The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.

We examined the phosphorylation and acetylation of histone H3 in ovarian granulosa cells stimulated to differentiate by follicle-stimulating hormone (FSH). We found that protein kinase A (PKA) mediates H3 phosphorylation on serine 10, based on inhibition exclusively by PKA inhibitors. FSH-stimulated H3 phosphorylation in granulosa cells is not downstream of mitogenactivated protein kinase/extracellular signal-regulated kinase, ribosomal S6 kinase-2, mitogen-and stressactivated protein kinase-1, p38 MAPK, phosphatidylinositol-3 kinase, or protein kinase C. Transcriptional activation-associated H3 phosphorylation on serine 10 and acetylation of lysine 14 leads to activation of serum glucocorticoid kinase, inhibin ␣, and c-fos genes. We propose that phosphorylation of histone H3 on serine 10 by PKA in coordination with acetylation of H3 on lysine 14 results in reorganization of the promoters of select FSH responsive genes into a more accessible configuration for activation. The unique role of PKA as the physiological histone H3 kinase is consistent with the central role of PKA in initiating granulosa cell differentiation.Maturation of ovarian follicles to a preovulatory stage requires follicle-stimulating hormone (FSH) 1 production by the pituitary gland. The FSH receptor is a member of the G protein-coupled seven-transmembrane receptor family and is coupled to adenylyl cyclase (1). It is expressed exclusively on ovarian granulosa cells in female mammals (2). Most of the actions of FSH are mediated by cAMP formation and activation of protein kinase A (PKA), based on the ability of cell-permeable cAMP analogs to mimic the known differentiation responses to FSH in granulosa cells (2) and on the ability of the PKA inhibitors H89 2 (3) and KT5720 2 to inhibit granulosa cell differentiation. The downstream consequences of FSH are well established and include, for example, the induction of receptors for luteinizing hormone (LH) and prolactin, induction of enzymes associated with the increased steroidogenic capacity of granulosa cells including P450 aromatase and cholesterol side chain cleavage, induction of proteins associated with PKA signaling including RII␤ (2, 4) and AKAP80 (5), and expression of the hormone inhibin (6). However, gene and/or protein induction for these responses to FSH is generally delayed by at least 24 h (2-4, 7). The more immediate responses to FSH, which lead to the induction of immediate early genes such as c-fos and serum glucocorticoid kinase (SGK) (3,8), are less well understood. Further elucidation of the FSH signaling pathways that lead to the induction of immediate early genes would be useful to understand how FSH initiates granulosa cell differentiation. We have previously shown that FSH (via PKA) promotes activation of the p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (9). FSH also activates the p38 MAPK pathway (10) and downstream phosphorylation of the heat shock protein (HSP) 27, leading to granulosa cell rounding (10). We 3 and ot...

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca 2؉ channels are already partially activated in vehicletreated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicletreated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca 2؉ channel blocker, and chelation of extracellular Ca 2؉ . These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.The cytoplasmic p42/p44 mitogen-activated protein kinase (MAPK) 1 /extracellular signal-regulated kinases (ERKs) comprise a critical convergence point in the signaling pathways initiated by a variety of receptor agonists that promote cellular differentiation or proliferation. For the classic receptor tyrosine kinase-initiated pathway, growth factors like epidermal growth factor (EGF) induce the autophosphorylation of their receptors and create specific binding sites for Src homology 2-containing proteins such as Grb2 (1). Grb2 complexed to Sos associates with the receptor tyrosine kinase, and Sos stimulates GDP release from Ras, leading to Ras activation. Active Ras then binds to Raf-1, leading to its activation, and Raf-1 in turn catalyzes the serine phosphorylation and activation of the MAPK/ERK kinase MEK. MEK then catalyzes the phosphorylation of ERK on regulatory Thr and Tyr residues, resulting in ERK activation.Guanine nucleotide-binding protein-coupled receptors (GPCRs) are also well known activators of ERK; however, there are a variety of pathways by which GPCRs promote ERK activation. Often, GPCRs such as those activated by lysophosphatidic acid or angiotensin II promote the transactivation of a receptor tyrosine kinase as evidenced by its increased tyrosine phosphorylation (2). Receptor tyrosine kinase transactivation directs the tyrosine phosphorylation of adaptor proteins such as Shc, recruitment of the Grb2-Sos complex, and subsequent Ras activation. It is less clear how GPCRs promote the tyrosine phosphorylation of the receptor tyrosine kinase, although Src activation downstream of the G␤␥ has been implicated in some cells (3, 4). For those GPCRs whose activated G␣ subunits promote...

Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC (NCT03214250). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal expression profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dpp3a, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dpp3a compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.