Abstract Background: The VEGF/VEGFR and HGF/cMet pathways are implicated in tumor survival, growth, angiogenesis, invasion and metastasis. DARPins (designed ankyrin repeat proteins) are small genetically engineered proteins that bind to specific targets with high affinity. MP0250 is a first-in-class, tri-specific DARPin with the ability to simultaneously neutralize the activities of VEGF and HGF and also to bind to human serum albumin (HSA) to give an increased plasma half-life and potentially enhanced tumor penetration. MP0250 is currently being explored in a Phase I study. Methods: A phase I, open-label, repeated-dose, dose escalation, multi-center study to assess safety, tolerability and pharmacokinetics of MP0250 is in progress in patients with advanced solid tumors who have progressed on at least one prior standard therapy. Using a 3+3 design, eligible patients are being enrolled into dose escalation cohorts receiving MP0250 by intravenous infusion every other week until disease progression or unacceptable toxicity. Results: Twelve patients have been enrolled in the first three cohorts dosed with MP0250 at 0.5 (n = 3), 1.5 (n = 3) and 4 mg/kg (n = 6). MP0250 has been well tolerated and a maximum tolerated dose has not been reached. A single dose-limiting toxicity was observed at 4mg/kg (significant reduction in cardiac ejection fraction after 1st infusion in a patient with multiple cardiac risk factors). The most frequent adverse events (AEs, CTC version 4.03) were transient hypertension (42%), diarrhea (33%), fatigue (25%) and nausea (25%). With the exception of hypertension (grade 3 in 33% of all patients), all AEs were grade 1 or 2. Interim pharmacokinetic analyses indicated linear behavior between doses 1.5 and 4 mg/kg with a mean half-life of approximately 11 days (range 9-15 days). Sustained exposure was observed for all patients throughout the treatment periods analyzed, the longest to-date being 9 months. Stable disease for 10 months (treatment ongoing) has been observed in one patient with a head and neck tumor and for 8 months in a patient with a cervical adenocarcinoma. Conclusion: Preliminary data from the Phase I study of the first-in-class, tri-specific VEGF, HGF and HSA binding DARPin MP0250 shows it to be well tolerated, to have a mean half-life around 11 days and to have sustained exposure on repeated dosing. In addition, there was disease stabilization exceeding 8 months in two patients suggestive of anti-tumor activity. Further dose escalation is ongoing. Citation Format: Jordi Rodon, Aurelius Omlin, Karin H. Herbschleb, Javier Garcia-Corbacho, Jan Steiner, Ignacio Dolado, Christof Zitt, Daniel Feurstein, Dascha Turner, Keith M. Dawson, Michael T. Stumpp, Patrick Gilboy, Andreas Harstrick, Analía Azaro, Christoph J. Ackermann, Mark R. Middleton, Richard D. Baird. First-in-human Phase I study to evaluate MP0250, a DARPin blocking HGF and VEGF, in patients with advanced solid tumors. [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 B25.

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor-c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.

The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H(2)O(2)) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/β1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H(2)O(2)-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H(2)O(2)-induced ASK1-karyopherin α2/β1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H(2)O(2)-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/β1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress.

The design and synthesis of a potent series of c-jun N-terminal kinase (JNK2) inhibitors is described. The development and optimization of the 2,4-diaminopyrimidines series was carried out from an earlier in-house kinase inhibitor program. Through the optimization of the scaffold 2, several cell potent compounds with good in vivo profiles were discovered.

The apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed serine/threonine protein kinase and one of more than 20 members that make up the triple MAP kinase (MAP3K) family of enzymes. Over the past decade, genetic studies have revealed that ASK1 plays a pivitol role in the cellular response to a wide variety of environmental and biological stressors including; reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress caused by protein aggregation, influx of calcium ions, and receptor-mediated signals transduced via lipopolysaccharides (LPS), Fas ligand, cytokines (TNFα) and certain G protein-coupled receptor (GPCR) agonists [1-5]. In addition, exogenous expression of ASK1 in cells has shown that ASK1 signaling engages the intrinsic apoptosis pathway promoting cytochrome c release from mitochodria and subsequent activation of caspase 3 and 9 [1, 6, 7]. Conversely, ASK1 deficient cells are resistant to cell death induced by oxidative and ER stress, indicating that ASK1 acts as the lynch pin in certain forms of stress-induced cell death [8]. Once activated, ASK1 relays cellular stress signals via the classical three tierd mitogen activated protein kinase (MAPK) signaling cascade, whereby a MAP3K phosphorylates and activates a MAP2K, that in turn phosphorylates and activates a MAPK [9] (Figure 1). More specifically, the ASK1 signaling axis activates the p38 and the c-jun NH2-terminal kinases (JNK) family of MAPKs, via activation of MKK3/MKK6 and MKK4/MKK7 respectively [1, 2, 4]. In addition to its role in the cellular stress response, ASK1 also regulates physiological processes including neuronal differentiation, synaptic plasticity and the innate immune response [10-13]. Thus, ASK1 acts as an important regulator of several important biological processess and not surprisingly, ASK1 activation is under tight regulatory control. Regulation of ASK1 activity is accomplished via a number of mechanisms including; protein-protein interactions as well as both spatial and temporal control. Firstly, more than 30 ASK1 interacting partners have been shown to regulate ASK1 activity (either positively or negatively) by posttranslational modifications and/or by inducing conformational changes through protein-protein interactions. Secondly, ASK1 signaling complexes are located in both the cytoplasm and mitochondria [14], with nuclear translocation observed upon stress induction indicating that ASK1 localization might also dictate the biological outcome [15,16] and thirdly, duration of ASK1 signaling can influence the nature of the

