The P2Y14 receptor (P2Y14R), one of eight P2Y G protein-coupled receptors (GPCR), is involved in inflammatory, endocrine, and hypoxic processes and is an attractive pharmaceutical target. The goal of this research is to develop high-affinity P2Y14R fluorescent probes based on the potent and highly selective antagonist 4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid (6, PPTN). A model of hP2Y14R based on recent hP2Y12R X-ray structures together with simulated antagonist docking suggested that the piperidine ring is suitable for fluorophore conjugation while preserving affinity. Chain-elongated alkynyl or amino derivatives of 6 for click or amide coupling were synthesized, and their antagonist activities were measured in hP2Y14R-expressing CHO cells. Moreover, a new Alexa Fluor 488 (AF488) containing derivative 30 (MRS4174, Ki = 80 pM) exhibited exceptionally high affinity, as compared to 13 nM for the alkyne precursor 22. A flow cytometry assay employing 30 as a fluorescent probe was used to quantify specific binding to P2Y14R. Known P2Y receptor ligands inhibited binding of 30 with properties consistent with their previously established receptor selectivities and affinities. These results illustrate that potency in this series of 2-naphthoic acid derivatives can be preserved by chain functionalization, leading to highly potent fluorescent molecular probes for P2Y14R. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands. This approach demonstrates the predictive power of GPCR homology modeling and the relevance of newly determined X-ray structures to GPCR medicinal chemistry.

In addition to their role in glycosylation reactions, UDP-sugars are released from cells and activate widely distributed cell surface P2Y 14 receptors (P2Y 14 R). However, the physiological/pathophysiological consequences of UDPsugar release are incompletely defined. Here, we report that UDP-glucose levels are abnormally elevated in lung secretions from patients with cystic fibrosis (CF) as well as in a mouse model of CF-like disease, the βENaC transgenic (Tg) mouse. Instillation of UDP-glucose into wild-type mouse tracheas resulted in enhanced neutrophil lung recruitment, and this effect was nearly abolished when UDP-glucose was coinstilled with the P2Y 14 R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid]. Importantly, administration of PPTN to βENaC-Tg mice reduced neutrophil lung inflammation. These results suggest that UDP-glucose released into the airways acts as a local mediator of neutrophil inflammation.

Burkitt lymphoma (BL) is caused by translocation of the MYC gene to an immunoglobulin locus resulting in its constitutive expression depending on the activity of the immunoglobulin (Ig) enhancer elements. Treatment of BL cell lines with epigenetic modifiers is known to repress B-cell-specific genes and to up-regulate B-cell-inappropriate genes including the transcription repressor ID2 expression. We found that the DNA methyltransferase inhibitor decitabine/5-aza-2-deoxycytidine (5-aza-dC) represses the MYC oncogene on RNA and protein levels by inducing ID2. Down-regulation of MYC was associated with repression of transcriptional activity of the Ig locus and with inhibition of proliferation. The induction of ID2 can be in part explained by activation of the transcription factor NF-κB. We conclude that up-regulation of ID2 contributes to anti-tumour activity of 5-aza-dC via repression of Ig locus activity and consequently MYC expression.

Key Points• FOXO1 directly activates PRDM1a, the master regulator of PC differentiation, and it enriches a PC signature in cHL cell lines. • PRDM1a is a tumor suppressor in cHL.The survival of classical Hodgkin lymphoma (cHL) cells depends on activation of NF-kB, JAK/STAT, and IRF4. Whereas these factors typically induce the master regulator of plasma cell (PC) differentiation PRDM1/BLIMP-1, levels of PRDM1 remain low in cHL. FOXO1, playing a critical role in normal B-cell development, acts as a tumor suppressor in cHL, but has never been associated with induction of PC differentiation.Here we show that FOXO1 directly upregulates the full-length isoform PRDM1a in cHL cell lines. We also observed a positive correlation between FOXO1 and PRDM1 expression levels in primary Hodgkin-Reed-Sternberg cells. Further, we show that PRDM1a acts as a tumor suppressor in cHL at least partially by blocking MYC. Here we provide a link between FOXO1 repression and PRDM1a downregulation in cHL and identify PRDM1a as a tumor suppressor in cHL. The data support a potential role for FOXO transcription factors in normal PC differentiation. (Blood. 2014;124(20):3118-3129) IntroductionClassical Hodgkin lymphoma (cHL) differs from other B-cell lymphomas by a unique phenotype of the malignant cells of cHL, Hodgkin and Reed-Sternberg (HRS) cells, characterized by loss of the B-cell program and by the formation of a reactive infiltrate harboring these cells. 1 The repression of the B-cell program in HRS cells is characterized by downregulation of critical B-cell transcription factors, 2 including BCL6. 3 The oncogenic program of cHL is based on the constitutive activation of JAK/STAT, NF-kB, ERK, and PI3K/AKT pathways. [4][5][6][7] Ultimate targets of these pathways are the protooncogenic transcription factors MYC and IRF4. 8,9 Of note, NF-kB, STAT3, and IRF4 converge to induce plasma cell (PC) differentiation in normal B cells by upregulation of the PC master transcription factor PRDM1/BLIMP-1. [10][11][12] Surprisingly, HRS cells express only low levels of PRDM1 and other PC markers. 3,13 PRDM1 acts as a tumor suppressor in diffuse large B-cell lymphoma of the activated B-cell type (ABC-DLBCL), whose oncogenic program resembles that of cHL, including dependency on constitutive NF-kB and JAK-STAT activation and high expression of IRF4. 1,8,14,15 Of note, NF-kB activation in B cells results in the development of lymphomas only when Prdm1 is deleted. 16 We found that FOXO1, which is highly expressed in B cells and in different types of non-Hodgkin lymphoma, is downregulated in cHL. Overexpression of a constitutively active FOXO1 protein induces growth arrest and apoptosis in cHL cell lines.17 FOXO1 belongs to the forkhead box (Fhbox) family of transcription factors that regulates different physiological processes including cell death, differentiation, and oxidative stress.18 FOXO1 plays a central role in the early stages of B-cell differentiation, 19 and it is essential for the expression of B-cell-specific genes such as BCL6, AICDA, an...

Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative MedB-1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL.