and STAT serine phosphorylation may play an important role in leukemias as well. Thus, the STAT signaling pathway is an attractive target for therapeutic intervention, and strategies designed to inhibit STAT activation and STAT mediated gene transcription may play an important role in the next generation of anti-leukemia therapies. Oncogene (2000).

Fludarabine is a nucleoside analog used in the treatment of hematologic malignancies that can induce severe and prolonged immunosuppression. Although it can be incorporated into the DNA of dividing cells, fludarabine is also a potent inhibitor of cells with a low growth fraction, thus it must have other mechanisms of action. STAT1, which is activated in response to many lymphocyte-activating cytokines including the interferons, is essential for cell-mediated immunity, as the absence of this protein is associated with prominent defects in the ability to control viral infections. Here we show that fludarabine, but not the immunosuppressant cyclosporine A, inhibits the cytokine-induced activation of STAT1 and STAT1-dependent gene transcription in normal resting or activated lymphocytes. Fludarabine caused a specific depletion of STAT1 protein (and mRNA) but not of other STATs. This loss of STAT1 was also seen in cells from patients treated with fludarabine in vivo. Brief exposure to fludarabine led to a sustained loss of STAT1, analogous to the prolonged period of immunosuppression induced by exposure to the drug in vivo. Thus, STAT1 may be a useful target in the development of new immunosuppressive and antineoplastic agents.

To explore the pathogenesis of chronic lymphocytic leukemia (CLL), we examined whether phosphorylation of one or more signal transducer and activator of transcription (STAT) factors was abnormal in cells from CLL patients. No constitutive tyrosine phosphorylation was detected on any STAT in CLL cells. To assess the phosphorylation of serine residues of STAT1 and STAT3 in CLL cells, we raised antibodies that specifically recognize the form of STAT1 phosphorylated on ser-727 and the form of STAT3 phosphorylated on ser-727. We found that in 100% of patients with CLL (n = 32), STAT1 and STAT3 were constitutively phosphorylated on serine. This was in contrast to normal peripheral blood B lymphocytes or CD5+) B cells isolated from tonsils, in which this phosphorylation was absent. Serine phosphorylation of STAT1 and STAT3 was seen occasionally in other leukemias, but it was a universal finding only in CLL. The serine phosphorylation of these STATs was a continuous process, as incubation of CLL cells with the kinase inhibitor H7 led to the dephosphorylation of these serine residues. The STAT serine kinase in CLL cells has not been identified, and appears to be neither mitogen-activated protein kinase nor pp70(s6k). In summary, the constitutive serine phosphorylation of STAT1 and STAT3 is present in all CLL samples tested to date, although the physiologic significance of this modification remains to be determined.