Fluorinated piperidine acetic acids as gamma-secretase modulators.

We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.

Purine derivatives as potent gamma-secretase modulators.

The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.

Characterization of murine CEACAM1 in vivo reveals low expression on CD8+ T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

Transferring Luminex® cytokine assays to a wall-less plate technology: Validation and comparison study with plasma and cell culture supernatants.

Luminex® technology provides a powerful methodology for multiplex cytokine detection but remains constrained by high costs and a minimum of 25-50µL sample volume requirement per assay-well often hindering analysis of limited biological samples. Here we compare the results of Luminex-based cytokine multiplexing assay performed using conventional 96-well microtiter plates and a particular 96-well wall-less plate based on Droparray® technology ("DA-Bead"). The application of the DA-Bead plate allows 80% reduction of sample and reagent volume, thus an opportunity for significant cost savings in Luminex reagents with no change to the workflow. To compare the DA-Bead method to the conventional method, two different types of samples were tested with two different commercially available Luminex kits and the results for each method were compared. The first type was splenocyte culture supernatants from murine spleens which were harvested from mice immunized with Ascaris suum protein As24 and followed by cell stimulation ex vivo at various time points with this same antigen. Cytokine levels in these supernatants were evaluated using a Bio-Plex® TH1/TH2 8-plex kit. The second sample type was plasma from mice from an experimental autoimmune encephalomyelitis (EAE) study, and these samples were evaluated using a Milliplex® TH17 25-plex kit. The data showed that the DA-Bead method for analysis was comparable to, if not superior to, the conventional method in terms of consistency/precision, accuracy, sensitivity and dynamic range and these results are not specific to sample type, reagents, or commercial vendor.

MCL1 and BCL-xL levels in solid tumors are predictive of dinaciclib-induced apoptosis.

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors.

The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical efficacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway. Here, we describe the identification and characterization of SCH772984, a novel and selective inhibitor of ERK1/2 that displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency in tumor cells with mutations in BRAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor-resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and MEK inhibitors. These data support the clinical development of ERK inhibitors for tumors refractory to MAPK inhibitors.

Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors.

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

PDK1 attenuation fails to prevent tumor formation in PTEN-deficient transgenic mouse models.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.

Splenectomy normalizes hematocrit in murine polycythemia vera.

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2(V617)) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2(V617F)-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2(V617F) transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2(V617)) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2(V617F)-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen.

A quantitative volumetric micro-computed tomography method to analyze lung tumors in genetically engineered mouse models.

Two genetically engineered, conditional mouse models of lung tumor formation, K-ras(LSL-G12D) and K-ras(LSL-G12D)/p53(LSL-R270H), are commonly used to model human lung cancer. Developed by Tyler Jacks and colleagues, these models have been invaluable to study in vivo lung cancer initiation and progression in a genetically and physiologically relevant context. However, heterogeneity, multiplicity and complexity of tumor formation in these models make it challenging to monitor tumor growth in vivo and have limited the application of these models in oncology drug discovery. Here, we describe a novel analytical method to quantitatively measure total lung tumor burden in live animals using micro-computed tomography imaging. Applying this methodology, we studied the kinetics of tumor development and response to targeted therapy in vivo in K-ras and K-ras/p53 mice. Consistent with previous reports, lung tumors in both models developed in a time- and dose (Cre recombinase)-dependent manner. Furthermore, the compound K-ras(LSL-G12D)/p53(LSL-R270H) mice developed tumors faster and more robustly than mice harboring a single K-ras(LSL-G12D) oncogene, as expected. Erlotinib, a small molecule inhibitor of the epidermal growth factor receptor, significantly inhibited tumor growth in K-ras(LSL-G12D)/p53(LSL-R270H) mice. These results demonstrate that this novel imaging technique can be used to monitor both tumor progression and response to treatment and therefore supports a broader application of these genetically engineered mouse models in oncology drug discovery and development.

Inhibition of tumor growth progression by antiandrogens and mTOR inhibitor in a Pten-deficient mouse model of prostate cancer.

Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients.