This study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation. Additional supportive evidence for nuclear accumulation of zyxin-enhancing cardiomyocyte survival includes the following: (a) promotion of zyxin nuclear localization by cardioprotective stimuli; (b) zyxin association with phospho-Akt 473 induced by cardioprotective stimuli; and (c) recruitment of zyxin to the nucleus by activated nuclear-targeted Akt as well as recruitment of Akt by nuclear-targeted zyxin. Nuclear accumulation of zyxin requires both Akt activation and nuclear localization. Potentiation of cell survival is sensitive to stimulation intensity with high-level induction by ANP or cGMP signaling leading to apoptotic cell death rather than enhancing resistance to apoptotic stimuli. Myocardial nuclear accumulation of zyxin and Akt responds similarly in vivo following treatment of mice with ANP or cGMP. Thus, zyxin and activated Akt participate in a cGMP-dependent signaling cascade leading from ANP receptors to nuclear accumulation of both molecules. Nuclear accumulation of zyxin and activated Akt may represent a fundamental mechanism that facilitates nuclear-signal transduction and potentiates cell survival.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG(1)) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122) and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the antitumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, ␣B-crystallin (␣BC), in cultured cardiac myocytes, we examined ␣BC levels in the mouse hearts. The level of ␣BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in ␣BC levels in the mitochondrial fractions of TG mouse hearts but no increase in ␣BC levels in any of the other fractions analyzed. These alterations in ␣BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts.

. Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6. Am J Physiol Heart Circ Physiol 291: H2462-H2472, 2006. First published June 9, 2006; doi:10.1152/ajpheart.01311.2005.-Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.ischemia-reperfusion; mitochondrial complex proteins; mitogenactivated protein THE MYOCARDIUM CAN BE STRESSED by chronic increases in blood pressure, changes in neurohumoral substances, and ischemia followed by reperfusion (I/R). Numerous signaling pathways, including the mitogen-activated protein kinases (MAPK), are activated in stressed cardiac myocytes; in some cases, those signals foster protection, while in others they mediate damage (5,22,24,42,46). All three members of the MAPK family are activated during most myocardial stresses; however, the roles played by each in contributing to protection or damage are not entirely clear. For example, p38 MAPK has been reported to have both protective and damaging effects in the myocardium; this conundrum is addressed in recent reviews (1, 24, 31-33) and will, therefore, not be discussed in detail here. However, such apparently conflicting findings raise the possibility that p38 might serve either protective or damaging roles, depending on conditions, such as the cellular and temporal context and differential activation ...

SGN-2FF, an orally bioavailable small molecule inhibitor of glycoprotein fucosylation, demonstrates encouraging preclinical antitumor activity in mouse models with suggested multiple mechanisms of action, including direct and indirect effects on immune cells, tumor cells, and the tumor microenvironment. The effects of SGN-2FF were evaluated on tumors implanted in multiple strains of mice to determine how differences in the immune repertoire affect the antitumor activity. SGN-2FF treatment of nude mice, which maintain functional B cells and antibody production, resulted in a delay in LS174T tumor growth compared with untreated mice, while LS174T tumors in SCID mice, which lack B cells, were unaffected by SGN-2FF. These data suggest that activity of SGN-2FF in nude mice may be dependent on residual B cells and circulating antibodies. The antitumor effect of SGN-2FF in syngeneic mouse models with intact immune systems also appears to be dependent on T cell activity. Transfer of T cells isolated from SGN-2FF-treated tumor-bearing mice to naïve tumor-bearing mice was sufficient to delay tumor growth. T cells isolated from untreated tumor-bearing mice did not have the same effect. These results demonstrate that afucosylated immune cells play a key role in the preclinical activity of SGN-2FF. Various preclinical assays were used to detect SGN-2FF-mediated changes in cellular and IgG fucosylation important for biological activity. These assays are being applied in evaluating patient samples in the ongoing phase 1, multicenter, dose-escalation study investigating the safety, tolerability, PK, and biomarkers of antitumor activity of SGN-2FF administered orally to adult patients with advanced solid tumors (NCT# 02952989). Changes in peripheral IgG fucosylation, absolute neutrophil count, and immune cell surface fucosylation were identified as initial biomarkers for proof of pharmacodynamic activity. Preliminary data following daily doses of SGN-2FF demonstrate that cell surface fucosylation on granulocytes was significantly reduced and neutrophil count was significantly increased in 6 of 7 treated subjects; additionally, IgG fucosylation was significantly decreased in 7 of 7 subjects. PK have been characterized, and preliminary results are within the expected range as predicted from preclinical studies. Following daily administration of SGN-2FF, accumulation of the active metabolite, GDP-2FF, was observed intracellularly, while no accumulation of SGN-2FF was observed in plasma. Collectively, these data demonstrate robust biological effects of SGN-2FF. The pharmacodynamic biomarkers and PK analysis are informing next steps in identifying an optimal dose and dosing schedule for SGN-2FF. Citation Format: Nicole M. Okeley, Ryan A. Heiser, Weiping Zeng, Shawna Mae Hengel, Jason Wall, Peter C. Haughney, Timothy Anthony Yap, Francisco Robert, Rachel E. Sanborn, Howard Burris, Laura Q. Chow, Khanh T. Do, Martin Gutierrez, Karen Reckamp, Amy Weise, D Ross Camidge, John Strickler, Conor Steuer, Zejing Wang, Megan M. O'Meara, Stephen C. Alley, Shyra J. Gardai. SGN-2FF: A small-molecule inhibitor of fucosylation modulates immune cell activity in preclinical models and demonstrates pharmacodynamic activity in early phase 1 analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5551.