Abstract The structure-specific flap endonuclease 1 (FEN-1) is a multifunctional enzyme that participates in various activities such as DNA replication, DNA repair and apoptotic DNA fragmentation. Several mechanisms, including protein-protein interactions, sub-cellular compartmentalization and posttranslational modifications have been proposed to explain how FEN1 accomplishes these various activities. For example, the C-terminus of FEN1 has been shown to be acetylated by the histone acetyl transferase protein (HAT) p300 which is known to result in a significant reduction in the DNA binding and nuclease activities of FEN1. We now demonstrate that the NAD-dependent class III histone deacetylase SIRT1 physically interacts with FEN1 and that the opposing deacetylation by SIRT1 subsequently restores the endonuclease activity of FEN1. Moreover, clonogenicity experiments suggest that genetic knockdown of FEN1 or SIRT1 in cancer cells sensitizes those cells to irradiation treatment. This is of significance as it has been observed that increased SIRT1 levels protect mice from irradiation-induced cancer. Furthermore, we have also identified two nuclear shuttling proteins, Karyopherin α2 and Karyopherin β1 as interacting partners of both SIRT1 and FEN1. Using mass spectrometry, immunoprecipitation combined with subsequent western-blot analysis and confocal microscopy we demonstrate that, under basal conditions, a significant amount of both SIRT1 and FEN1 is located in the cytoplasm of cells. However, after induction of DNA damage by the methylating agent MMS, Karyopherin α2 and Karyopherin β1 mediate the translocation of SIRT1 and FEN1 into the nucleus in a time dependent manner. In summary we describe a novel interaction between SIRT1 and FEN1, demonstrate that acetylation/deacetylation regulates FEN1 activity and that the Karyopherins α2 and β1 mediate the nuclear import of SIRT1 and FEN1 following DNA damage. Our results suggest that inhibiting the nuclear import or activation of FEN1 or SIRT1 could represent potential anticancer strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2011-4193

Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine kinase that responds to a plethora of stress-inducing signals. In turn, activation of ASK1 is associated with a number of human pathological conditions, including neurodegenerative disease, inflammation, and heart failure. In response to oxidative stress, ASK1 activates the cell death-associated p38 MAPK pathway by phosphorylating MKK6. Here, we investigated the regulation of oxidative stress-induced ASK1-catalyzed phosphorylation of MKK6. MKK6 phosphorylation levels increased immediately after H(2)O(2) treatment in intact cells and decreased following treatment for 30 min. When expressed in HEK293T cells, ASK1 was reproducibly purified within a high-molecular mass complex ( approximately 1500 kDa) known as the ASK1 signalosome. Measurement of the in vitro kinetic parameters revealed that the catalytic efficiency (k(cat)/K(m)) of ASK1 was 4000-fold greater in cells treated with H(2)O(2) for 3 min than in untreated cells. Interestingly, although the K(m(ATP)) values were found to be unchanged, the K(m(MKK6)) was dramatically decreased ( approximately 1000-fold). The increased affinity was specific for MKK6 and short-lived, as the K(m(MKK6)) returned to basal levels 30 min after treatment. Consistently, endogenous MKK6 was found within the ASK1 signalosome in intact cells and in addition copurified with ASK1 following treatment for 3 min. In contrast, proteins modulating ASK1 activity and degradation were found to interact with the ASK1 signalosome once MKK6 activation was completed. Taken together, these data suggest that oxidative stress rapidly increases ASK1 catalytic efficiency for MKK6 phosphorylation by increasing MKK6 binding affinity within the ASK1 signalosome prior to induction of inactivation and degradation of the complex.